BACKGROUND
shigella flexneri is a gram-negative bacterium that causes dysentery, an acute human rectocolitis that usually results in destruction of the intestinal mucosa and bloody diarrhoea.
the ability of this pathogen to invade epithelial cells at the colonic and rectal mucosal surface is a key determinant in the establishment of the disease.
this is mediated by a type iii secretion system  encoded on the large shigella virulence plasmid  <cit> .
the t3ss acts like a molecular syringe that delivers molecules directly from the bacterial cytoplasm into host cells via a needle-like structure  <cit> .
however, before the bacterium reaches the large intestine and invades mucosal epithelial cells, shigella must successfully survive the hostile conditions found in the gastrointestinal tract.
therefore the capacity of the bacterium to adapt to anaerobiosis, changes in ph, resist antimicrobial peptides, and acquire nutrients is essential for its pathogenesis  <cit> .
anaerobiosis is known to influence the virulence of several enteric pathogens including shigella, escherichia coli, salmonella spp., vibrio cholerae and yersinia enterocolitica.
in particular, s. flexneri has been shown to be primed for invasion in anaerobic conditions, in which it expresses longer t3ss needles while reducing ipa  effector secretion; this results from fnr-mediated repression of the virulence plasmid genes, spa <dig> and spa <dig> <cit> .
fnr is a major regulator of anaerobic metabolism that is inactivated by the presence of oxygen.
its function depends on the integrity of its o2-sensitive  cluster, which is required for fnr dimerization and thence site-specific dna binding and transcriptional regulation  <cit> .
one rna deep sequencing  and several microarray studies have been performed to characterise the extent of the fnr regulon in e. coli and other gram negative pathogens such as salmonella enterica and neisseria gonorrhoeae.
in e. coli, there were significant discrepancies between studies even when the same strain was examined.
however some differences could be attributed to the use of media containing high levels of glucose, which represses expression from some fnr-activated promoters, and the delayed growth rate of mutants lacking fnr compared with wild-type strains under anaerobic conditions  <cit> .
here we define the regulatory role of oxygen and fnr in s. flexneri.
we have applied two powerful whole-transcriptome approaches, rna-seq complemented with flow cell reverse transcription sequencing , in which there is no amplification during library preparation, to quantify differences in gene expression induced by anaerobiosis and to define the contribution of fnr in this process.
we found that shigella grown anaerobically exhibits global transcriptional changes compared to when grown aerobically, with marked changes in metabolic and transport genes, as well as those involved in regulatory and virulence functions.
importantly, transcription from the shigella virulence plasmid is extensively modified in anaerobiosis, with most of t3ss-related genes being down regulated in the absence of oxygen in an fnr-dependent manner, demonstrating that this highly conserved regulator of metabolism also controls the horizontally-acquired virulence genes on the plasmid, but not on the chromosome, in this important human pathogen.
RESULTS
growth conditions and rna sequencing strategies
to determine the response of shigella to anaerobiosis and the role of fnr in this process, we employed rna-seq to compare the transcriptional profiles of wild type s. flexneri m90t and its Δfnr mutant grown in luria-bertani  medium in the presence and absence of oxygen.
constantinidou et al.
designed a supplemented, minimal salts medium  in which an e. coli fnr mutant exhibited similar growth as the parental strain in the absence of oxygen  <cit> .
however, this medium did not support the growth of s. flexneri m90t.
on the other hand, enriched-glucose media have been shown to repress some fnr-activated promoters  <cit> .
therefore, we chose lb with no added glucose for our experiments.
particular attention was paid to ensure that the culture volume, agitation, temperature and the growth stage of bacteria did not differ in aerobic and anaerobic conditions.
cultures were grown to an optical density at 600 nm  of  <dig>  to avoid a reduction in the concentration of dissolved oxygen tension and total depletion of sugars that occurs during exponential growth  <cit> .
furthermore until reach od <dig> of  <dig>  under anaerobiosis, there was no obvious delay in growth rate of the Δfnr mutant in relation to the wild-type strain .
three biological replicates were performed per strain in each condition, and differential expression between conditions was analysed with the deseq r statistical package.
to assess the reproducibility of results obtained with rna-seq data and to further characterise the role of fnr, the shigella fnr regulon under anaerobiosis was also examined using frt-seq, an alternative sequencing approach in which cdna synthesis is performed on the sequencing flowcell thereby avoiding the possible pcr biases generated during library preparation using standard rna-seq methods  <cit> .
