BACKGROUND
the human body is home to a vast number of different bacterial species, and the overwhelming complexity of the human microbiome has only very recently been fully uncovered  <cit> .
although the majority of these colonizing bacteria seem to be harmless or even beneficial commensals, some have long been known to be janus-faced, and neisseria meningitidis is a particularly prominent example in this respect.
on the one hand, this β-proteobacterium is an exclusively human-adapted commensal that is carried in the nasopharynx of about 20% of the healthy population  <cit> .
on the other hand, n. meningitidis is also a ferocious pathogen that can cause life-threatening invasive meningococcal disease , and “no other infection so quickly slays”  <cit> .
after crossing the mucosal barrier and entering the bloodstream, meningococci can cause septicemia, and by crossing the blood–brain barrier and multiplying in the cerebrospinal fluid  also acute bacterial meningitis, both often within less than 24 h  <cit> .
in many commensal pathogens like escherichia coli, often the only difference between a pathogenic and a non-pathogenic strain is a small set of so called virulence genes  <cit> .
by definition, a virulence gene is a gene whose loss specifically impairs virulence but not viability in rich media and which should be associated exclusively with pathogenic but not with non-pathogenic strains of a species  <cit> .
however, with respect to gene content meningococcal strains isolated from healthy carriers and imd patients are almost indistinguishable  <cit> , and many of the so called meningococcal virulence genes have also been found in purely commensal neisserial species  <cit> .
the analysis of meningococcal population genetic structure by multilocus sequence typing  demonstrated that disease-causing meningococci do belong to particular groups of related sequence types , termed clonal complexes , which are overrepresented in disease isolates relative to their carriage prevalences and are responsible for most disease  <cit> .
accordingly, these data indicate that the propensity to cause invasive disease is somehow associated with the genetic make-up of hyperinvasive lineages.
experimental observations along with epidemiological models further indicate that genetic differences in metabolic genes might have a central role in the observed virulence differences among different lineages in a yet to define manner  <cit> .
alongside the well-established significance of gene content variation in creating genetic diversity in the bacterial world, regulatory evolution is increasingly acknowledged to substantially contribute to this diversity  <cit> .
mutations affecting gene expression regulation encompass differences in the coding sequences of transcription factors  acting in trans and thus affecting the expression of entire regulons  <cit> , as well as sequence differences in the regulatory regions acting in cis on the expression of downstream genes .
by acquiring functionally divergent homologous promoter regions through horizontal transfer bacterial genes were shown to rapidly shift between multiple regulatory modes affecting, for example, up to 15% of the meningococcal core genome  <cit> .
in addition, also mutations in metabolic genes can indirectly cause compensatory changes in gene expression regulation of other housekeeping genes to maintain cellular homeostasis.
the ensuing differential regulation of conserved genes can mediate phenotypic traits that distinguish closely related bacterial species  <cit>  or even strains of the same species  <cit> .
in consequence, also the expression of a virulence-associated gene could be epistatic and thus depend on the genetic background of the respective strain   <cit>  and/or the environment   <cit> .
given the high genetic diversity of n. meningitidis  <cit> , a virulence gene candidate should consequently be differently expressed between an invasive and a commensal strain under disease mimicking conditions but not under conditions mimicking the commensal state.
in addition, although the correlation between when genes are important for fitness and when those genes are upregulated was shown to be small  <cit> , the fitness of a knock-out strain should differ between an invasive and a commensal strain  under conditions mimicking invasive infection .
the strict tropism of n. meningitidis for humans has so far impeded the development of a suitable animal model to analyze the course of meningococcal infection in vivo, and therefore alternative experimental approaches such as ex vivo models have been established to study meningococcal infection biology.
for example, human whole blood served as an ex vivo model to analyze how meningococci regulate gene expression to permit survival in human bloodstream during septicemia  <cit> .
likewise, meningococcal resistance to human complement was studied in an ex vivo model using human csf  <cit> , and human saliva has already been used to study ex vivo the transcriptional response which enables meningococci to adapt to this relevant host niche  <cit> .
however, most of these ex vivo studies analyzed gene expression only under a single condition and/or used only a single strain from a hyperinvasive lineage.
consequently, our knowledge about how this commensal pathogen adapts during the transition from colonization to an invasive infection is still very limited, and nothing is known so far about gene expression or phenotypic variability between carriage and hyperinvasive strains in conditions mimicking invasive infection.
in a systems biological approach we used different ex vivo conditions as environmental perturbation and natural genetic variation as genetic perturbation of the meningococcal gene expression network and considered gene expression as quantitative intermediate phenotype  <cit> .
based on prior population genetic information we selected two genetically related meningococcal serogroup b strains from the same phylogenetic clade pc32/ <dig>  <cit>  with yet markedly different epidemiology   <cit> .
strain mc <dig> belonging to the hyperinvasive st- <dig> cc was chosen as a reference as this strain has already served as a model system to experimentally study meningococcal infection biology in vitro and a large body of transcriptomic data is thus available .
the carriage strain α <dig> belongs to the carriage st- <dig> cc which is a four locus variant of the st- <dig> cc sharing about 96% of its genes with strain mc <dig>  <cit> .
we combined phenotypic, genomic and transcriptomic comparisons with mutagenesis studies to seek genetic variants that influence meningococcal gene expression in human saliva, whole blood and csf mimicking commensal and virulence compartments, respectively.
gene expression was analyzed in a strain- and condition-dependent manner with particular emphasis on virulence-associated genes and genes involved in gene expression regulation.
the combined data show that transcriptomic buffering of cryptic genetic variation, which is the genetic variation present in the meningococcal population that is not phenotypically expressed under commensal conditions but visible upon environmental or genetic perturbations such as growth in human blood  <cit> , contributes to the regulatory evolution of meningococcal virulence.
we further demonstrate that it is likely affected by the differential presence of a short, non-coding inverted-repeat transposable-element in the promoter region of rela encoding the guanosine 3'-diphosphate 5-'diphosphate ppgpp) synthetase of the stringent response pathway.table  <dig> strains used for ex vivo transcriptome comparisons
phylogenetic clade
frequency of cc in carriers
disease/carriage ratio
serum resistance
adhesion to epithelial cells
invasion of epithelial cells
in vitro logarithmic growth rates
ex vivo growth rates
according to ref.
<cit>
according to ref.
<cit>
according to ref.
<cit>
ratio in percent of viable bacteria after incubation for 30 min in the presence of 10% human serum and viable bacteria incubated without serum.
given are the average and standard deviation from four independent experiments with pooled human serum
ratio in percent of adherent and invasive bacteria, respectively, to total bacteria.
given are the average and standard deviation from at least four independent experiments
given are the mean and standard deviation of the logarithmic growth rate k according to log/od
600) = kt for t ∈  as depicted in fig.
<dig> using linear regression
given are the mean and standard deviation of the growth rate k according to log/n) = kt for t ∈  as depicted in fig.
<dig> using linear regression
RESULTS
mc <dig> and α <dig> have similar gene content including most genes invovled in host interactions but with function-dependent sequence variation among orthologs
in order to comprehensively analyze genetic differences between both strains we generated a draft sequence of the α <dig> genome for comparative genome expression analyses .
the common genomic backbone of both strains comprises  <dig>  mbp and encodes  <dig> orthologous proteins with an average blastp bit score ratio  of  <dig>   = ), corresponding to an average amino acid identity of  <dig> % .
notably, there was a significant variation of the bsrps of orthologous proteins with respect to the cog functional category  <cit>   .
the 10% most divergent orthologous genes  were significantly enriched for genes involved in cell motility  =  <dig> , false discovery rate  =  <dig> , fisher’s exact test with benjamini-hochberg multiple testing correction) as well as secretion and transport .
the latter included numerous surface antigens involved in host interactions such as the major type iv pilus subunit protein pile, the major outer membrane proteins pora and porb, the autotransporters app  and nalp , as well as proteins involved in iron acquisition like the transferrin-binding protein b and the lactoferrin-binding protein b. compared to the 10% most divergent orthologous genes, identical genes  were significantly enriched for genes involved in energy production and conversion , carbohydrate transport and metabolism , amino acid biosynthesis  and translation, ribosomal structure and biogenesis  .
of the  <dig> genes coding for putative virulence factors or involved in meningococcal host interactions in strain mc <dig>  only  <dig> are missing in strain α <dig>  and strain α <dig> lacks in particular large parts of the islands of horizontal transfer b and c that code for a two-partner secretion system involved in host cell adhesion  <cit>  .
it further lacks almost the entire repeat-in-toxin island  <dig> encoding frpa/c-like proteins which induce high levels of serum antibodies during invasive disease in humans  <cit> .
in addition, downstream of glnb encoding the signal-transducing nitrogen regulatory protein pii strain α <dig> also lacks nada which codes for a minor adhesin that was found to promote bacterial adhesion to and penetration into human epithelial cells in vitro  <cit> .
mc <dig> further harbors a 30 kb duplication spanning  <dig> coding sequences involving the cysghdnji genes for sulfur acquisition which is missing in α <dig> as in most other meningococcal genomes.
both strains display similar phenotypes in ex vivo fitness and in vitro cell culture assays indicative of phenotypic buffering
despite the genetic differences described above, both strains were phenotypically similar with respect to resistance against human serum, adhesion to and invasion of nasopharyngeal cell lines, respectively, as well as in ex vivo growth .
surprisingly, although meningococci are part of the human oral microbiome both strains were not able to grow in human saliva.
since strain mc <dig> was able to grow in meningococcal minimal medium , the observed growth inhibitory effect of human saliva was probably not due to nutrient limitation but more likely caused by the presence of growth inhibiting substances known to be present in human saliva like antimicrobial peptides  <cit> .
of note, in their ecological niche, the human nasopharynx, meningococci are attached to host cells in the form of microcolonies and are unlikely to grow in a planktonic state as in the ex vivo situation  <cit> .
the fact that bacteria in biofilms are less susceptible to antimicrobial agents and host immune responses thereby becoming persistent colonizers  <cit>  might explain the unexpected ex vivo growth phenotype in saliva.
furthermore, both strains grew equally well in human blood and csf and thus under disease mimicking conditions despite their about 7-fold difference in the disease/carriage ratio .
these data indicate that the ability to survive in human blood is obviously necessary but per se not sufficient to cause imd.
we therefore focused on compensatory mechanisms which might buffer the genotypic differences described above under ex vivo conditions and considered the transcriptome as an intermediate phenotype  <cit> .
we hypothesized that both strains differ in the way they accomplish growth under disease mimicking conditions by differential transcriptional activation of metabolic and stress response pathways.
for convenience, comparisons of the mc <dig> transcriptomes between saliva, whole blood and csf, respectively, will be called cross-condition comparisons and discussed only briefly in the main text and in more detail in the additional file  <dig>  likewise, comparisons of the transcriptomes of strain mc <dig> and α <dig> in saliva, whole blood and csf, respectively, will be called cross-strain comparisons .
genes differently expressed between two conditions or between both strains in a given condition will be called differently expressed genes, and genes differently regulated between both strains between two conditions will shortly be called differently regulated genes.
throughout the following analyses, we further discern directional and non-directional classes of gene sets.
the non-directional class of significantly differently expressed genes contains genes where the information about direction of differential expression is omitted, so that significant gene sets can be interpreted as affected by differential expression in general.
the directional class aims to identify gene sets that are significantly affected by regulation in a distinct direction, i. e. condition or strain.
if a gene set contains significantly expressed genes in both directions, they will cancel out and the directional fdr-value will not be significant.
in turn, if a gene set is not significantly enriched for differently expressed genes in general it might nonetheless comprise mainly genes with a significant expression bias in one direction.
it consequently will have a non-significant non-directional fdr-value but a significant directional fdr-value.
condition-dependent expression changes affect 24% of the mc <dig> transcriptome
of the  <dig> genes assayed in the cross-condition comparisons in strain mc <dig>   <dig> genes were differently expressed between saliva and blood and  <dig> between blood and csf, respectively .
gene set enrichment analysis  showed that genes differently expressed between saliva and blood were significantly enriched for genes coding for nucleotide transport and metabolism  , whereas genes differently expressed between blood and csf were just slightly enriched for genes involved in posttranslational modification, protein turnover and chaperones  .
gsa further showed significant differences also in the directionality of gene expression changes .
the  <dig> genes that showed higher expression levels in saliva compared to blood were enriched for genes involved in energy metabolism and conversion , posttranslational modification, protein turnover and chaperones  and cell envelope and outer membrane biogenesis .
however, genes that were more highly expressed in blood than in saliva and csf, respectively, were significantly enriched predominantly for genes coding for proteins without any cog functional annotation so far, suggesting that our knowledge about the mechanisms employed by meningococci to survive in human blood are still quite limited.fig.
<dig> genes significantly differently expressed and/or regulated in cross-condition and/or cross-strain comparisons.
a venn diagram comparing sets of genes in strain mc <dig> differently expressed between conditions as indicated.
the total number of genes compared was  <dig>  b venn diagrams comparing sets of genes differently expressed between strains as indicated with each diagram.
the total number of genes compared in each panel was  <dig>  c heatmap depicting cross-condition and cross-strain gene expression differences and hierarchical clustering of significantly differently expressed genes.
average linkage clustering based on the spearman rank correlation of all  <dig> genes significantly differently expressed and/or regulated in at least one cross-condition and/or cross-strain comparison .
grey lines correspond to genes that were absent in the α <dig> genome sequence and therefore excluded from the cross-strain comparisons
comparison of differently versus non-differently expressed genes to identify cog categories that are affected by differential expression in general
comparison of differentially expressed genes to identify cog categories that are significantly affected by regulation in a distinct direction
odds ratios based on fisher’s exact test.
values greater than  <dig> indicate that significantly differentially expressed genes are enriched for genes from the corresponding cog functional class
false discovery rate based on p-values from fisher’s exact test and the benjamini-hochberg multiple testing correction with a significance cut-off of fdr <  <dig>
for cross-condition comparisons, values greater than  <dig> indicate that genes highly expressed in blood were enriched for the respective cog category.
in cross-strain comparisons, values greater than  <dig> indicate that the respective cog category is significantly highly expressed or upregulated in strain mc58
fig.