frt-seq confirmed 77% of the genes found differentially expressed by rna-seq, showing a robust concordance between the two techniques.
due to its higher sensitivity, frt-seq detected more genes whose transcription was significantly influenced by the absence of fnr than rna-seq .
a complete catalogue of significant differences is shown in additional material  as well as a summary of the mapping statistics .
to confirm the results obtained by global analysis of the transcriptional profile, we performed strand-specific qrt-pcr to analyse mrna levels of several genes found to be differentially expressed under anaerobic and aerobic growth conditions.
identification of novel chromosomal genes influenced by the absence of oxygen in s. flexneri
analysis of the rna-seq data revealed that  <dig> chromosomal genes were differentially expressed by wild-type s. flexneri m90t grown under anaerobic conditions compared with aerobic conditions, with  <dig> genes being up-regulated, and  <dig> genes down-regulated.
additional file 1: table s <dig> shows these genes classified into functional categories based on the database of clusters of orthologous groups   <cit> .
as expected, most of the genes differentially expressed were related to energy production and metabolism .
the remaining genes were involved in cellular processes and signalling , information storage and processing  or were poorly characterized .
rna-seq data also showed that from the above  <dig> differentially expressed genes,  <dig> genes  were influenced by the absence of fnr under anaerobic conditions .
importantly the majority of genes that we found to be anaerobically induced/repressed have been identified in previous microarray studies with other enteric pathogens examining the effect of oxygen on the transcriptome and/or the two main anaerobic regulators, fnr and arca .
consistent with previous work, we found increased expression of genes involved in anoxic carbon metabolism , respiratory pathways , production of hydrogenases , fermentation  and acid response  under anaerobiosis  .
our analysis also identified several anaerobically repressed genes that have been previously characterised .
these genes encode enzymes of the tricarboxylic acid cycle , aerobic dehydrogenases , transhydrogenases  and iron acquisition systems , and others  .
the sensitivity of the direct sequencing approaches, rna-seq and frt-seq, compared with array-based methods enabled us to extend the repertoire of shigella genes modulated by ambient oxygen.
table  <dig> shows all genes influenced by the presence of oxygen and not detected in previous microarray studies on e. coli and s. flexneri.
the effect of fnr mutation on the transcription of previous genes under anaerobiosis  is also shown in table  <dig>  several members of the phosphoenolpyruvate–carbohydrate phosphotransferase system , involved in the transport and phosphorylation of sugars, were up-regulated under anaerobic conditions.
examples include ptshi, which encode the general pts components phosphohistidine carrier protein  and enzyme i  respectively, and sugar-specific pts components like ptsg and manxyz , trebc , mtla  and murqp that contribute to the uptake and catabolism of n-acetylmuramic acid .
of note, the murqp operon, which is also involved in peptidoglycan recycling, showed an fnr-dependent expression pattern   <cit> .table  <dig>