<dig> gene set enrichment analysis of differently expressed genes.
a non-directional comparison of significantly differently expressed gene sets according to the cog functional classification scheme for cross-condition and cross-strain comparisons, respectively, indicating significantly overrepresented functional categories among the significantly differently expressed genes.
b directional comparison of significantly differently expressed gene sets according to the cog functional classification scheme for cross-condition and cross-strain comparisons, indicating significant gene expression asymmetries.
in both panels, the heat map depicts significantly enriched cog functional categories in red coloring.
the kind of comparison  is indicated for each column of the heat maps, and the corresponding fdrs are color coded and given in the respective inserts.
the associated tree is based on average linkage clustering of the functional categories using the spearman rank correlation coefficient
both strains differ in the expression of over  <dig> genes in a condition and strain dependent manner
based on the genome comparisons we further selected a sub-set of  <dig> single-copy orthologous genes for cross-strain gene expression comparisons.
of these,  <dig> were expressed and/or regulated at significantly different levels between both strains .
notably, proteins differently expressed in at least one ex vivo condition were not significantly more variable between both strains as indicated by their bsrps than proteins that were not , confirming that there was no significant sequence bias in the expression data.
of all the  <dig> genes that showed cross-condition and/or cross-strain expression differences  <dig> were differently expressed only in cross-strain and  <dig> only in cross-condition comparisons, respectively, and the overlap between cross-condition and cross-strain expression comparisons was thus surprisingly small .
the total number of significantly differently expressed genes between both strains was small in rich medium   and csf  and not significantly greater than the number expected by chance , and there were only  <dig> and  <dig> genes that showed significant expression level differences between both strains exclusively in csf and ppm+, respectively.
the number of differently expressed genes was significantly higher for saliva  and blood   than expected by chance , although both strains were not able to grow in saliva.
this further suggests that gene expression differences were not due to growth rate differences but were specific responses of both strains to these two ex vivo conditions.
in line with this hypothesis, the transcriptomes differed significantly among both strains in response to the ex vivo condition tested .
of note, the pattern of transcriptionally activated genes upon transition from saliva to blood was opposite to the pattern of transcriptionally activated genes upon transition from blood to csf , indicating that transcriptional changes are likely in response to components present in human blood but neither in saliva nor csf.
since these two ex vivo compounds were both free of any phagocytic cells, differences in transcriptional responses in both strains might be triggered by blood phagocytes.
cross-strain gene expression differences in saliva comprise numerous stress response genes
a total of  <dig> genes were differently expressed between both strains in saliva, with a slight yet not significant enrichment of genes involved in anorganic ion transport and metabolism  .
functionally, around one third coded each for poorly characterized proteins , proteins involved in cellular processes  or proteins involved in metabolism or information storage and processing.
among the  <dig> genes highly expressed in strain α <dig> were, amongst others, seven for anorganic ion transport and metabolism proteins  including a putative multidrug resistance protein , the lactoferrin-binding protein a  and the putative ammonium transporter amtb, as well as six genes involved in cell envelope biogenesis  including genes for the two sialic acid capsule biosynthesis proteins synx/siaa/cssa and siab/cssb.
genes involved in translation and ribosomal biogenesis  comprised the single largest group  among the genes that were in turn highly expressed in strain mc <dig> in saliva, next to genes in amino acid transport and metabolism  and cell envelope biogenesis .
since both strains were not able to grow in human saliva these gene expression differences likely reflect differences in the stress response between both strains when exposed to this hostile environment.
both strains differed in the expression of virulence-associated genes involved in the pathogenesis of imd
numerous genes involved in the interaction of meningococci with its human host have so far been studied in order to understand the genetic and mechanistic basis of meningococcal virulence, i.e.
host damage, and were consequently of special interest.
accordingly, of the  <dig> virulence-associated genes used for cross-strain comparisons  ,  <dig> were differently expressed and/or regulated among both strains under at least one of the ex vivo conditions tested which is significantly more than in the cross-condition comparisons for strain mc <dig>  .
in addition, genes differently expressed and/or regulated between both strains in at least one cross-strain comparison were significantly enriched for virulence-associated genes , and the expression profiles were significantly different between virulence-associated and not virulence-associated genes .
of the  <dig> virulence-associated genes that were differently expressed between both strains in blood,  <dig> were highly expressed in α <dig> and comprised genes involved in cell envelope biogenesis and in particular genes for capsule and los biosynthesis.
los was shown to be the dominant molecule in meningococci inducing organ inflammation in human patients  <cit> , and differences in los biosynthesis might therefore have an immediate impact on the extent of host damage caused by both strains.
in turn, genes involved in type iv pilus biosynthesis iron homeostasis, the stress response genes as well as genes encoding adhesins such as opc or the hemagglutinin/hemolysin-related protein tpsa <dig> or nspa  <cit>  were all highly expressed in mc <dig>  besides opc, we could at best detect only very small expression level differences for other recently introduced vaccine antigens that were conserved in both strains .
likewise, of the  <dig> genes that have been described so far as being involved in neutrophil interactions in neisseria and which were part of the gene expression comparisons  <cit> ,  <dig> were differently expressed between both strains in blood .
genes differently expressed between both strains were therefore significantly enriched for neutrophil response genes .
these included a number of virulence-associated genes like capsule synthesis genes or genes coding for efflux pump components , as well as genes not so far associated with meningococcal virulence like the dna damage repair genes uvra and uvrb.
this finding indicates that neutrophils might have an important role in shaping the meningococcal transcriptional response to human blood.
of note, ania  encoding nitrite reductase and norb coding for no reductase  <cit>  showed the largest blood-specific expression differences between both strains  and were among the highest expressed genes in mc <dig> as was also confirmed by qrt-pcr .
the gene products ania and norb constitute a pathway that enables the organism to grow under conditions of low oxygen in the presence of nitrite.
amongst others, meningococcal derived no was recently shown to play an essential role in the pathophysiology of septicemic meningococcal infection in humans by inhibiting platelet aggregation  <cit>  and modifying the release of cytokines and chemokines by human macrophages  <cit> .
therefore, a number of virulence-associated genes with an also experimentally established role in the pathogenesis of imd like ania or los biosynthesis genes were differently expressed in both strains particularly in human blood.
transcriptomic differences between both strains associated with the transition from saliva to blood are enriched for metabolic, information processing and cell envelope biogenesis genes
overall, the number of differently expressed genes between both strains was highest in blood , and also the number of genes differently regulated between both strains between two conditions was highest between saliva and blood  .
both gene sets were functionally enriched for genes required for energy production and conversion  .
these data thus clearly demonstrate differences in the environment-dependent gene-expression regulation between both strains.
the  <dig> genes highly expressed in strain α <dig> in human blood were significantly enriched for genes involved in cell wall/membrane biogenesis  , including los and peptidoglycan biosynthesis genes like murb, murd, mure and ddl.
in contrast, the  <dig> genes highly expressed in strain mc <dig> were significantly enriched for metabolic genes  including genes for oxidative phosphorylation  and nitrogen respiration such as ania and norb described above.
with respect to differences in the direction of gene expression regulation in both strains, genes required for energy production and conversion  were also strongly upregulated in strain mc <dig> between saliva and blood, whereas genes for cell envelope biogenesis  and translation  were in turn strongly upregulated in strain α <dig>
based on differences in the directionality of gene expression levels and regulation , the functional categories cog e, c and m form a cluster of co-regulated genes which is part of a larger cluster including also the functional categories cog j, cog k and cog l. this finding suggests a regulatory link between metabolism, the biosynthesis of the cell envelope, and genes for the gene expression machinery which is differently activated in both strains particularly in human blood.
integrative network analysis of differently expressed genes identifies subnetworks of co-regulated genes
in order to identify differentially expressed functional subnetworks, we combined the transcriptomic with protein-protein interaction  network data of strain mc <dig> as deposited in the string database and used an algorithm which optimally identifies responsive subnetworks  <cit> .
this integrative network analysis revealed a densly connected subnetwork comprising mainly genes that are highly expressed in strain mc <dig> in blood  involved particularly in energy and carbohydrate metabolism .
this subnetwork comprised genes of the tricarboxylic acid  cycle , for the metabolism of pyruvate , glycine , leucine  and fatty acids .
the second subnetwork contained significantly more genes highly expressed in strain α <dig>  coding for a significantly different array of cellular functions .
it comprised genes involved in the biosynthesis of the capsule , peptidoglycan , and los , respectively, as well as genes for trafficking, secretion and vesicular transport  or information storage and processing including in particular translation and ribosomal biogenesis genes .
a few genes in this second subnetwork were highly expressed in mc <dig> yet and included genes for glycolysis , the genes for the na+-translocating nadh-quinone reductase subunit b, d and f  along with virulence-associated genes mentioned above like dsba- <dig>  pilg, pilt- <dig>  sodc .
transcriptomic differences in both strains in blood were thus organized in two subnetworks consisting mainly of genes that were either highly expressed in α <dig> or in mc <dig> and that coded for different biological functions.fig.
<dig> analysis of gene expression data based on protein-protein interaction networks.
a integrative network analysis of differently expressed genes between strain mc <dig> and α <dig> in human whole blood based on the string protein-protein interaction network for strain mc <dig> .
a subnetwork that consists predominantly  of genes that were expressed at higher levels in mc <dig> than in α <dig> and that code for metabolic genes and in particular for genes involved in energy production and conversion  is shaded in orange.
the remaining part of the network comprising  <dig> protein-coding genes is shaded in light blue.
b integrative network analysis of gene regulation differences between both strains upon transition from saliva to blood .
the two modules consisting predominantly of genes either upregulated in α <dig>  or mc <dig>  upon transition from saliva to blood are shaded in green and orange, respectively.
only 21% of the genes in the left subnetwork consisting mainly of genes that were upregulated in α <dig> code for proteins involved in  metabolism compared to over 66% of the genes that were upregulated in mc <dig> upon transition from saliva to blood.
for each gene, the respective expression differences between conditions and strains, respectively, are color coded and indicated in each panel.
white boxes indicate genes that were not differently expressed but are part of a subnetwork as identified by the integrative network analysis.
pie charts next to the sub-networks in each panel show the distribution of proteins in the respective subnetwork over the different cog functional classes
saliva vs. blood
miss
misr
siac
siab
siaa
ctrb
lipb
kdta
lpxc
lpxa
lpxd
lpxb
lgtf
lgtb
rfac
pilt-1
pilf
pilg
pilt-2
pilp
pglb
pglc
fara
mtre
mtrd
-
omp85
rmpm
tpsa2
nspa
opc
tpsa3
-
nalp
tbp2
-
-
brfb
brfa
lbpa
dsba-1
dsba-2
znub
znuc
sodc
-
-
fabz
lctp
iga
hemh
purc
pgm
-
-
prc
-
-
-
-
norb
ania
-
-
-
-
virulence-associated genes were compiled from table  <dig> in ref.
<cit> , table  <dig> in ref.
<cit> , table  <dig>  in ref.
<cit> , and additional file  <dig> from ref.
<cit>  .
only those genes are included in the transcriptome comparisons that showed significant expression differences in at least one type of comparison
positive values indicate that the gene is expressed at higher levels in blood than in saliva
positive values indicate that the gene is expressed at higher levels in csf than in blood
positive values indicate that the gene is expressed at higher levels in strain mc <dig> than in α522
positive values indicate that the gene expression difference between saliva and blood is greater in strain mc <dig> than it is in α522
positive values indicate that the gene expression difference between blood and csf is greater in strain mc <dig> than it is in α522
n. s., not significant
n. d., no data due to missing α <dig> genome sequence data
along with these cross-strain differences in the directionality of gene expression levels also cross-strain differences in the directionality of gene regulation between saliva and blood were organized in two functionally differing subnetworks  .
the first subnetwork consisted almost exclusively of genes found to be upregulated in mc <dig> in blood compared to saliva , and over 70% of the genes in this subnetwork code for metabolic functions compared to only 24% in the other subnetwork .
it comprised genes for tca cycle enzymes , for 2-oxocarboxylic acid metabolism , oxidative phosphorylation , fatty acid metabolism , for thioredoxin-fold proteins  required for oxidative stress response as well as the oxidoreductase genes nqrb, nqrd, nqrf and ania, the latter playing a major role in the pathogenesis of imd as described above.
the second subnetwork consisted of genes that were strongly upregulated in strain α <dig> in blood compared to saliva  and included predominantly genes involved in information storage and processing and in particular for the translation machinery .
in line with the gsa results, integrative network analysis thus indicated that in contrast to strain α <dig> strain mc <dig> allocates transcriptional resources predominantly in the expression of metabolic genes in human blood.
elementary mode analysis of blood transcriptomic data indicate compensatory flux differences between both strains particularly in energy, glutamine and cysteine metabolism
in order to assess the possible impact of the transcriptomic differences on metabolism in blood in more detail, we reconstructed a condensed metabolic network based on the nmb_itm <dig> model for strain mc <dig>  <cit>  comprising  <dig> enzymes  and  <dig> metabolites.
the  <dig> elementary metabolic modes and pathways as identified by elementary mode analysis  <cit>  are given in the additional file 3: s <dig>
computation of metabolic fluxes  <cit>  based on the observed growth rates of both strains in human blood , the corresponding gene expression data  and the average composition of human blood as taken from  <cit>  showed that the flux activities in mc <dig> were 25-50% higher than in α <dig> with major differences in the metabolism of some amino acids  .
of note, one flux mode  displayed an opposite direction between mc <dig> and α <dig>  indicating that α <dig> uses a large amount of external glutamine  from human blood to produce gutamate .fig.
<dig> analysis of gene expression data based on a metabolic model for strain mc <dig>  a comparison of elementary mode activities in mc <dig> and α <dig>  the histogram depicts differences in the elementary mode activities  for each of the elementary metabolic modes  as defined in the additional file 3: s <dig> for strain mc <dig>  and α <dig>  based on gene expression data in human blood.
b inferred differences in metabolic fluxes between strain mc <dig> and strain α <dig> in blood based on a metabolic model for strain mc <dig>  internal metabolites which are considered to have balanced concentrations are given by dark green spheres, external metabolites which are allowed to accumulate or to be consumed by green cones, and reactions together with their corresponding numbers as light green boxes.
the reactions for all reaction numbers are given in the additional file 3: s <dig>  arrows connect reaction with metabolites.
red coloring indicates higher fluxes in mc <dig> compared to strain α <dig>  whereas blue colouring indicates that the flux is slightly enhanced in α <dig>  asterisks along with pink colouring indicate that the reaction has an opposite direction in both strains
for more detailed information about differences in important biochemical pathways, we converted the flux activities into enzyme and enzyme complex activities, respectively.
the major differences are given in table  <dig> and fig.
4b.
only very few fluxes were slightly stronger in α <dig> such as r <dig>  r <dig> and r <dig> which are all involved in sulfur metabolism.
on the other hand, there were numerous reactions that were even more than tenfold stronger in mc <dig> than in α <dig> such as the acetate synthesis reactions r <dig>  r <dig> and r <dig> suggesting that mc <dig> compared to α <dig> may accumulate acetate as intermediate metabolite in this environment.
in meningococci, such metabolic stimulation has been shown to result from the consumption of lactate which in the presence of glucose is used as a source of additional energy  <cit> .
of note, lactate is a by-product of neutrophil glycolysis and enhances bacterial consumption of molecular oxygen, which depletes the substrate for neutrophil nadph oxidase and thus blunts its oxidative burst  <cit> .
since both strains have almost identical growth rates in human blood  mc <dig> probably requires this additional metabolic energy for other, not growth related processes such as, e.g., defense against the neutrophil oxidative burst.
other reactions that were particularly stronger in mc <dig> included r <dig>  r <dig> and r <dig> leading from phosphoenolpyruvate  to α-ketoglutarate  as well as some reactions involved in amino acid metabolism like r <dig> and r <dig> resulting in the synthesis of serine  from glu and glycine , respectively, or r <dig>  r <dig> and r <dig> resulting in the synthesis of leucine , phenylalanine  and tyrosine  from glu, respectively.
since the reactions r <dig>  r <dig>  r <dig> and r <dig> are all transamination reactions resulting in the consumption of glu and concomitant synthesis of akg, these findings further suggest that mc <dig> and α <dig> might differ in their intracellular levels of glu and akg in human blood.
finally, reactions r <dig> and r <dig> have opposite directions in both strains in human blood, which indicates that α <dig> relies mostly on gln to produce glu with some glu being further converted into akg, whereas mc <dig> is capable to produce enough glu from the tca cycle and convert it further to gln.
likewise, also reaction r <dig> which is the production of cysteine  from serine  has an opposite direction in both strains in human blood, and α <dig> consequently seems to rely on external cys to produce ser whereas mc <dig> is capable of producing cys from ser.table  <dig> inferred reaction activity differences between mc <dig> and α <dig> in human blood based on gene cross-strain expression differences
ratio of the reaction activity in mc <dig> divided by the reaction activity in α <dig>  a ratio larger than one indicates that the reaction has a higher activity in mc <dig>  and a negative ratio that the reaction occurs in opposite directions both strains
strain α <dig> differs from strain mc <dig> in gln and cys auxotrophy in vitro
in order to experimentally validate the transcriptomic results with respect to possible differences in gln and cys metabolism, we assessed the growth of both strains in mmm supplemented with different amino acids as well as ppm+ .
whereas both strains were equally able to grow in ppm+ and mmm supplemented with all  <dig> proteinogenic amino acids at milimolar concentrations, strain α <dig> was not able to grow in mmm without amino acids.
in addition to glucose or lactose as carbon source  it requires cys and gln for growth .
contrary to α <dig>  the growth of mc <dig> was slightly suppressed by these two amino acids.
the ability of mc <dig> but not α <dig> to grow in the absence of cys and gln indicates strain specific differences in the respective metabolic pathways, and the requirement of some meningococcal strains for cys and its growth inhibiting effect on others has already been reported  <cit> .
in parallel with these phenotypic differences, genome comparisons revealed also striking differences in cys and gln biosynthesis genes that might contribute to the phenotypic finding, although the repertoire of enzymes required for the biosynthesis of amino acids is otherwise highly conserved in both genomes.
in addition to the duplication of cysghdnji genes in mc <dig> , these include large sequence differences in the phosphoadenosine phosphosulfate reductase cysh and the glutamate-ammonia-ligase adenylyltransferase glne which are among the least conserved genes involved in the biosynthesis of amino acids .
cysh is required for the reduction of sulfate into hydrogen sulfide and thus for sulfur acquisition in n. meningitidis  <cit> , and glne is a key regulatory enzyme in nitrogen assimilation in e. coli  <cit> .
furthermore, the intergenic region between purl and glnb differs in both strains due to the insertion of coding sequence in strain α <dig> upstream of glnb resulting in entirely different glnb promoter regions .
along with glne glnb is involved in the regulation of nitrogen assimilation in e. coli via affecting the activity of glna, an enzyme that lies at the heart of the nitrogen assimilation network.
glna is involved in the complex regulation of the interconversion of glu to gln in response to the intracellular concentration of ammonium, glu, the gln/akg ratio, the redox  and the free energy state of the cell  <cit> .
sequence variation at these loci is therefore likely to have pleiotropic effects, and a detailed experimental analysis of the biochemical consequences of these genetic differences is subject to ongoing work.fig.
<dig> growth phenotypes of n. meningitidis mc <dig> and α <dig> wild-type and mutant strains.
a in vitro growth phenotypes.
growth as quantified by the optical density  is given on the ordinate and the time in hours on the abscissa.
b ex vivo growth phenotypes.
the number of colony forming units for each time point ) relative to the initial number ) is given on the ordinate and the time in minutes on the abscissa.
for each strain and condition the respective growth curves are coded as indicated in the insert in each panel, and the genotypes of the respective strains compared are shown along with the corresponding growth curves.
in each experiment rich medium  was used as growth control.
the arrow at the top of panel b indicates the time when total rna was extracted for microarray analysis
expression changes in numerous regulatory genes are likely compensatory
the large transcriptome differences observed particularly in blood could be caused by differences in regulatory elements acting either in cis or in trans, or be compensatory to ensure cellular homeostasis.
among the  <dig> orthologous proteins those involved in gene expression regulation  were no more different between both strains than proteins involved in other functions .
also the 200 bp upstream regions were not less conserved than the downstream orthologous genes .
of the  <dig> single-copy orthologous genes used for transcriptomic comparisons  <dig> differed in the presence of putative mobile genetic elements within their 200 bp upstream regions .
these comprised various so called minimal mobile elements  <cit>  as well correia repeats and correia repeat enclosed elements which have previously been shown to affect gene expression in a polar manner .
however, this class of genes was not enriched for genes differently expressed in at least one ex vivo condition.
although genetic differences in gene regulatory elements might contribute to the transcriptomic differences these data do not provide evidence yet that they are the sole reason of the large cross-strain expression differences observed particularly in human blood.
they might rather orchestrate different compensatory gene expression adaptations in both strains in response to differences in the interaction of both strains with human blood components.
both strains activate different sets of regulatory genes in response to human blood
of the  <dig> genes with significant expression differences in cross-condition or cross-strain comparisons involved in signal transduction or transcription ,  <dig> showed significant expression level differences between both strains in blood .
the eight regulatory genes that were highly expressed in mc <dig> specifically in blood included in particular csta annotated as carbon starvation protein a, nmb <dig> coding for an arsr family transcriptional regulator, rela encoding the guanosine 3'-diphosphate 5-'diphosphate ppgpp) synthetase of the stringent response pathway, as well as misr  coding for a phop-family response regulator of a two component signal transduction system.
of note,  <dig> genes of the  <dig> genes differently expressed in blood belong to the misr regulon  <cit> , and misr was shown to be involved in the oxidative stress response in meningococci  <cit> , required for colonization of host cells  <cit>  and meningococcal survival in mice  <cit> .
in addition,  <dig> differently expressed genes which are part of the fur regulon  <cit>  were almost all highly expressed in mc <dig>  since fur senses cellular iron concentrations and since iron in general acts as a co-repressor, these data indicate that strain mc <dig> might experience more pronounced iron starvation in blood compared to strain α <dig>  likewise, another  <dig> genes are known to be regulated by fnr  <cit> , the master regulator involved in the adaptation to oxygen-limited conditions, of which the majority  were also highly expressed in mc <dig> table  <dig> differentially expressed genes involved in transcription and signal transduction
saliva vs. blood
miss
misr
ptsn
-
narp
bass
fnr
-
-
-
-
-
-
-
-
-
-
-
rpoh
rpoe
-
dksa
nusg
-
-
rho
rnc
-
-
-
grea
csta
-
nusa
-
rpoz
rela
-
-
-
positive values indicate that the gene is highly expressed in blood
positive values indicate that the gene is highly expressed in csf
positive values indicate that the gene is highly expressed in strain mc58
n. s. not significant
n. a. not applicable due to high sequence divergence between bot hhomologs
n. d. no data due to missing α <dig> genome sequence data
in turn, the ten regulatory genes highly expressed in α <dig> included nmb <dig> encoding a exoribonuclease and rpoe  which codes for the alternative sigma factor e  and in n. gonorrhoeae is activated in response to oxidative stress  <cit> .
since phagocytic cells are a major source of reactive oxygen species and were present only in the blood assay, these data further suggests that σe might be involved in meningococcal interaction with human phagocytes.
in line with previous findings that the expression of ania and norb are under the negative control of σe  <cit> , both strains also showed significantly different expression levels of ania and norb especially in blood as mentioned above .
along with the finding that nitric oxide  generated by ania inhibits platelet aggregation  <cit>  this observation provides a direct link between the oxidative stress response and the pathophysiology of imd.
these data indicate that the complex gene expression differences between mc <dig> and α <dig> specifically in blood are caused by the activation of different sets of regulatory genes including misr, fur, fnr, rela and σe.
in contrast to the expression of rpoe  <cit>  and misr  <cit>  which in both cases was found to be auto regulated, fur and fnr did not differ in their expression between both strains, suggesting strain-dependent differences in the post-transcriptional activation of these regulators especially in blood.
differences in promoter regions demonstrate an important role for the stringent response and hfq mediated differences in gene expression regulation among both strains in blood
although sequence analyses of the 200 bp upstream regions of the  <dig> genes that were differently expressed between both strains in cross-strain comparisons failed to identify any consistent sequence differences or overrepresented bona fide tf binding sites for any of the cross-strain comparisons, the analysis of gc content variation yet revealed a 6 bp region immediately upstream of the predicted ribosome binding site  having a significantly lower gc content in genes that were expressed at higher levels in mc <dig> than in α <dig> in blood  .
at the mrna level, such au-rich elements next to the rbs are often targets for the hfq-mediated binding of small non-coding rnas which thus post-transcriptionally regulate the degradation and/or translation efficiency of the corresponding mrna  <cit> .
in strain mc <dig>  the rna chaperone hfq was already shown to be involved in the regulation of amino acid and energy metabolism, the oxidative stress response and required for survival in human blood  <cit> .
the comparison of the differently expressed genes showed that of the  <dig> genes that are part of the hfq regulon and that were included in this study,  <dig> were differently expressed in both strains, all higher in mc <dig>  therefore, these data suggest that hfq contributes to gene regulation differences between both strains in blood.fig.
<dig> gc content variation in potential promoter regions based on the mc <dig> genome sequence.
a scatter plot of the gc content variation averaged over a 5-bp sliding window within 100 bp upstream regions for genes highly expressed in mc <dig>  or α <dig>  in human blood.
the black line gives the gc content of the respective upstream regions for genes not differently expressed.
regulatory regions are indicated at the top of the panel based on the average length of 5’-untranslated regions in n. gonorrhoeae  <cit> .
the insert gives the number of genes in each gene set.
b box-and-whiskers plot depicting differences in the mean gc content of the putative discriminator  and hfq-binding regions  between genes highly expressed in mc <dig>  or α <dig>  in human blood as depicted in panel .
the line within each box gives the median and the upper and lower margins the upper and the lower quartile, respectively.
the whiskers denote the highest and the lowest values, respectively, and the open circles outliers.
*: p <  <dig> , **: p <  <dig>
furthermore, genes differently expressed between both strains in blood had also significant gc content differences in a 10 bp region 40 bp upstream of the predicted rbs  , and there was a negative correlation between expression differences in blood and the deviation from the average genomic gc content .
since the average length of 5’-utrs in neisseria was shown to be between  <dig> and 50 bp  <cit>  this region corresponds to the transcriptional start site.
the length of this region, its pattern of gc content variation and its location close to the presumed transcriptional start site are hallmark features of so called discriminator regions  <cit>  which in γ-proteobacteria determine whether the adjacent gene is activated or repressed during the stringent response.
accordingly, of the  <dig> genes differently expressed between both strains in human blood,  <dig> have a discriminator gc content higher than the genome-wide average of 50% and were highly expressed in α <dig>  and  <dig> genes with a discriminator gc content lower than the average were highly expressed in mc <dig> .
together, these genes account for 56% of all genes differently expressed in blood.
since activated targets typically have an at-rich discriminator whereas repressed targets have a gc-rich discriminator, these data along with the higher expression of rela in mc <dig> indicate that in blood the stringent response pathway is comparatively more activated in mc <dig> than in α <dig>  genes highly expressed in mc <dig> and with a low gc discriminator region were predominantly involved in energy production and conversion  and comprised genes involved in carbohydrate  and energy metabolism , in aerobic energy generation , ccoo ), the genes for cytochrome c <dig>  and c <dig> ) as well genes required for the oxidative/nitrosative stress responses  along with surface proteins like nspa, lip and laz.
genes highly expressed in α <dig> and having a high gc discriminator region were predominantly involved in cell envelope and outer membrane biosynthesis  and included genes for los  and peptidoglycan biosynthesis  as well as regulatory genes including rpoe.
rela and the stringent response pathway contribute to meningococcal ex vivo fitness in a condition and strain dependent manner
although the stringent response pathway was already shown to be crucial for virulence in a number of bacterial pathogens  <cit> , nothing is known about its contribution to meningococcal ex vivo or in vitro fitness so far.
therefore, the observation that rela was differently expressed between both strains exclusively in blood along with the finding that genes differently expressed between both strains in blood had significant gc content differences in their putative discriminator regions prompted us to further assess the contribution of the stringent response and in particulate of rela to meningococcal fitness ex vivo.
the machinery of the stringent response pathway comprises several enzymes involved in the turnover of ppgpp which is a signaling nucleotide that coordinates a variety of cellular activities in response to changes in nutritional abundance  <cit> .
in e. coli, rela is activated upon amino acid starvation and together with spot is able to catalyze pyrophosphoryl transfer from atp to gtp or gdp to synthesize ppgpp.
together with dnak suppressor , ppgpp directs transcription initiation at particular gene promoters through binding to the interface between the two rna polymerase subunits β’ and ω  <cit> .
in part, ppgpp and dksa act by promoting the interaction of rna polymerase with alternative σ-factors such as σe or σh.
when metabolic precursors are plentiful, spot instead degrades ppgpp, and the vegetative σ-factor, σ <dig>  directs rna polymerase to genes that are crucial for bacterial replication.
whereas β’, ω, spot and dksa were identical in both strains they differed in the coding sequences and promoter region of rela , and gene expression analyses via qrt-pcr further confirmed particular large and blood-specific cross-strain expression differences for rela but not for spot or dksa .fig.