chromosomal genes differentially expressed in response to anaerobic conditions not previously published in
e. coli
and
s. flexneri
microarray analysis
orf id
ab
c
c
c
2
/o
2
fnr
/wt no o
2
fnr
/wt no o
2
metabolism
<dig>
- <dig>
- <dig>
yiay
<dig>
- <dig>
- <dig>
<dig>
- <dig>
pcka
<dig>
<dig>
yiak
<dig>
<dig>
<dig>
ugpq
<dig>
hmpa
<dig>
<dig>
<dig>
caib
<dig>
atpf
<dig>
atpe
<dig>
ppc
<dig>
<dig>
galt
<dig>
ydja
- <dig>
<dig>
<dig>
ygaf
- <dig>
<dig>
<dig>
gltp
- <dig>
rnfb
- <dig>
fldb
- <dig>
rnfa
- <dig>
ruta
- <dig>
treb
<dig>
trec
<dig>
manx
<dig>
many
<dig>
manz
<dig>
rbsd
<dig>
ptsg
<dig>
tref
<dig>
murp
<dig>
- <dig>
pfka
<dig>
<dig>
<dig>
<dig>
agp
<dig>
<dig>
<dig>
rpia
<dig>
pgk
<dig>
gapa
<dig>
yegt
<dig>
fba
<dig>
ptsh
<dig>
mtla
<dig>
ydhc
<dig>
<dig>
<dig>
<dig>
tpia
<dig>
ptsi
<dig>
fuci
<dig>
bgla
<dig>
malp
<dig>
<dig>
ycfo
<dig>
talb
<dig>
gnd
<dig>
galm
<dig>
<dig>
<dig>
eda
<dig>
galk
<dig>
<dig>
tkta
<dig>
frub
- <dig>
fruk
- <dig>
ptso
- <dig>
- <dig>
<dig>
frua
- <dig>
arga
<dig>
fliy
<dig>
ybgh
<dig>
- <dig>
- <dig>
pepd
<dig>
<dig>
<dig>
gcvt
<dig>
<dig>
proa
<dig>
potd
<dig>
- <dig>
cysd
<dig>
- <dig>
- <dig>
dcp
<dig>
<dig>
prob
<dig>
- <dig>
glya
<dig>
gsp
<dig>
<dig>
edd
<dig>
- <dig>
<dig>
hisj
<dig>
ybik
<dig>
potc
<dig>
- <dig>
- <dig>
pepp
<dig>
gcvp
<dig>
<dig>
<dig>
asna
- <dig>
- <dig>
lysc
- <dig>
<dig>
<dig>
trpe
- <dig>
cyca
- <dig>
yeda
- <dig>
yifk
- <dig>
livj
- <dig>
lysa
- <dig>
<dig>
prop
- <dig>
<dig>
<dig>
ytff
- <dig>
<dig>
udp
<dig>
<dig>
nupc
<dig>
nupg
<dig>
ybek
<dig>
adk
<dig>
- <dig>
purc
<dig>
cpdb
<dig>
gpt
<dig>
- <dig>
add
<dig>
pure
- <dig>
menb
<dig>
- <dig>
- <dig>
<dig>
mend
<dig>
- <dig>
- <dig>
menc
<dig>
- <dig>
- <dig>
ispb
<dig>
pdxh
<dig>
visc
<dig>
ribb
- <dig>
yigc
- <dig>
ygfa
- <dig>
bira
- <dig>
- <dig>
thim
- <dig>
acpp
<dig>
- <dig>
mene
<dig>
- <dig>
- <dig>
ucpa
<dig>
- <dig>
sbma
- <dig>
<dig>
ycbo
<dig>
cysp
<dig>
- <dig>
- <dig>
sodb
<dig>
<dig>
<dig>
<dig>
copa
<dig>
<dig>
<dig>
yjce
<dig>
cysn
<dig>
- <dig>
- <dig>
ybal
- <dig>
- <dig>
mnth
- <dig>
<dig>
<dig>
tauc
- <dig>
shif
- <dig>
ygjt
- <dig>
fhue
- <dig>
ydie
- <dig>
- <dig>
- <dig>
mdti
- <dig>
<dig>
taud
- <dig>
<dig>
cellular processes and signalling
ycib
<dig>
- <dig>
ampc
<dig>
- <dig>
emrd
<dig>
<dig>
- <dig>
- <dig>
shia
<dig>
ampd
<dig>
ybhf
<dig>
<dig>
ybhs
<dig>
ybhr
<dig>
mdla
- <dig>
yehu
<dig>
uspa
<dig>
yfea
- <dig>
- <dig>
crec
- <dig>
<dig>
nmpc
<dig>
- <dig>
- <dig>
slyb
<dig>
- <dig>
- <dig>
ompa
<dig>
tsx
<dig>
gtrb
<dig>
- <dig>
rfbc
<dig>
- <dig>
slt
<dig>
<dig>
<dig>
muri
<dig>
- <dig>
rfad
<dig>
tonb
- <dig>
murb
- <dig>
- <dig>
fliq
<dig>
sece
<dig>
- <dig>
- <dig>
tatc
<dig>
- <dig>
yiio
- <dig>
<dig>
mopb
<dig>
slyd
<dig>
ybbn
<dig>
clpp
<dig>
ibpa
- <dig>
yegd
- <dig>
- <dig>
information storage and processing
yfia
<dig>
gltx
<dig>
frr
<dig>
glns
<dig>
glyq
<dig>
- <dig>
yjea
<dig>
- <dig>
- <dig>
glys
<dig>
ygip
<dig>
- <dig>
- <dig>
murr
<dig>
rhar
<dig>
mali
<dig>
yeht
<dig>
cspc
<dig>
- <dig>
malt
<dig>
<dig>
ompr
<dig>
yiag
<dig>
<dig>
gatr
<dig>
cra
<dig>
yjdc
<dig>
<dig>
<dig>
rfah
- <dig>
- <dig>
yjeb
- <dig>
csph
- <dig>
enda
<dig>
- <dig>
- <dig>
ygjf
<dig>
<dig>
hupb
<dig>
rhle
- <dig>
dead
- <dig>
dbpa
- <dig>
poorly characterized
stpa
<dig>
- <dig>
- <dig>
<dig>
muq
<dig>
- <dig>
<dig>
- <dig>
<dig>
<dig>
<dig>
yfbt