<dig> genomic distribution of atrs and the rela locus in n. meningitidis.
a the intergenic region between grxb and rela.
the integration site of a copy of an atr repeat element upstream of rela  in strain α <dig> is indicated with respect to the mc <dig> locus.
the transcriptional start sites as determined by 5’-race in both strains are indicated along with the deduced − <dig> and − <dig> boxes and the computationally predicted promoter regions using ppp  <cit> .
dr: direct repeat.
b alignment of both the mc <dig>  and α <dig>  genomes as visualized with the artemis comparison tool based on a blastn comparison.
the linearized mc <dig> and α <dig> genomes are shown in the upper and lower panel as gray bars, and regions syntenic in both genomes are connected via red and inverted regions via blue lines, respectively.
the location of atrs is indicated by small arrows in each genome, and the rela region is highlighted in yellow
to test whether these differences in the coding sequences of rela affected its catalytic activity we assessed ppgpp levels in wild-type, ∆rela::kmr as well as ∆rela::kmr ∆spot::cmr mutants in both strains during growth in ppm+ medium.
as can be seen in additional file 1: figure s5a, both alleles were catalytically active and the catalytic activity was affected by the bacterial growth phase in a similar manner.
similar to the α <dig> wild-type strain, the mc <dig> ∆rela::kmr mutant could further not grow in minimal medium, and in both genetic backgrounds the addition of all  <dig> proteinogenic amino acids could compensate for the loss of functional rela but not the addition of only cys and/or gln to minimal medium .
in addition, similar to e. coli ppgpp <dig> strains the growth defect in minimal medium was less severe in meningococcal ∆rela::kmr ∆spot::cmr double mutants.
in support of the notion that  high levels of ppgpp might be toxic for the meningococcal cell we could not obtain viable isogenic spot single deletion mutants, and a presumed spot knock-out mutant had a compensatory frame-shift mutation prior the catalytic domain of rela resulting in reduced ppgpp levels .
with respect to ex vivo fitness the deletion of rela had no effect in mc <dig> on growth in saliva, blood or csf whereas the α <dig> ∆rela::kmr mutant was severely impaired exclusively in blood .
the ex vivo fitness defect was less severe in a α <dig> ∆rela::kmr ∆spot::cmr double mutant.
unfortunately, since all attempts to clone full-length rela and spot, respectively, in n. meningitidis for cis/trans complementation assays failed, the possibility that the observed phenotypes are, at least in part, caused by polar effects cannot be ruled out entirely.
however, as depicted in additional file 1: figure s <dig>  there are rho-independent transcriptional terminators at the 3′ ends of the rela as well as the spot gene which have not been altered in the respective mutants.
furthermore, since the phenotype of the atrrela mutants particularly in blood resembles the phenotype of the rela mutants in both strains, and since both are also different from the phenotype of the grxb mutants, it seems rather unlikely that the rela phenotype is due to a polar effect on grxb expression  and not due to the decreased ppgpp levels .
together, these data show that that the stringent response pathway is functional in both meningococcal strains despite their different rela alleles.
the deletion of rela is conditional lethal and it is differently expressed between both strains under virulence-mimicking conditions in human blood.
the effect of rela on ex vivo fitness is thus dependent on the environment and the genetic background.
a non-coding mobile element affects meningococcal fitness in a strain- and condition-dependent manner
in addition to differences in the rela coding sequences, the intergenic region between rela and the upstream grxb encoding the redox enzyme glutaredoxin differed substantially in both strains due to the integration of an at-rich  repeat element in strain α <dig> .
atr repeat elements occur  <dig> and  <dig> times in the genomes of α <dig> and mc <dig>  respectively, of which only  <dig> have the same position in both genomes .
they have a conserved length of 181 bp with ends forming a perfect 13–bp inverted repeat and belong to a class of non-autonomous dna transposons also known as miniature inverted-repeat transposable-elements   <cit> .
atrs are almost exclusively located in intergenic regions in both genomes and are almost ten times more frequently found in intergenic regions flanked by convergently transcribed genes than expected by chance .
this indicates that the location in potential promoter regions might be under negative selection possibly due to adverse effects on the expression of neighboring genes.
as experimentally determined by 5’-race, the − <dig> boxes of the rela promoter differ between both strains due to the insertion of atrrela in strain α <dig> between the − <dig> and − <dig> boxes .
furthermore, growth experiments demonstrated that the fitness of an atrrela knock-out strain was impaired only in α <dig> and only in human blood thus resembling the phenotype of the rela deletion mutant .
in contrast to the deletion of atrrela or rela, the effect of deleting the neighboring grxb gene was condition- but not strain-dependent.
these findings thus suggest that via affecting the expression of rela, differences in the binding of transcriptional regulators might contribute to the observed epistatic effects of atrrela on ex vivo fitness.
the conditional essentiality of grxb for ex vivo fitness further supports the hypothesis that the oxidative stress response is required for blood survival especially in human blood.
in support of a background- and condition-dependent effect of atrrela on meningococcal fitness, we could not observe any ex vivo fitness differences between the atrrela knock-in mutant strain mc <dig> relap::atrrela and the corresponding mc <dig> wild-type strain.
surprisingly, the integration of atrrela into the rela promoter region abolished however the inhibitory effect of cys and gln on growth in mmm in strain mc <dig>  whereas the deletion of atrrela had no effect on the in vitro growth of α <dig> .
this suggests a link between cys and gln metabolism and the stringent response regulation in a yet to define epistatic manner.
although the role of mites in meningococcal infection biology has already been established  <cit>  this is the first time that a biological function has been shown for the atr class of mites in an infection process.
discussion
it has recently been shown that transcriptional regulation in prokaryotes is more flexible than the genetic component of the organisms and that its complexity and structure plays an important role in phenotypic adaptation  <cit> .
however, little is known so far about the significance of regulatory evolution that might underlie bacterial virulence.
accordingly, we used a hypothesis-generating systems biological approach  <cit>  to analyse gene-expression differences between two meningococcal strains from a hyperinvasive and a carriage clonal complex, respectively, under infection-mimicking conditions .
despite the substantial genetic differences between both strains affecting surface antigens as well as metabolic genes likely affecting cys and gln biosynthesis , both were surprisingly similar in a variety of in vitro virulence assays and in their growth behavior under infection mimicking conditions .
in particular, the finding that both strains have the same fitness in human blood and csf despite the large differences in the disease/carriage ratios between cc st- <dig> and cc st- <dig> strains further indicates that the ability to grow under infection mimicking conditions might be necessary but not sufficient for explaining the invasive property of certain meningococcal lineages.
virulence, i.e.
host damage, might rather be related to the way how meningococci accomplish growth in this environment.
in line with this hypothesis, the large transcriptome differences observed particularly in human blood  indicate that different transcriptional programs probably compensate for the differences in the genetic backgrounds of both strains in response to host components.
this so called phenotypic buffering is a general property of complex gene-regulatory networks  <cit> .fig.
<dig> graphical summary and hypothesis relating major findings of this work and published data.
the figure is not intended to give a comprehensive overview of the entire metabolism and stress responses in n. meningitidis but to illustrate pathways that link metabolism, protein sequence and gene expression differences of selected  genes and the pathogenesis of imd as described in the main text.
accordingly, genes and pathways that were highly expressed in mc <dig> in blood and/or that are strongly upregulated between saliva and blood in mc <dig> are depicted in red, and genes and pathways that are highly expressed in α <dig> or that are strongly upregulated between saliva and blood in α <dig> are depicted in green.
asterisks next to enzyme or protein names indicate that the corresponding proteins have a less than average sequence similarity  or are entirely missing in strain α <dig>  arrows with plus signs indicate  activating regulatory interactions, and arrows with minus signs  inhibitory regulatory interactions.
for further details and abbreviations see main text.
the literature cited in the figure is indicated by bracketed numerals next to the respective arrows:  newcombe et al.
<cit> ,  delany et al.
<cit> ,  fantappie et al.
<cit> ,  monaco et al.
<cit> ,  huis in’t veld et al.
<cit> ,  tala et al.
<cit> ,  takahashi et al.
<cit> ,  gunesekere et al.
<cit> ,  criss and seifert   <cit> ,  seib et al.
<cit> ,  schmitt et al.
<cit> ,  stevanin et al.
<cit> ,  kobsar et al.
<cit> ,  coureuil et al.
<cit> ,  virji   <cit> ,  hellerud et al.
<cit>
upon transition from commensal to invasive behavior meningococci have to adapt to the accompanying large environmental changes caused largely by differences in nutritional and innate immunity in these different compartments.
these comprise, e.g., differences in the presence of professional phagocytes or the availability and concentration and of key nutrients such as amino acids or iron .
accordingly, it has already been shown that the oxidative burst, which is one of the most prominent effector mechanisms in human neutrophils, is modulated by neisserial porins, and – although to a lesser extent – also opc has been described to be involved in n. meningitidis – neutrophil interaction .
the observed differences in the repertoire and/or expression of, e.g., opc, pora and porb, could lead to differences in the phagocytic uptake by neutrophils and/or the activation of the oxidative burst .
phagocytosis and the activation of the neutrophil oxidative burst in turn exert nutritional and oxidative/nitrosative stresses on the bacterial cell  <cit> .
the resulting damage to the bacterial cell triggers bacterial stress responses which in pathogenic bacteria have already been considered as virulence factors.
in particular, lactate, a by-product of neutrophil glycolysis, was shown to enhance bacterial consumption of molecular oxygen in the presence of glucose, which depletes the substrate for neutrophil nadph oxidase and thus blunts its oxidative burst  <cit> .
accordingly, our transcriptomic data analyses outlined above suggest a particularly strong metabolic activation in strain mc <dig> in blood .
another important component in the oxidative/nitrosative stress responses in neisseria is glutathione   <cit> .
it is synthesized from cys, gln/glu and gly which are either taken up from the environment or synthesized from precursors generated in the entner–doudoroff  pathway and tca cycle, respectively  <cit>  .
via the gsh cycle, the biosynthesis of cys and gln/glu are thus linked to the oxidative/nitrosative stress response  <cit> .
we therefore hypothesize that the observed sequence variations in metabolic genes such as cysh, glne or glnb involved in cys and gln/glu biosynthesis, respectively, lead to the activation of compensatory transcriptional programs to allow survival upon exposure to human blood phagocytes.
our data further indicate that rela contributes to the homeostatic transcriptional response to nutritional and oxidative/nitrosative stresses as it is required for amino acid biosynthesis in n. meningitidis .
consequently, via variation in the gc content of the discriminator regions, the accumulation of ppgpp during the stringent response might directly or indirectly contribute to the adapative regulation of genes required for energy metabolism, cell envelope biogenesis, translation and ribosome biogenesis and thus for large parts of the meningococcal transcriptome .
furthermore, as the educt for glu biosynthesis akg provides a potential metabolic link between energy metabolism and the gsh cycle  and was shown in n. meningitidis to contribute to, amongst others, the regulation of σe expression  <cit> .
since the expression of ania was in turn shown to be under the negative control of σe  <cit> , the finding that no generated by ania inhibits platelet aggregation  <cit>  provides an exemplary link between oxidative stress, glu/gln metabolism and the pathophysiology of meningococcal disease.
in this picture, virulence, i.e.
host damage, occurs coincidentally due to the transcriptional compensation of metabolic deficiencies by including genes with an established role in the pathogenesis of imd.
in consequence, meningococcal virulence is a byproduct of global stress and metabolic responses potentially triggered by human neutrophils and is dependent on the genetic background of the invading strain.
in line with the recently proposed damage-response framework of microbial pathogenesis  <cit> , we further hypothesize that strain-dependent differences in the interaction of meningococci with human neutrophils have a central role in explaining meningococcal virulence differences.
however, a better systems biological understanding of the interaction of human phagocytes with meningococcal cells under infection relevant conditions has yet to be reached, and in addition to transcription  <cit>  many other layers such as post-transcriptional modifications, allosteric regulation  <cit>  and non-coding small rnas  <cit>  are likely to play an important role in metabolic regulation in meningococci.
the phenotypic similarity of both strains under the ex vivo conditions tested further indicate that most of genotypic variation observed in the genomic comparisons  is likely cryptic for selection under conditions encountered by the species during its commensal life-cycle, and only upon an environmental perturbation these genotypic differences do result in different phenotypes.
in evolutionary genetics, this so called cryptic genetic variation  describes the part of the genetic diversity that has the potential to affect the phenotype but that is not expressed under the current genotypic or environmental conditions which limits the opportunities for selection to act on the variation  <cit> .
however, under atypical conditions, rare in the history of a population, cgv can generate phenotypic variation.
human blood is a condition that is normally not encountered by meningococci during its commensal life cycle, and since bacteria replicating in the bloodstream are not transmitted to new hosts it has not been encountered by any ancestral meningococcal strain before.
cgv might thus contribute to the high standing genetic variation observed in meningococcal population since the conditions that induce such allelic effects are rare in the history of the population, and imd would therefore result from the accidental unmasking of meningococcal cgv in human blood.
furthermore, cgv is a subclass of variation with conditional effects, either in form of g × g or in g × e interaction.
accordingly, not only by environmental perturbation discussed above but also by genetic perturbation such as deletion of rela we could experimentally uncover cgv likely in genes required for amino acid biosynthesis and ex vivo fitness .
loci with such pleiotropic effects known as genetic hubs or buffers are a common feature of the genetics of gene expression  <cit>  and their existence emerges from the concept of cgv  <cit> .
these genes are important for buffering both environmental change and stochastic variation thus ensuring environmental and stochastic resilience.
rather surprisingly, there have been only very few verified genetic hubs in published genetical genomics studies to date.
based on our observations and the published data on the physiology of the stringent response in, e.g., e. coli we hypothesize that it contributes to the phenotypic buffering of cgv in metabolic genes and that rela in particular might constitute such a regulatory hub coincidentally affecting also meningococcal virulence.
therefore, the elucidation of the genetic variability and molecular mechanisms of stress responses in n. meningitidis will be important for the understanding of virulence evolution in this commensal pathogen.
of note, whereas the variability in the complement and/or sequence of genes coding for surface antigens and metabolic functions, respectively, among different meningococcal lineages and their potential role in meningococcal virulence have already been well established , the extent of sequence variability in stress response genes and their possible contribution to virulence differences in meningococci have not been addressed so far.
in addition, differences in intergenic regions affecting gene expression regulation  <cit>  are often overlooked genetic determinants in the search for bacterial virulence factors.
the impaired fitness of the α <dig> atrrela deletion mutant in human blood  provides an example of how regulatory evolution via the integration of non-coding mites  <cit>  into promoter regions might contribute to fitness and consequently virulence differences among bacterial strains in a condition-dependent manner.
as all experimental approaches for studying meningococcal infection biology this study has also its limitations.
first, the ex vivo conditions chosen might not be truly representative for the in vivo situation.
in particular, although meningococci have been found to be part of the normal flora of the oral cavity  <cit>  they actually colonize the human nasopharynx where they immerse in the liquid produced by nasopharyngeal epithelium and constantly interact with epithelial cells.
therefore, saliva produced by saliva glands may not be truly representative of this commensal environment.
second, given their high genetic diversity  <cit>  more meningococcal strains need to be compared in order to see how generalizable the transcriptomic results are with respect to the entire species.
furthermore, any microarray-based approach restricts the number of genes being compared to those represented on the microarray.
comparative transcriptome sequencing  approaches circumvent this limitation and will allow not only for the detection of expression differences in non-coding small rnas  <cit>  but, due to the higher dynamic range compared to microarrays  <cit> , also for the detection of differences in genes expressed at very low and very high levels, respectively.
finally, as it was experimentally not possible to generate rela and spot complemented strains for cis/trans complementation assays, the phenotypes of the rela and rela spot deletion strains could in principle be also affected by polar effects of the gene disruption on adjacent genes.
this needs to be addressed in further experimental studies.
in summary, the data presented in this work allow novel hypotheses to be generated regarding the genetic basis of meningococcal virulence differences.
they in particular warrant detailed analyses of the interaction between meningococci and human neutrophils, the physiological consequences of sequence differences in cysh, glne or glnbp, the strain-dependent regulation of the stringent response in meningococci and how atr might affect the expression of adjacent genes.
the large transcriptomic data set provides per se an ex vivo gene expression compendium and as such a valuable resource for the meningococcal research community.
finally, our experimental approach further allows to challenge these findings in a larger panel of strains from carriage as well as hyperinvasive lineages and to seek for other genetic determinants affecting ex vivo fitness.
CONCLUSIONS
in the present work, we showed that despite identical ex vivo phenotypes two genetically similar strains of n. meningitidis displayed large differences in their transcriptomes including numerous virulence genes and subject to environmental conditions.
consequently, the often employed “model strain” approach might give misleading results in genetically diverse species like n. meningitidis which might not be representative for the entire species.
furthermore, the finding that rela is conditionally essential and likely contributes to the transcriptional buffering of cryptic genetic variation in metabolic genes potentially limits the universality of rela as a novel drug target  <cit> .
finally, beyond the variation in the set of virulence genes observed in “model” bacterial pathogens such as e. coli, the finding that a short, non-coding repeat element affected meningococcal fitness in a strain- and condition-dependent manner highlights the importance also of regulatory evolution in the emergence of virulence in commensal pathogens.
together, these findings demonstrate that “to generalize results across genetic backgrounds, experiments must be carried out across genetic backgrounds”  <cit> , and because the pathogenic nature of a microbe is a quantitative trait resulting from multiple interacting loci, with allelic effects that are sensitive to the environmental conditions, best within a systems biological framework.
in this respect our experimental approach is generic for the identification of loci that are associated with the invasive phenotype also in other genetically diverse commensal pathogens.

 the human body is home to a vast number of different bacterial species, and the overwhelming complexity of the human microbiome has only very recently been fully uncovered. although the majority of these colonizing bacteria seem to be harmless or even beneficial commensals, some have long been known to be janus-faced, and neisseria meningitidis is a particularly prominent example in this respect. on the one hand, this β-proteobacterium is an exclusively human-adapted commensal that is carried in the nasopharynx of about 20% of the healthy population. on the other hand, n. meningitidis is also a ferocious pathogen that can cause life-threatening invasive meningococcal disease, and “no other infection so quickly slays”. after crossing the mucosal barrier and entering the bloodstream, meningococci can cause septicemia, and by crossing the blood–brain barrier and multiplying in the cerebrospinal fluid also acute bacterial meningitis, both often within less than 24 h. in many commensal pathogens like escherichia coli, often the only difference between a pathogenic and a non-pathogenic strain is a small set of so called virulence genes. by definition, a virulence gene is a gene whose loss specifically impairs virulence but not viability in rich media and which should be associated exclusively with pathogenic but not with non-pathogenic strains of a species. however, with respect to gene content meningococcal strains isolated from healthy carriers and imd patients are almost indistinguishable, and many of the so called meningococcal virulence genes have also been found in purely commensal neisserial species. the analysis of meningococcal population genetic structure by multilocus sequence typing demonstrated that disease-causing meningococci do belong to particular groups of related sequence types, termed clonal complexes, which are overrepresented in disease isolates relative to their carriage prevalences and are responsible for most disease. accordingly, these data indicate that the propensity to cause invasive disease is somehow associated with the genetic make-up of hyperinvasive lineages. experimental observations along with epidemiological models further indicate that genetic differences in metabolic genes might have a central role in the observed virulence differences among different lineages in a yet to define manner. alongside the well-established significance of gene content variation in creating genetic diversity in the bacterial world, regulatory evolution is increasingly acknowledged to substantially contribute to this diversity. mutations affecting gene expression regulation encompass differences in the coding sequences of transcription factors acting in trans and thus affecting the expression of entire regulons, as well as sequence differences in the regulatory regions acting in cis on the expression of downstream genes. by acquiring functionally divergent homologous promoter regions through horizontal transfer bacterial genes were shown to rapidly shift between multiple regulatory modes affecting, for example, up to 15% of the meningococcal core genome. in addition, also mutations in metabolic genes can indirectly cause compensatory changes in gene expression regulation of other housekeeping genes to maintain cellular homeostasis. the ensuing differential regulation of conserved genes can mediate phenotypic traits that distinguish closely related bacterial species or even strains of the same species. in consequence, also the expression of a virulence-associated gene could be epistatic and thus depend on the genetic background of the respective strain and/or the environment. given the high genetic diversity of n. meningitidis, a virulence gene candidate should consequently be differently expressed between an invasive and a commensal strain under disease mimicking conditions but not under conditions mimicking the commensal state. in addition, although the correlation between when genes are important for fitness and when those genes are upregulated was shown to be small, the fitness of a knock-out strain should differ between an invasive and a commensal strain under conditions mimicking invasive infection. the strict tropism of n. meningitidis for humans has so far impeded the development of a suitable animal model to analyze the course of meningococcal infection in vivo, and therefore alternative experimental approaches such as ex vivo models have been established to study meningococcal infection biology. for example, human whole blood served as an ex vivo model to analyze how meningococci regulate gene expression to permit survival in human bloodstream during septicemia. likewise, meningococcal resistance to human complement was studied in an ex vivo model using human csf, and human saliva has already been used to study ex vivo the transcriptional response which enables meningococci to adapt to this relevant host niche. however, most of these ex vivo studies analyzed gene expression only under a single condition and/or used only a single strain from a hyperinvasive lineage. consequently, our knowledge about how this commensal pathogen adapts during the transition from colonization to an invasive infection is still very limited, and nothing is known so far about gene expression or phenotypic variability between carriage and hyperinvasive strains in conditions mimicking invasive infection. in a systems biological approach we used different ex vivo conditions as environmental perturbation and natural genetic variation as genetic perturbation of the meningococcal gene expression network and considered gene expression as quantitative intermediate phenotype. based on prior population genetic information we selected two genetically related meningococcal serogroup b strains from the same phylogenetic clade pc32/ with yet markedly different epidemiology. strain mc belonging to the hyperinvasive st- cc was chosen as a reference as this strain has already served as a model system to experimentally study meningococcal infection biology in vitro and a large body of transcriptomic data is thus available. the carriage strain α belongs to the carriage st- cc which is a four locus variant of the st- cc sharing about 96% of its genes with strain mc. we combined phenotypic, genomic and transcriptomic comparisons with mutagenesis studies to seek genetic variants that influence meningococcal gene expression in human saliva, whole blood and csf mimicking commensal and virulence compartments, respectively. gene expression was analyzed in a strain- and condition-dependent manner with particular emphasis on virulence-associated genes and genes involved in gene expression regulation. the combined data show that transcriptomic buffering of cryptic genetic variation, which is the genetic variation present in the meningococcal population that is not phenotypically expressed under commensal conditions but visible upon environmental or genetic perturbations such as growth in human blood, contributes to the regulatory evolution of meningococcal virulence. we further demonstrate that it is likely affected by the differential presence of a short, non-coding inverted-repeat transposable-element in the promoter region of rela encoding the guanosine 3'-diphosphate 5-'diphosphate ppgpp) synthetase of the stringent response pathway.table strains used for ex vivo transcriptome comparisons  phylogenetic clade  frequency of cc in carriers  disease/carriage ratio  serum resistance  adhesion to epithelial cells  invasion of epithelial cells in vitro logarithmic growth rates ex vivo growth rates according to ref. according to ref. according to ref. ratio in percent of viable bacteria after incubation for 30 min in the presence of 10% human serum and viable bacteria incubated without serum. given are the average and standard deviation from four independent experiments with pooled human serum ratio in percent of adherent and invasive bacteria, respectively, to total bacteria. given are the average and standard deviation from at least four independent experiments given are the mean and standard deviation of the logarithmic growth rate k according to log/od 600) = kt for t ∈ as depicted in fig.  using linear regression given are the mean and standard deviation of the growth rate k according to log/n) = kt for t ∈ as depicted in fig.  using linear regression  mc and α have similar gene content including most genes invovled in host interactions but with function-dependent sequence variation among orthologs in order to comprehensively analyze genetic differences between both strains we generated a draft sequence of the α genome for comparative genome expression analyses. the common genomic backbone of both strains comprises mbp and encodes orthologous proteins with an average blastp bit score ratio of  = ), corresponding to an average amino acid identity of %. notably, there was a significant variation of the bsrps of orthologous proteins with respect to the cog functional category. the 10% most divergent orthologous genes were significantly enriched for genes involved in cell motility  = , false discovery rate  = , fisher’s exact test with benjamini-hochberg multiple testing correction) as well as secretion and transport. the latter included numerous surface antigens involved in host interactions such as the major type iv pilus subunit protein pile, the major outer membrane proteins pora and porb, the autotransporters app and nalp, as well as proteins involved in iron acquisition like the transferrin-binding protein b and the lactoferrin-binding protein b. compared to the 10% most divergent orthologous genes, identical genes were significantly enriched for genes involved in energy production and conversion, carbohydrate transport and metabolism, amino acid biosynthesis and translation, ribosomal structure and biogenesis. of the genes coding for putative virulence factors or involved in meningococcal host interactions in strain mc only are missing in strain α and strain α lacks in particular large parts of the islands of horizontal transfer b and c that code for a two-partner secretion system involved in host cell adhesion. it further lacks almost the entire repeat-in-toxin island encoding frpa/c-like proteins which induce high levels of serum antibodies during invasive disease in humans. in addition, downstream of glnb encoding the signal-transducing nitrogen regulatory protein pii strain α also lacks nada which codes for a minor adhesin that was found to promote bacterial adhesion to and penetration into human epithelial cells in vitro. mc further harbors a 30 kb duplication spanning coding sequences involving the cysghdnji genes for sulfur acquisition which is missing in α as in most other meningococcal genomes. both strains display similar phenotypes in ex vivo fitness and in vitro cell culture assays indicative of phenotypic buffering despite the genetic differences described above, both strains were phenotypically similar with respect to resistance against human serum, adhesion to and invasion of nasopharyngeal cell lines, respectively, as well as in ex vivo growth. surprisingly, although meningococci are part of the human oral microbiome both strains were not able to grow in human saliva. since strain mc was able to grow in meningococcal minimal medium, the observed growth inhibitory effect of human saliva was probably not due to nutrient limitation but more likely caused by the presence of growth inhibiting substances known to be present in human saliva like antimicrobial peptides. of note, in their ecological niche, the human nasopharynx, meningococci are attached to host cells in the form of microcolonies and are unlikely to grow in a planktonic state as in the ex vivo situation. the fact that bacteria in biofilms are less susceptible to antimicrobial agents and host immune responses thereby becoming persistent colonizers might explain the unexpected ex vivo growth phenotype in saliva. furthermore, both strains grew equally well in human blood and csf and thus under disease mimicking conditions despite their about 7-fold difference in the disease/carriage ratio. these data indicate that the ability to survive in human blood is obviously necessary but per se not sufficient to cause imd. we therefore focused on compensatory mechanisms which might buffer the genotypic differences described above under ex vivo conditions and considered the transcriptome as an intermediate phenotype. we hypothesized that both strains differ in the way they accomplish growth under disease mimicking conditions by differential transcriptional activation of metabolic and stress response pathways. for convenience, comparisons of the mc transcriptomes between saliva, whole blood and csf, respectively, will be called cross-condition comparisons and discussed only briefly in the main text and in more detail in the additional file likewise, comparisons of the transcriptomes of strain mc and α in saliva, whole blood and csf, respectively, will be called cross-strain comparisons. genes differently expressed between two conditions or between both strains in a given condition will be called differently expressed genes, and genes differently regulated between both strains between two conditions will shortly be called differently regulated genes. throughout the following analyses, we further discern directional and non-directional classes of gene sets. the non-directional class of significantly differently expressed genes contains genes where the information about direction of differential expression is omitted, so that significant gene sets can be interpreted as affected by differential expression in general. the directional class aims to identify gene sets that are significantly affected by regulation in a distinct direction, i. e. condition or strain. if a gene set contains significantly expressed genes in both directions, they will cancel out and the directional fdr-value will not be significant. in turn, if a gene set is not significantly enriched for differently expressed genes in general it might nonetheless comprise mainly genes with a significant expression bias in one direction. it consequently will have a non-significant non-directional fdr-value but a significant directional fdr-value. condition-dependent expression changes affect 24% of the mc transcriptome of the genes assayed in the cross-condition comparisons in strain mc genes were differently expressed between saliva and blood and between blood and csf, respectively. gene set enrichment analysis showed that genes differently expressed between saliva and blood were significantly enriched for genes coding for nucleotide transport and metabolism, whereas genes differently expressed between blood and csf were just slightly enriched for genes involved in posttranslational modification, protein turnover and chaperones. gsa further showed significant differences also in the directionality of gene expression changes. the genes that showed higher expression levels in saliva compared to blood were enriched for genes involved in energy metabolism and conversion, posttranslational modification, protein turnover and chaperones and cell envelope and outer membrane biogenesis. however, genes that were more highly expressed in blood than in saliva and csf, respectively, were significantly enriched predominantly for genes coding for proteins without any cog functional annotation so far, suggesting that our knowledge about the mechanisms employed by meningococci to survive in human blood are still quite limited.fig. genes significantly differently expressed and/or regulated in cross-condition and/or cross-strain comparisons. a venn diagram comparing sets of genes in strain mc differently expressed between conditions as indicated. the total number of genes compared was b venn diagrams comparing sets of genes differently expressed between strains as indicated with each diagram. the total number of genes compared in each panel was c heatmap depicting cross-condition and cross-strain gene expression differences and hierarchical clustering of significantly differently expressed genes. average linkage clustering based on the spearman rank correlation of all genes significantly differently expressed and/or regulated in at least one cross-condition and/or cross-strain comparison. grey lines correspond to genes that were absent in the α genome sequence and therefore excluded from the cross-strain comparisons comparison of differently versus non-differently expressed genes to identify cog categories that are affected by differential expression in general comparison of differentially expressed genes to identify cog categories that are significantly affected by regulation in a distinct direction odds ratios based on fisher’s exact test. values greater than indicate that significantly differentially expressed genes are enriched for genes from the corresponding cog functional class false discovery rate based on p-values from fisher’s exact test and the benjamini-hochberg multiple testing correction with a significance cut-off of fdr <  for cross-condition comparisons, values greater than indicate that genes highly expressed in blood were enriched for the respective cog category. in cross-strain comparisons, values greater than indicate that the respective cog category is significantly highly expressed or upregulated in strain mc58 fig. gene set enrichment analysis of differently expressed genes. a non-directional comparison of significantly differently expressed gene sets according to the cog functional classification scheme for cross-condition and cross-strain comparisons, respectively, indicating significantly overrepresented functional categories among the significantly differently expressed genes. b directional comparison of significantly