<dig>
ybhg
<dig>
<dig>
hns
<dig>
- <dig>
yfbb
<dig>
- <dig>
- <dig>
yrda
<dig>
hfq
<dig>
- <dig>
gph
<dig>
yeir
<dig>
yede
<dig>
- <dig>
- <dig>
yhfc
<dig>
- <dig>
- <dig>
yejk
<dig>
yegh
<dig>
yhgh
<dig>
yram
<dig>
ybip
- <dig>
yejm
- <dig>
yhbe
- <dig>
- <dig>
ycca
- <dig>
- <dig>
yqab
- <dig>
bax
- <dig>
- <dig>
yhhx
- <dig>
<dig>
aslb
- <dig>
- <dig>
<dig>
- <dig>
<dig>
<dig>
<dig>
yead
<dig>
ybjo
<dig>
- <dig>
dsrb
<dig>
- <dig>
yfbu
<dig>
ybak
<dig>
- <dig>
- <dig>
<dig>
- <dig>
yaaa
<dig>
<dig>
yiiu
<dig>
- <dig>
<dig>
yjgd
<dig>
- <dig>
<dig>
- <dig>
yhbn
<dig>
ybbf
- <dig>
rtn
- <dig>
yban
- <dig>
- <dig>
- <dig>
- <dig>
ymcd
- <dig>
yjba
- <dig>
<dig>
ycfj
- <dig>
<dig>
- <dig>
yoho
- <dig>
- <dig>
- <dig>
- <dig>
- <dig>
ybaa
- <dig>
relf
<dig>
<dig>
- <dig>
<dig>
- <dig>
agenomes used as reference are: s. flexneri 5a str.
m90t, s. flexneri 2a str.
<dig>  s. flexneri  <dig>  shigella sp.
d <dig> and s. flexneri 2457 t with genbank accession numbers agnm <dig>  nc_ <dig>  nc_ <dig>  nz_gg <dig> and nc_ <dig> respectively.
bgenes are classified in functional categories based on the database of clusters of orthologous groups .
http://www.ncbi.nlm.nih.gov/cog/.
inside each subgroup, genes are arranged in descending order in relation to log <dig> of fold change values of wt no o2/wt o <dig> comparison.
clog <dig> of fold change values of wt no o2/wt o <dig> and Δfnr no o2/wt no o <dig> comparisons are presented.
only values considered differentially expressed are shown .
qrt-pcr verification of
s. flexneri
chromosomal genes induced under anaerobic growth conditions and the role of fnr in the process.
strand specific qrt-pcr analysis of mrna levels of s. flexneri m90t chromosomal genes shown to be induced under anaerobiosis in rna-seq analysis.
panel a shows transport and metabolic genes, and panel b acid resistance, omp and regulatory genes.
data were calculated as the n-fold difference relative to pola .
results are shown in relation to the wild-type strain 2-Δct levels under aerobic conditions, here referred to as  <dig>  thus, values greater than  <dig> indicate increased transcription under anaerobiosis, and lower than  <dig> indicate the opposite.
significant differences were detected when wild-type 2-Δct levels under aerobic and anaerobic conditions, or wild-type vs. Δfnr 2-Δct levels under anaerobiosis were compared.
ns = non-significant, p <  <dig> , *; p < <dig> , **; n = 4; mann–whitney test.
error bars show standard deviation .
the expression of other genes involved in transport displayed altered expression in anaerobiosis.
for instance, emrd, coding for a drug transporter, cysp, involved in the binding and uptake of sulfate and thiosulfate, yjce, coding for a na+/h+ exchanger, ybgh, which encodes a peptide transporter and genes involved in nucleoside transport and catabolism  are induced in anaerobiosis  .
we found several metabolic genes induced under anaerobic growth such as cra, coding for the catabolite repressor/activator protein, cra, tpia, encoding a key enzyme of the gluconeogenic and glycolytic pathways, gapa, involved in glycolysis, yehu/yeht, coding for a two component system involved in responses to carbon starvation, malt, the transcriptional activator of the genes responsible for uptake and metabolism of maltodextrins and proa, which encodes an enzyme in proline biosynthesis .