differently expressed gene sets according to the cog functional classification scheme for cross-condition and cross-strain comparisons, indicating significant gene expression asymmetries. in both panels, the heat map depicts significantly enriched cog functional categories in red coloring. the kind of comparison is indicated for each column of the heat maps, and the corresponding fdrs are color coded and given in the respective inserts. the associated tree is based on average linkage clustering of the functional categories using the spearman rank correlation coefficient both strains differ in the expression of over genes in a condition and strain dependent manner based on the genome comparisons we further selected a sub-set of single-copy orthologous genes for cross-strain gene expression comparisons. of these, were expressed and/or regulated at significantly different levels between both strains. notably, proteins differently expressed in at least one ex vivo condition were not significantly more variable between both strains as indicated by their bsrps than proteins that were not, confirming that there was no significant sequence bias in the expression data. of all the genes that showed cross-condition and/or cross-strain expression differences were differently expressed only in cross-strain and only in cross-condition comparisons, respectively, and the overlap between cross-condition and cross-strain expression comparisons was thus surprisingly small. the total number of significantly differently expressed genes between both strains was small in rich medium and csf and not significantly greater than the number expected by chance, and there were only and genes that showed significant expression level differences between both strains exclusively in csf and ppm+, respectively. the number of differently expressed genes was significantly higher for saliva and blood than expected by chance, although both strains were not able to grow in saliva. this further suggests that gene expression differences were not due to growth rate differences but were specific responses of both strains to these two ex vivo conditions. in line with this hypothesis, the transcriptomes differed significantly among both strains in response to the ex vivo condition tested. of note, the pattern of transcriptionally activated genes upon transition from saliva to blood was opposite to the pattern of transcriptionally activated genes upon transition from blood to csf, indicating that transcriptional changes are likely in response to components present in human blood but neither in saliva nor csf. since these two ex vivo compounds were both free of any phagocytic cells, differences in transcriptional responses in both strains might be triggered by blood phagocytes. cross-strain gene expression differences in saliva comprise numerous stress response genes a total of genes were differently expressed between both strains in saliva, with a slight yet not significant enrichment of genes involved in anorganic ion transport and metabolism. functionally, around one third coded each for poorly characterized proteins, proteins involved in cellular processes or proteins involved in metabolism or information storage and processing. among the genes highly expressed in strain α were, amongst others, seven for anorganic ion transport and metabolism proteins including a putative multidrug resistance protein, the lactoferrin-binding protein a and the putative ammonium transporter amtb, as well as six genes involved in cell envelope biogenesis including genes for the two sialic acid capsule biosynthesis proteins synx/siaa/cssa and siab/cssb. genes involved in translation and ribosomal biogenesis comprised the single largest group among the genes that were in turn highly expressed in strain mc in saliva, next to genes in amino acid transport and metabolism and cell envelope biogenesis. since both strains were not able to grow in human saliva these gene expression differences likely reflect differences in the stress response between both strains when exposed to this hostile environment. both strains differed in the expression of virulence-associated genes involved in the pathogenesis of imd numerous genes involved in the interaction of meningococci with its human host have so far been studied in order to understand the genetic and mechanistic basis of meningococcal virulence, i.e. host damage, and were consequently of special interest. accordingly, of the virulence-associated genes used for cross-strain comparisons, were differently expressed and/or regulated among both strains under at least one of the ex vivo conditions tested which is significantly more than in the cross-condition comparisons for strain mc. in addition, genes differently expressed and/or regulated between both strains in at least one cross-strain comparison were significantly enriched for virulence-associated genes, and the expression profiles were significantly different between virulence-associated and not virulence-associated genes. of the virulence-associated genes that were differently expressed between both strains in blood, were highly expressed in α and comprised genes involved in cell envelope biogenesis and in particular genes for capsule and los biosynthesis. los was shown to be the dominant molecule in meningococci inducing organ inflammation in human patients, and differences in los biosynthesis might therefore have an immediate impact on the extent of host damage caused by both strains. in turn, genes involved in type iv pilus biosynthesis iron homeostasis, the stress response genes as well as genes encoding adhesins such as opc or the hemagglutinin/hemolysin-related protein tpsa or nspa were all highly expressed in mc besides opc, we could at best detect only very small expression level differences for other recently introduced vaccine antigens that were conserved in both strains. likewise, of the genes that have been described so far as being involved in neutrophil interactions in neisseria and which were part of the gene expression comparisons, were differently expressed between both strains in blood. genes differently expressed between both strains were therefore significantly enriched for neutrophil response genes. these included a number of virulence-associated genes like capsule synthesis genes or genes coding for efflux pump components, as well as genes not so far associated with meningococcal virulence like the dna damage repair genes uvra and uvrb. this finding indicates that neutrophils might have an important role in shaping the meningococcal transcriptional response to human blood. of note, ania encoding nitrite reductase and norb coding for no reductase showed the largest blood-specific expression differences between both strains and were among the highest expressed genes in mc as was also confirmed by qrt-pcr. the gene products ania and norb constitute a pathway that enables the organism to grow under conditions of low oxygen in the presence of nitrite. amongst others, meningococcal derived no was recently shown to play an essential role in the pathophysiology of septicemic meningococcal infection in humans by inhibiting platelet aggregation and modifying the release of cytokines and chemokines by human macrophages. therefore, a number of virulence-associated genes with an also experimentally established role in the pathogenesis of imd like ania or los biosynthesis genes were differently expressed in both strains particularly in human blood. transcriptomic differences between both strains associated with the transition from saliva to blood are enriched for metabolic, information processing and cell envelope biogenesis genes overall, the number of differently expressed genes between both strains was highest in blood, and also the number of genes differently regulated between both strains between two conditions was highest between saliva and blood. both gene sets were functionally enriched for genes required for energy production and conversion. these data thus clearly demonstrate differences in the environment-dependent gene-expression regulation between both strains. the genes highly expressed in strain α in human blood were significantly enriched for genes involved in cell wall/membrane biogenesis, including los and peptidoglycan biosynthesis genes like murb, murd, mure and ddl. in contrast, the genes highly expressed in strain mc were significantly enriched for metabolic genes including genes for oxidative phosphorylation and nitrogen respiration such as ania and norb described above. with respect to differences in the direction of gene expression regulation in both strains, genes required for energy production and conversion were also strongly upregulated in strain mc between saliva and blood, whereas genes for cell envelope biogenesis and translation were in turn strongly upregulated in strain α based on differences in the directionality of gene expression levels and regulation, the functional categories cog e, c and m form a cluster of co-regulated genes which is part of a larger cluster including also the functional categories cog j, cog k and cog l. this finding suggests a regulatory link between metabolism, the biosynthesis of the cell envelope, and genes for the gene expression machinery which is differently activated in both strains particularly in human blood. integrative network analysis of differently expressed genes identifies subnetworks of co-regulated genes in order to identify differentially expressed functional subnetworks, we combined the transcriptomic with protein-protein interaction network data of strain mc as deposited in the string database and used an algorithm which optimally identifies responsive subnetworks. this integrative network analysis revealed a densly connected subnetwork comprising mainly genes that are highly expressed in strain mc in blood involved particularly in energy and carbohydrate metabolism. this subnetwork comprised genes of the tricarboxylic acid cycle, for the metabolism of pyruvate, glycine, leucine and fatty acids. the second subnetwork contained significantly more genes highly expressed in strain α coding for a significantly different array of cellular functions. it comprised genes involved in the biosynthesis of the capsule, peptidoglycan, and los, respectively, as well as genes for trafficking, secretion and vesicular transport or information storage and processing including in particular translation and ribosomal biogenesis genes. a few genes in this second subnetwork were highly expressed in mc yet and included genes for glycolysis, the genes for the na+-translocating nadh-quinone reductase subunit b, d and f along with virulence-associated genes mentioned above like dsba- pilg, pilt- sodc. transcriptomic differences in both strains in blood were thus organized in two subnetworks consisting mainly of genes that were either highly expressed in α or in mc and that coded for different biological functions.fig. analysis of gene expression data based on protein-protein interaction networks. a integrative network analysis of differently expressed genes between strain mc and α in human whole blood based on the string protein-protein interaction network for strain mc. a subnetwork that consists predominantly of genes that were expressed at higher levels in mc than in α and that code for metabolic genes and in particular for genes involved in energy production and conversion is shaded in orange. the remaining part of the network comprising protein-coding genes is shaded in light blue. b integrative network analysis of gene regulation differences between both strains upon transition from saliva to blood. the two modules consisting predominantly of genes either upregulated in α or mc upon transition from saliva to blood are shaded in green and orange, respectively. only 21% of the genes in the left subnetwork consisting mainly of genes that were upregulated in α code for proteins involved in metabolism compared to over 66% of the genes that were upregulated in mc upon transition from saliva to blood. for each gene, the respective expression differences between conditions and strains, respectively, are color coded and indicated in each panel. white boxes indicate genes that were not differently expressed but are part of a subnetwork as identified by the integrative network analysis. pie charts next to the sub-networks in each panel show the distribution of proteins in the respective subnetwork over the different cog functional classes saliva vs. blood miss misr siac siab siaa ctrb lipb kdta lpxc lpxa lpxd lpxb lgtf lgtb rfac pilt-1 pilf pilg pilt-2 pilp pglb pglc fara mtre mtrd - omp85 rmpm tpsa2 nspa opc tpsa3 - nalp tbp2 - - brfb brfa lbpa dsba-1 dsba-2 znub znuc sodc - - fabz lctp iga hemh purc pgm - - prc - - - - norb ania - - - - virulence-associated genes were compiled from table in ref., table in ref., table in ref., and additional file from ref.. only those genes are included in the transcriptome comparisons that showed significant expression differences in at least one type of comparison positive values indicate that the gene is expressed at higher levels in blood than in saliva positive values indicate that the gene is expressed at higher levels in csf than in blood positive values indicate that the gene is expressed at higher levels in strain mc than in α522 positive values indicate that the gene expression difference between saliva and blood is greater in strain mc than it is in α522 positive values indicate that the gene expression difference between blood and csf is greater in strain mc than it is in α522 n. s., not significant n. d., no data due to missing α genome sequence data along with these cross-strain differences in the directionality of gene expression levels also cross-strain differences in the directionality of gene regulation between saliva and blood were organized in two functionally differing subnetworks. the first subnetwork consisted almost exclusively of genes found to be upregulated in mc in blood compared to saliva, and over 70% of the genes in this subnetwork code for metabolic functions compared to only 24% in the other subnetwork. it comprised genes for tca cycle enzymes, for 2-oxocarboxylic acid metabolism, oxidative phosphorylation, fatty acid metabolism, for thioredoxin-fold proteins required for oxidative stress response as well as the oxidoreductase genes nqrb, nqrd, nqrf and ania, the latter playing a major role in the pathogenesis of imd as described above. the second subnetwork consisted of genes that were strongly upregulated in strain α in blood compared to saliva and included predominantly genes involved in information storage and processing and in particular for the translation machinery. in line with the gsa results, integrative network analysis thus indicated that in contrast to strain α strain mc allocates transcriptional resources predominantly in the expression of metabolic genes in human blood. elementary mode analysis of blood transcriptomic data indicate compensatory flux differences between both strains particularly in energy, glutamine and cysteine metabolism in order to assess the possible impact of the transcriptomic differences on metabolism in blood in more detail, we reconstructed a condensed metabolic network based on the nmb_itm model for strain mc comprising enzymes and metabolites. the elementary metabolic modes and pathways as identified by elementary mode analysis are given in the additional file 3: s computation of metabolic fluxes based on the observed growth rates of both strains in human blood, the corresponding gene expression data and the average composition of human blood as taken from showed that the flux activities in mc were 25-50% higher than in α with major differences in the metabolism of some amino acids. of note, one flux mode displayed an opposite direction between mc and α indicating that α uses a large amount of external glutamine from human blood to produce gutamate.fig. analysis of gene expression data based on a metabolic model for strain mc a comparison of elementary mode activities in mc and α the histogram depicts differences in the elementary mode activities for each of the elementary metabolic modes as defined in the additional file 3: s for strain mc and α based on gene expression data in human blood. b inferred differences in metabolic fluxes between strain mc and strain α in blood based on a metabolic model for strain mc internal metabolites which are considered to have balanced concentrations are given by dark green spheres, external metabolites which are allowed to accumulate or to be consumed by green cones, and reactions together with their corresponding numbers as light green boxes. the reactions for all reaction numbers are given in the additional file 3: s arrows connect reaction with metabolites. red coloring indicates higher fluxes in mc compared to strain α whereas blue colouring indicates that the flux is slightly enhanced in α asterisks along with pink colouring indicate that the reaction has an opposite direction in both strains for more detailed information about differences in important biochemical pathways, we converted the flux activities into enzyme and enzyme complex activities, respectively. the major differences are given in table  and fig. 4b. only very few fluxes were slightly stronger in α such as r r and r which are all involved in sulfur metabolism. on the other hand, there were numerous reactions that were even more than tenfold stronger in mc than in α such as the acetate synthesis reactions r r and r suggesting that mc compared to α may accumulate acetate as intermediate metabolite in this environment. in meningococci, such metabolic stimulation has been shown to result from the consumption of lactate which in the presence of glucose is used as a source of additional energy. of note, lactate is a by-product of neutrophil glycolysis and enhances bacterial consumption of molecular oxygen, which depletes the substrate for neutrophil nadph oxidase and thus blunts its oxidative burst. since both strains have almost identical growth rates in human blood mc probably requires this additional metabolic energy for other, not growth related processes such as, e.g., defense against the neutrophil oxidative burst. other reactions that were particularly stronger in mc included r r and r leading from phosphoenolpyruvate to α-ketoglutarate as well as some reactions involved in amino acid metabolism like r and r resulting in the synthesis of serine from glu and glycine, respectively, or r r and r resulting in the synthesis of leucine, phenylalanine and tyrosine from glu, respectively. since the reactions r r r and r are all transamination reactions resulting in the consumption of glu and concomitant synthesis of akg, these findings further suggest that mc and α might differ in their intracellular levels of glu and akg in human blood. finally, reactions r and r have opposite directions in both strains in human blood, which indicates that α relies mostly on gln to produce glu with some glu being further converted into akg, whereas mc is capable to produce enough glu from the tca cycle and convert it further to gln. likewise, also reaction r which is the production of cysteine from serine has an opposite direction in both strains in human blood, and α consequently seems to rely on external cys to produce ser whereas mc is capable of producing cys from ser.table inferred reaction activity differences between mc and α in human blood based on gene cross-strain expression differences ratio of the reaction activity in mc divided by the reaction activity in α a ratio larger than one indicates that the reaction has a higher activity in mc and a negative ratio that the reaction occurs in opposite directions both strains strain α differs from strain mc in gln and cys auxotrophy in vitro in order to experimentally validate the transcriptomic results with respect to possible differences in gln and cys metabolism, we assessed the growth of both strains in mmm supplemented with different amino acids as well as ppm+. whereas both strains were equally able to grow in ppm+ and mmm supplemented with all proteinogenic amino acids at milimolar concentrations, strain α was not able to grow in mmm without amino acids. in addition to glucose or lactose as carbon source it requires cys and gln for growth. contrary to α the growth of mc was slightly suppressed by these two amino acids. the ability of mc but not α to grow in the absence of cys and gln indicates strain specific differences in the respective metabolic pathways, and the requirement of some meningococcal strains for cys and its growth inhibiting effect on others has already been reported. in parallel with these phenotypic differences, genome comparisons revealed also striking differences in cys and gln biosynthesis genes that might contribute to the phenotypic finding, although the repertoire of enzymes required for the biosynthesis of amino acids is otherwise highly conserved in both genomes. in addition to the duplication of cysghdnji genes in mc, these include large sequence differences in the phosphoadenosine phosphosulfate reductase cysh and the glutamate-ammonia-ligase adenylyltransferase glne which are among the least conserved genes involved in the biosynthesis of amino acids. cysh is required for the reduction of sulfate into hydrogen sulfide and thus for sulfur acquisition in n. meningitidis, and glne is a key regulatory enzyme in nitrogen assimilation in e. coli. furthermore, the intergenic region between purl and glnb differs in both strains due to the insertion of coding sequence in strain α upstream of glnb resulting in entirely different glnb promoter regions. along with glne glnb is involved in the regulation of nitrogen assimilation in e. coli via affecting the activity of glna, an enzyme that lies at the heart of the nitrogen assimilation network. glna is involved in the complex regulation of the interconversion of glu to gln in response to the intracellular concentration of ammonium, glu, the gln/akg ratio, the redox and the free energy state of the cell. sequence variation at these loci is therefore likely to have pleiotropic effects, and a detailed experimental analysis of the biochemical consequences of these genetic differences is subject to ongoing work.fig. growth phenotypes of n. meningitidis mc and α wild-type and mutant strains. a in vitro growth phenotypes. growth as quantified by the optical density is given on the ordinate and the time in hours on the abscissa. b ex vivo growth phenotypes. the number of colony forming units for each time point ) relative to the initial number ) is given on the ordinate and the time in minutes on the abscissa. for each strain and condition the respective growth curves are coded as indicated in the insert in each panel, and the genotypes of the respective strains compared are shown along with the corresponding growth curves. in each experiment rich medium was used as growth control. the arrow at the top of panel b indicates the time when total rna was extracted for microarray analysis expression changes in numerous regulatory genes are likely compensatory the large transcriptome differences observed particularly in blood could be caused by differences in regulatory elements acting either in cis or in trans, or be compensatory to ensure cellular homeostasis. among the orthologous proteins those involved in gene expression regulation were no more different between both strains than proteins involved in other functions. also the 200 bp upstream regions were not less conserved than the downstream orthologous genes. of the single-copy orthologous genes used for transcriptomic comparisons differed in the presence of putative mobile genetic elements within their 200 bp upstream regions. these comprised various so called minimal mobile elements as well correia repeats and correia repeat enclosed elements which have previously been shown to affect gene expression in a polar manner. however, this class of genes was not enriched for genes differently expressed in at least one ex vivo condition. although genetic differences in gene regulatory elements might contribute to the transcriptomic differences these data do not provide evidence yet that they are the sole reason of the large cross-strain expression differences observed particularly in human blood. they might rather orchestrate different compensatory gene expression adaptations in both strains in response to differences in the interaction of both strains with human blood components. both strains activate different sets of regulatory genes in response to human blood of the genes with significant expression differences in cross-condition or cross-strain comparisons involved in signal transduction or transcription, showed significant expression level differences between both strains in blood. the eight regulatory genes that were highly expressed in mc specifically in blood included in particular csta annotated as carbon starvation protein a, nmb coding for an arsr family transcriptional regulator, rela encoding the guanosine 3'-diphosphate 5-'diphosphate ppgpp) synthetase of the stringent response pathway, as well as misr coding for a phop-family response regulator of a two component signal transduction system. of note, genes of the genes differently expressed in blood belong to the misr regulon, and misr was shown to be involved in the oxidative stress response in meningococci, required for colonization of host cells and meningococcal survival in mice. in addition, differently expressed genes which are part of the fur regulon were almost all highly expressed in mc since fur senses cellular iron concentrations and since iron in general acts as a co-repressor, these data indicate that strain mc might experience more pronounced iron starvation in blood compared to strain α likewise, another genes are known to be regulated by fnr, the master regulator involved in the adaptation to oxygen-limited conditions, of which the majority were also highly expressed in mc table differentially expressed genes involved in transcription and signal transduction saliva vs. blood miss misr ptsn - narp bass fnr - - - - - - - - - - - rpoh rpoe - dksa nusg - - rho rnc - - - grea csta - nusa - rpoz rela - - - positive values indicate that the gene is highly expressed in blood positive values indicate that the gene is highly expressed in csf positive values indicate that the gene is highly expressed in strain mc58 n. s. not significant n. a. not applicable due to high sequence divergence between bot hhomologs n. d. no data due to missing α genome sequence data in turn, the ten regulatory genes highly expressed in α included nmb encoding a exoribonuclease and rpoe which codes for the alternative sigma factor e and in n. gonorrhoeae is activated in response to oxidative stress. since phagocytic cells are a major source of reactive oxygen species and were present only in the blood assay, these data further suggests that σe might be involved in meningococcal interaction with human phagocytes. in line with previous findings that the expression of ania and norb are under the negative control of σe, both strains also showed significantly different expression levels of ania and norb especially in blood as mentioned above. along with the finding that nitric oxide generated by ania inhibits platelet aggregation this observation provides a direct link between the oxidative stress response and the pathophysiology of imd. these data indicate that the complex gene expression differences between mc and α specifically in blood are caused by the activation of different sets of regulatory genes including misr, fur, fnr, rela and σe. in contrast to the expression of rpoe and misr which in both cases was found to be auto regulated, fur and fnr did not differ in their expression between both strains, suggesting strain-dependent differences in the post-transcriptional activation of these regulators especially in blood. differences in promoter regions demonstrate an important role for the stringent response and hfq mediated differences in gene expression regulation among both strains in blood although sequence analyses of the 200 bp upstream regions of the genes that were differently expressed between both strains in cross-strain comparisons failed to identify any consistent sequence differences or overrepresented bona fide tf binding sites for any of the cross-strain comparisons, the analysis of gc content variation yet revealed a 6 bp region immediately upstream of the predicted ribosome binding site having a significantly lower gc content in genes that were expressed at higher levels in mc than in α in blood. at the mrna level, such au-rich elements next to the rbs are often targets for the hfq-mediated binding of small non-coding rnas which thus post-transcriptionally regulate the degradation and/or translation efficiency of the corresponding mrna. in strain mc the rna chaperone hfq was already shown to be involved in the regulation of amino acid and energy metabolism, the oxidative stress response and required for survival in human blood. the comparison of the differently expressed genes showed that of the genes that are part of the hfq regulon and that were included in this study, were differently expressed in both strains, all higher in mc therefore, these data suggest that hfq contributes to gene regulation differences between both strains in blood.fig. gc content variation in potential promoter regions based on the mc genome sequence. a scatter plot of the gc content variation averaged over a 5-bp sliding window within 100 bp upstream regions for genes highly expressed in mc or α in human blood. the black line gives the gc content of the respective upstream regions for genes not differently expressed. regulatory regions are indicated at the top of the panel based on the average length of 5’-untranslated regions in n. gonorrhoeae. the insert gives the number of genes in each gene set. b box-and-whiskers plot depicting differences in the mean gc content of the putative discriminator and hfq-binding regions between genes highly expressed in mc or α in human blood as depicted in panel. the line within each box gives the median and the upper and lower margins the upper and the lower quartile, respectively. the whiskers denote the highest and the lowest values, respectively, and the open circles outliers. *: p < , **: p <  furthermore, genes differently expressed between both strains in blood had also significant gc content differences in a 10 bp region 40 bp upstream of the predicted rbs, and there was a negative correlation between expression differences in blood and the deviation from the average genomic gc content. since the average length of 5’-utrs in neisseria was shown to be between and 50 bp this region corresponds to the transcriptional start site. the length of this region, its pattern of gc content variation and its location close to the presumed transcriptional start site are hallmark features of so called discriminator regions which in γ-proteobacteria determine whether the adjacent gene is activated or repressed during the stringent response. accordingly, of the genes differently expressed between both strains in human blood, have a discriminator gc content higher than the genome-wide average of 50% and were highly expressed in α and genes with a discriminator gc content lower than the average were highly expressed in mc. together, these genes account for 56% of all genes differently expressed in blood. since activated targets typically have an at-rich discriminator whereas repressed targets have a gc-rich discriminator, these data along with the higher expression of rela in mc indicate that in blood the stringent response pathway is comparatively more activated in mc than in α genes highly expressed in mc and with a low gc discriminator region were predominantly involved in energy production and conversion and comprised genes involved in carbohydrate and energy metabolism, in aerobic energy generation, ccoo ), the genes for cytochrome c and c ) as well genes required for the oxidative/nitrosative stress responses along with surface proteins like nspa, lip and laz. genes highly expressed in α and having a high gc discriminator region were predominantly involved in cell envelope and outer membrane biosynthesis and included genes for los and peptidoglycan biosynthesis as well as regulatory genes including rpoe. rela and the stringent response pathway contribute to meningococcal ex vivo fitness in a condition and strain dependent manner although the stringent response pathway was already shown to be crucial for virulence in a number of bacterial pathogens, nothing is known about its contribution to meningococcal ex vivo or in vitro fitness so far. therefore, the observation that rela was differently expressed between both strains exclusively in blood along with the finding that genes differently expressed between both strains in blood had significant gc content differences in their putative discriminator regions prompted us to further assess the contribution of the stringent response and in particulate of rela to meningococcal fitness ex vivo. the machinery of the stringent response pathway comprises several enzymes involved in the turnover of ppgpp which is a signaling nucleotide that coordinates a variety of cellular activities in response to changes in nutritional abundance. in e. coli, rela is activated upon amino acid starvation and together with spot is able to catalyze pyrophosphoryl transfer from atp to gtp or gdp to synthesize ppgpp. together with dnak suppressor, ppgpp directs transcription initiation at particular gene promoters through binding to the interface between the two rna polymerase subunits β’ and ω. in part, ppgpp and dksa act by promoting the interaction of rna polymerase with alternative σ-factors such as σe or σh. when metabolic precursors are plentiful, spot instead degrades ppgpp, and the vegetative σ-factor, σ directs rna polymerase to genes that are crucial for bacterial replication. whereas β’, ω, spot and dksa were identical in both strains they differed in the coding sequences and promoter region of rela, and gene expression analyses via qrt-pcr further confirmed particular large and blood-specific cross-strain expression differences for rela but not for spot or dksa.fig. genomic distribution of atrs and the rela locus in n. meningitidis. a the intergenic region between grxb and rela. the integration site of a copy of an atr repeat element upstream of rela in strain α is indicated with respect to the mc locus. the transcriptional start sites as determined by 5’-race in both strains are indicated along with the deduced − and − boxes and the computationally predicted promoter regions using ppp. dr: direct repeat. b alignment of both the mc and α genomes as visualized with the artemis comparison tool based on a blastn comparison. the linearized mc and α genomes are shown in the upper and lower panel as gray bars, and regions syntenic in both genomes are connected via red and inverted regions via blue lines, respectively. the location of atrs is indicated by small arrows in each genome, and the rela region is highlighted in yellow to test whether these differences in the coding sequences of rela affected its catalytic activity we assessed ppgpp levels in wild-type, ∆rela::kmr as well as ∆rela::kmr ∆spot::cmr mutants in both strains during growth in ppm+ medium. as can be seen in additional file 1: figure s5a, both alleles were catalytically active and the catalytic activity was affected by the bacterial growth phase in a similar manner. similar to the α wild-type strain, the mc ∆rela::kmr mutant could further not grow in minimal medium, and in both genetic backgrounds the addition of all proteinogenic amino acids could compensate for the loss of functional rela but not the addition of only cys and/or gln to minimal medium. in addition, similar to e. coli ppgpp strains the growth defect in minimal medium was less severe in meningococcal ∆rela::kmr ∆spot::cmr double mutants. in support of the notion that high levels of ppgpp might be toxic for the meningococcal cell we could not obtain viable isogenic spot single deletion mutants, and a presumed spot knock-out mutant had a compensatory frame-shift mutation prior the catalytic domain of rela resulting in reduced ppgpp levels. with respect to ex vivo fitness the deletion of rela had no effect in mc on growth in saliva, blood or csf whereas the α ∆rela::kmr mutant was severely impaired exclusively in blood. the ex vivo fitness defect was less severe in a α ∆rela::kmr ∆spot::cmr double mutant. unfortunately, since all attempts to clone full-length rela and spot, respectively, in n. meningitidis for cis/trans complementation assays failed, the possibility that the observed phenotypes are, at least in part, caused by polar effects cannot be ruled out entirely. however, as depicted in additional file 1: figure s there are rho-independent transcriptional terminators at the 3′ ends of the rela as well as the spot gene which have not been altered in the respective mutants. furthermore, since the phenotype of the atrrela mutants particularly in blood resembles the phenotype of the rela mutants in both strains, and since both are also different from the phenotype of the grxb mutants, it seems rather unlikely that the rela phenotype is due to a polar effect on grxb expression and not due to the decreased ppgpp levels. together, these data show that that the stringent response pathway is functional in both meningococcal strains despite their different rela alleles. the deletion of rela is conditional lethal and it is differently expressed between both strains under virulence-mimicking conditions in human blood. the effect of rela on ex vivo fitness is thus dependent on the environment and the genetic background. a non-coding mobile element affects meningococcal fitness in a strain- and condition-dependent manner in addition to differences in the rela coding sequences, the intergenic region between rela and the upstream grxb encoding the redox enzyme glutaredoxin differed substantially in both strains due to the integration of an at-rich repeat element in strain α. atr repeat elements occur and times in the genomes of α and mc respectively, of which only have the same position in both genomes. they have a conserved length of 181 bp with ends forming a perfect 13–bp inverted repeat and belong to a class of non-autonomous dna transposons also known as miniature inverted-repeat transposable-elements. atrs are almost exclusively located in intergenic regions in both genomes and are almost ten times more frequently found in intergenic regions flanked by convergently transcribed