the expression of these genes was not fnr-dependent .
in addition to metabolism, we observed anaerobic up-regulation of: genes involved in stress response such as cspc; genes coding for outer membrane proteins  such as nmpc, ompa and slyb; genes with global regulatory functions such as yjgd that codes for rrab, which interacts with the endonuclease rnase e; yfia, encoding a ribosome-associated protein that inhibits protein translation; and yejk, hns and its paralogue stpa coding for nucleoid-associated proteins responsible for chromosomal dna compaction and global gene regulation .
interestingly, anaerobic induction of cspc, nmpc, slyb, yjgd, hns and stpa was dependent, at least in part, on fnr .
anaerobiosis can also down-regulate transcription.
this is the case for frubka, encoding the fructose pts  <cit>  .figure  <dig>
qrt-pcr verification of
s. flexneri
chromosomal genes repressed under anaerobic growth conditions and the role of fnr in the repression.
strand specific qrt-pcr analysis of mrna levels of s. flexneri m90t chromosomal genes shown to be repressed in rna-seq analysis.
data were calculated as the n-fold difference relative to pola .
results are shown in relation to wild-type 2-Δct levels under aerobic conditions, here referred to as  <dig>  thus, values greater than  <dig> indicate increased transcription under anaerobiosis and lower than  <dig> indicates the opposite.
significant differences were detected when wild-type 2-Δct levels under aerobic and anaerobic conditions or wild-type vs. Δfnr 2-Δct levels under anaerobiosis were compared.
p < <dig> , **; n = 4; mann–whitney test.
error bars show standard deviation .
the analysis of genes known to be influenced by anaerobiosis revealed further functions of fnr.
this is the case for ygip, encoding a nucleoid-associated protein induced under anaerobic growth conditions, which we found is fnr-dependent  <cit> .
furthermore, we observed that mendbce, genes required for the biosynthesis of quinones with essential roles in anaerobic electron transport systems, are affected by the presence of fnr in contrast to e. coli  .
our study revealed extended regulatory roles for fnr, such as in the biosynthesis of l-cysteine.
previous work has demonstrated that cysk, which encodes an enzyme in l-cysteine biosynthesis, is subject to fnr regulation and identified an fnr-like domain in cysj, which encodes a component of the sulfite reductase  <cit> .
here, we found that loss of fnr affects the entire l-cysteine biosynthetic pathway including genes involved in the uptake and transport of sulfate , sulfate activation , reduction to sulfide  and transformation into l-cysteine   .
reprogramming of t3ss related genes under anaerobic conditions
analysis of genes involved in shigella virulence revealed that multiple genes on the shigella virulence plasmid, including ipa-mxi-spa genes, were repressed under anaerobic growth in an fnr-dependent manner .
in contrast, only seven genes on the plasmid  were up-regulated in the absence of oxygen; all of these are regulated by fnr except para and yigb .
figure  <dig> shows effect of oxygen on expression of genes on the virulence plasmid genes.
these findings were confirmed by strand specific qrt-pcr for several genes .
since excess para levels compared with parb can affect plasmid partitioning, we also examined the transcription profile of parb <cit> .
similar to para, mrna levels of parb are elevated during anaerobic growth .