genes than expected by chance. this indicates that the location in potential promoter regions might be under negative selection possibly due to adverse effects on the expression of neighboring genes. as experimentally determined by 5’-race, the − boxes of the rela promoter differ between both strains due to the insertion of atrrela in strain α between the − and − boxes. furthermore, growth experiments demonstrated that the fitness of an atrrela knock-out strain was impaired only in α and only in human blood thus resembling the phenotype of the rela deletion mutant. in contrast to the deletion of atrrela or rela, the effect of deleting the neighboring grxb gene was condition- but not strain-dependent. these findings thus suggest that via affecting the expression of rela, differences in the binding of transcriptional regulators might contribute to the observed epistatic effects of atrrela on ex vivo fitness. the conditional essentiality of grxb for ex vivo fitness further supports the hypothesis that the oxidative stress response is required for blood survival especially in human blood. in support of a background- and condition-dependent effect of atrrela on meningococcal fitness, we could not observe any ex vivo fitness differences between the atrrela knock-in mutant strain mc relap::atrrela and the corresponding mc wild-type strain. surprisingly, the integration of atrrela into the rela promoter region abolished however the inhibitory effect of cys and gln on growth in mmm in strain mc whereas the deletion of atrrela had no effect on the in vitro growth of α. this suggests a link between cys and gln metabolism and the stringent response regulation in a yet to define epistatic manner. although the role of mites in meningococcal infection biology has already been established this is the first time that a biological function has been shown for the atr class of mites in an infection process. discussion it has recently been shown that transcriptional regulation in prokaryotes is more flexible than the genetic component of the organisms and that its complexity and structure plays an important role in phenotypic adaptation. however, little is known so far about the significance of regulatory evolution that might underlie bacterial virulence. accordingly, we used a hypothesis-generating systems biological approach to analyse gene-expression differences between two meningococcal strains from a hyperinvasive and a carriage clonal complex, respectively, under infection-mimicking conditions. despite the substantial genetic differences between both strains affecting surface antigens as well as metabolic genes likely affecting cys and gln biosynthesis, both were surprisingly similar in a variety of in vitro virulence assays and in their growth behavior under infection mimicking conditions. in particular, the finding that both strains have the same fitness in human blood and csf despite the large differences in the disease/carriage ratios between cc st- and cc st- strains further indicates that the ability to grow under infection mimicking conditions might be necessary but not sufficient for explaining the invasive property of certain meningococcal lineages. virulence, i.e. host damage, might rather be related to the way how meningococci accomplish growth in this environment. in line with this hypothesis, the large transcriptome differences observed particularly in human blood indicate that different transcriptional programs probably compensate for the differences in the genetic backgrounds of both strains in response to host components. this so called phenotypic buffering is a general property of complex gene-regulatory networks.fig. graphical summary and hypothesis relating major findings of this work and published data. the figure is not intended to give a comprehensive overview of the entire metabolism and stress responses in n. meningitidis but to illustrate pathways that link metabolism, protein sequence and gene expression differences of selected genes and the pathogenesis of imd as described in the main text. accordingly, genes and pathways that were highly expressed in mc in blood and/or that are strongly upregulated between saliva and blood in mc are depicted in red, and genes and pathways that are highly expressed in α or that are strongly upregulated between saliva and blood in α are depicted in green. asterisks next to enzyme or protein names indicate that the corresponding proteins have a less than average sequence similarity or are entirely missing in strain α arrows with plus signs indicate activating regulatory interactions, and arrows with minus signs inhibitory regulatory interactions. for further details and abbreviations see main text. the literature cited in the figure is indicated by bracketed numerals next to the respective arrows: newcombe et al., delany et al., fantappie et al., monaco et al., huis in’t veld et al., tala et al., takahashi et al., gunesekere et al., criss and seifert, seib et al., schmitt et al., stevanin et al., kobsar et al., coureuil et al., virji, hellerud et al. upon transition from commensal to invasive behavior meningococci have to adapt to the accompanying large environmental changes caused largely by differences in nutritional and innate immunity in these different compartments. these comprise, e.g., differences in the presence of professional phagocytes or the availability and concentration and of key nutrients such as amino acids or iron. accordingly, it has already been shown that the oxidative burst, which is one of the most prominent effector mechanisms in human neutrophils, is modulated by neisserial porins, and – although to a lesser extent – also opc has been described to be involved in n. meningitidis – neutrophil interaction. the observed differences in the repertoire and/or expression of, e.g., opc, pora and porb, could lead to differences in the phagocytic uptake by neutrophils and/or the activation of the oxidative burst. phagocytosis and the activation of the neutrophil oxidative burst in turn exert nutritional and oxidative/nitrosative stresses on the bacterial cell. the resulting damage to the bacterial cell triggers bacterial stress responses which in pathogenic bacteria have already been considered as virulence factors. in particular, lactate, a by-product of neutrophil glycolysis, was shown to enhance bacterial consumption of molecular oxygen in the presence of glucose, which depletes the substrate for neutrophil nadph oxidase and thus blunts its oxidative burst. accordingly, our transcriptomic data analyses outlined above suggest a particularly strong metabolic activation in strain mc in blood. another important component in the oxidative/nitrosative stress responses in neisseria is glutathione. it is synthesized from cys, gln/glu and gly which are either taken up from the environment or synthesized from precursors generated in the entner–doudoroff pathway and tca cycle, respectively. via the gsh cycle, the biosynthesis of cys and gln/glu are thus linked to the oxidative/nitrosative stress response. we therefore hypothesize that the observed sequence variations in metabolic genes such as cysh, glne or glnb involved in cys and gln/glu biosynthesis, respectively, lead to the activation of compensatory transcriptional programs to allow survival upon exposure to human blood phagocytes. our data further indicate that rela contributes to the homeostatic transcriptional response to nutritional and oxidative/nitrosative stresses as it is required for amino acid biosynthesis in n. meningitidis. consequently, via variation in the gc content of the discriminator regions, the accumulation of ppgpp during the stringent response might directly or indirectly contribute to the adapative regulation of genes required for energy metabolism, cell envelope biogenesis, translation and ribosome biogenesis and thus for large parts of the meningococcal transcriptome. furthermore, as the educt for glu biosynthesis akg provides a potential metabolic link between energy metabolism and the gsh cycle and was shown in n. meningitidis to contribute to, amongst others, the regulation of σe expression. since the expression of ania was in turn shown to be under the negative control of σe, the finding that no generated by ania inhibits platelet aggregation provides an exemplary link between oxidative stress, glu/gln metabolism and the pathophysiology of meningococcal disease. in this picture, virulence, i.e. host damage, occurs coincidentally due to the transcriptional compensation of metabolic deficiencies by including genes with an established role in the pathogenesis of imd. in consequence, meningococcal virulence is a byproduct of global stress and metabolic responses potentially triggered by human neutrophils and is dependent on the genetic background of the invading strain. in line with the recently proposed damage-response framework of microbial pathogenesis, we further hypothesize that strain-dependent differences in the interaction of meningococci with human neutrophils have a central role in explaining meningococcal virulence differences. however, a better systems biological understanding of the interaction of human phagocytes with meningococcal cells under infection relevant conditions has yet to be reached, and in addition to transcription many other layers such as post-transcriptional modifications, allosteric regulation and non-coding small rnas are likely to play an important role in metabolic regulation in meningococci. the phenotypic similarity of both strains under the ex vivo conditions tested further indicate that most of genotypic variation observed in the genomic comparisons is likely cryptic for selection under conditions encountered by the species during its commensal life-cycle, and only upon an environmental perturbation these genotypic differences do result in different phenotypes. in evolutionary genetics, this so called cryptic genetic variation describes the part of the genetic diversity that has the potential to affect the phenotype but that is not expressed under the current genotypic or environmental conditions which limits the opportunities for selection to act on the variation. however, under atypical conditions, rare in the history of a population, cgv can generate phenotypic variation. human blood is a condition that is normally not encountered by meningococci during its commensal life cycle, and since bacteria replicating in the bloodstream are not transmitted to new hosts it has not been encountered by any ancestral meningococcal strain before. cgv might thus contribute to the high standing genetic variation observed in meningococcal population since the conditions that induce such allelic effects are rare in the history of the population, and imd would therefore result from the accidental unmasking of meningococcal cgv in human blood. furthermore, cgv is a subclass of variation with conditional effects, either in form of g × g or in g × e interaction. accordingly, not only by environmental perturbation discussed above but also by genetic perturbation such as deletion of rela we could experimentally uncover cgv likely in genes required for amino acid biosynthesis and ex vivo fitness. loci with such pleiotropic effects known as genetic hubs or buffers are a common feature of the genetics of gene expression and their existence emerges from the concept of cgv. these genes are important for buffering both environmental change and stochastic variation thus ensuring environmental and stochastic resilience. rather surprisingly, there have been only very few verified genetic hubs in published genetical genomics studies to date. based on our observations and the published data on the physiology of the stringent response in, e.g., e. coli we hypothesize that it contributes to the phenotypic buffering of cgv in metabolic genes and that rela in particular might constitute such a regulatory hub coincidentally affecting also meningococcal virulence. therefore, the elucidation of the genetic variability and molecular mechanisms of stress responses in n. meningitidis will be important for the understanding of virulence evolution in this commensal pathogen. of note, whereas the variability in the complement and/or sequence of genes coding for surface antigens and metabolic functions, respectively, among different meningococcal lineages and their potential role in meningococcal virulence have already been well established, the extent of sequence variability in stress response genes and their possible contribution to virulence differences in meningococci have not been addressed so far. in addition, differences in intergenic regions affecting gene expression regulation are often overlooked genetic determinants in the search for bacterial virulence factors. the impaired fitness of the α atrrela deletion mutant in human blood provides an example of how regulatory evolution via the integration of non-coding mites into promoter regions might contribute to fitness and consequently virulence differences among bacterial strains in a condition-dependent manner. as all experimental approaches for studying meningococcal infection biology this study has also its limitations. first, the ex vivo conditions chosen might not be truly representative for the in vivo situation. in particular, although meningococci have been found to be part of the normal flora of the oral cavity they actually colonize the human nasopharynx where they immerse in the liquid produced by nasopharyngeal epithelium and constantly interact with epithelial cells. therefore, saliva produced by saliva glands may not be truly representative of this commensal environment. second, given their high genetic diversity more meningococcal strains need to be compared in order to see how generalizable the transcriptomic results are with respect to the entire species. furthermore, any microarray-based approach restricts the number of genes being compared to those represented on the microarray. comparative transcriptome sequencing approaches circumvent this limitation and will allow not only for the detection of expression differences in non-coding small rnas but, due to the higher dynamic range compared to microarrays, also for the detection of differences in genes expressed at very low and very high levels, respectively. finally, as it was experimentally not possible to generate rela and spot complemented strains for cis/trans complementation assays, the phenotypes of the rela and rela spot deletion strains could in principle be also affected by polar effects of the gene disruption on adjacent genes. this needs to be addressed in further experimental studies. in summary, the data presented in this work allow novel hypotheses to be generated regarding the genetic basis of meningococcal virulence differences. they in particular warrant detailed analyses of the interaction between meningococci and human neutrophils, the physiological consequences of sequence differences in cysh, glne or glnbp, the strain-dependent regulation of the stringent response in meningococci and how atr might affect the expression of adjacent genes. the large transcriptomic data set provides per se an ex vivo gene expression compendium and as such a valuable resource for the meningococcal research community. finally, our experimental approach further allows to challenge these findings in a larger panel of strains from carriage as well as hyperinvasive lineages and to seek for other genetic determinants affecting ex vivo fitness.  in the present work, we showed that despite identical ex vivo phenotypes two genetically similar strains of n. meningitidis displayed large differences in their transcriptomes including numerous virulence genes and subject to environmental conditions. consequently, the often employed “model strain” approach might give misleading results in genetically diverse species like n. meningitidis which might not be representative for the entire species. furthermore, the finding that rela is conditionally essential and likely contributes to the transcriptional buffering of cryptic genetic variation in metabolic genes potentially limits the universality of rela as a novel drug target. finally, beyond the variation in the set of virulence genes observed in “model” bacterial pathogens such as e. coli, the finding that a short, non-coding repeat element affected meningococcal fitness in a strain- and condition-dependent manner highlights the importance also of regulatory evolution in the emergence of virulence in commensal pathogens. together, these findings demonstrate that “to generalize results across genetic backgrounds, experiments must be carried out across genetic backgrounds”, and because the pathogenic nature of a microbe is a quantitative trait resulting from multiple interacting loci, with allelic effects that are sensitive to the environmental conditions, best within a systems biological framework. in this respect our experimental approach is generic for the identification of loci that are associated with the invasive phenotype also in other genetically diverse commensal pathogens. 
our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. here, in a systems biological approach we compared gene expression of the invasive strain mc and the carriage strain α under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence. a model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. neisseria meningitidisvirulenceregulatory evolutionsystems biologymetabolismcryptic genetic variationstringent responsemiterelahttp://dx.doi.org/ /501100001659deutsche forschungsgemeinschaftscho 1322/1-1tr34/a8tr34/a8liang chunguang dandekar thomas schoen christoph issue-copyright-statement© the author 2017 they further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as n. meningitidis. commensal bacteria like neisseria meningitidis sometimes cause serious disease. in support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the ppgpp synthetase gene rela and of a short non-coding at-rich repeat element in its promoter region. these differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous los biosynthesis genes.
 commensal bacteria like neisseria meningitidis sometimes cause serious disease. however, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. here, in a systems biological approach we compared gene expression of the invasive strain mc and the carriage strain α under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence.  despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over genes under infection mimicking conditions. these differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous los biosynthesis genes. a model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. in support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the ppgpp synthetase gene rela and of a short non-coding at-rich repeat element in its promoter region.  our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. they further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as n. meningitidis. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords neisseria meningitidisvirulenceregulatory evolutionsystems biologymetabolismcryptic genetic variationstringent responsemiterelahttp://dx.doi.org/ /501100001659deutsche forschungsgemeinschaftscho 1322/1-1tr34/a8tr34/a8liang chunguang dandekar thomas schoen christoph issue-copyright-statement© the author 2017 