consistent with this finding, there was no significant difference in loss of the virulence plasmid from bacteria grown in aerobic and anaerobic conditions .table  <dig>
virulence plasmid genes differentially expressed in response to anaerobic conditions
orf id
ab
c
c
c
2
/o
2
fnr
/wt no o
2
fnr
/wt no o
2
yigb
<dig>
ospi
<dig>
- <dig>
shf
<dig>
- <dig>
- <dig>
rfbu
<dig>
- <dig>
- <dig>
virk
<dig>
- <dig>
- <dig>
para
<dig>
msbb
<dig>
- <dig>
- <dig>
sepa
- <dig>
<dig>
<dig>
- <dig>
<dig>
ipah <dig>
- <dig>
- <dig>
<dig>
- <dig>
<dig>
- <dig>
<dig>
<dig>
- <dig>
<dig>
- <dig>
<dig>
- <dig>
<dig>
<dig>
virg
- <dig>
<dig>
ipgf
- <dig>
<dig>
virf
- <dig>
- <dig>
ipge
- <dig>
<dig>
mxih
- <dig>
<dig>
<dig>
- <dig>
<dig>
<dig>
- <dig>
vira
- <dig>
<dig>
mxii
- <dig>
<dig>
<dig>
mkad
- <dig>
<dig>
<dig>
mxig
- <dig>
<dig>
mxij
- <dig>
<dig>
<dig>
- <dig>
- <dig>
<dig>
sopa
- <dig>
<dig>
- <dig>
ipgb
- <dig>
<dig>
spa15
- <dig>
<dig>
<dig>
shet2-2
- <dig>
<dig>
mxia
- <dig>
<dig>
spa32
- <dig>
<dig>
<dig>
- <dig>
<dig>
phon2
- <dig>
<dig>
<dig>
mxil
- <dig>
<dig>
<dig>
acp
- <dig>
<dig>
spa-orf10
- <dig>
<dig>
<dig>
spa47
- <dig>
<dig>
<dig>
icsb
- <dig>
<dig>
<dig>
spa24
- <dig>
<dig>
<dig>
spa13
- <dig>
<dig>
spa33
- <dig>
<dig>
<dig>
mxim
- <dig>
<dig>
<dig>
mxic
- <dig>
<dig>
<dig>
- <dig>
<dig>
- <dig>
<dig>
<dig>
spa9
- <dig>
<dig>
<dig>
ipgc
- <dig>
<dig>
<dig>
ipac
- <dig>
<dig>
<dig>
spa40
- <dig>
<dig>
ipga
- <dig>
<dig>
<dig>
mxid
- <dig>
<dig>
<dig>
mxie
- <dig>
<dig>
<dig>
mxid
- <dig>
<dig>
<dig>
ipad
- <dig>
<dig>
<dig>
spa-orf11
- <dig>
<dig>
<dig>
ipab
- <dig>
<dig>
<dig>
ipaa
- <dig>
<dig>
<dig>
- <dig>
<dig>
spa29
- <dig>
<dig>
<dig>
virb
- <dig>
<dig>
<dig>
a
s. flexneri 5a str.
m90t pwr <dig> virulence plasmid sequence was used as reference genbank accession numbers af <dig>
bgenes are arranged in descending order in relation to log <dig> of fold change values of wt no o2/wt o <dig> comparison.
clog <dig> of fold change values of wt no o2/wt o <dig> and Δfnr no o2/wt no o <dig> comparisons are presented.
only values considered differentially expressed are shown .
circular map of genes differentially expressed in the virulence plasmid under anaerobiosis.
outer ring shows orfs and their orientations.
genes differentially repressed and induced in the wild type m90t strain under anaerobiosis in relation to aerobic conditions were marked in deep blue and red respectively.
scale is in base pairs.
the figure was generated with dnaplotter.
qrt-pcr verification of
s. flexneri
virulence plasmid genes repressed under anaerobic growth conditions and the role of fnr in the repression.
strand specific qrt-pcr analysis of s. flexneri m90t virulence plasmid genes mrna levels shown to be repressed in rna-seq analysis.
data were calculated as the n-fold difference relative to pola .
results are shown in relation to the wild-type 2-Δct levels under aerobic conditions .
values greater than  <dig> indicate increased transcription under anaerobiosis, while lower than  <dig> indicate the opposite.
significant differences were detected with the wild-type strain 2-Δct levels under aerobic and anaerobic conditions, or wild-type vs. Δfnr 2-Δct levels under anaerobiosis were compared.
p < <dig> , **; n = 4; mann–whitney test.
error bars show standard deviation .
qrt-pcr verification of
s. flexneri
virulence plasmid genes induced under anaerobic growth conditions and the role of fnr in the induction.
strand specific qrt-pcr analysis of s. flexneri m90t virulence genes mrna levels shown to be induced in rna-seq analysis.
data were calculated as the n-fold difference relative to pola .
results are shown in relation to the wild-type 2-Δct levels under aerobic conditions .
values greater than  <dig> indicate increased transcription under anaerobiosis, while lower than  <dig> indicate the opposite.
significant differences were detected with the wild-type strain 2-Δct levels under aerobic and anaerobic conditions, or wild-type vs. Δfnr 2-Δct levels under anaerobiosis were compared.
p <  <dig> , *; p < <dig> , **; n = 4; mann–whitney test.
error bars show standard deviation .
the shigella pathogenicity island shi- <dig> is not present in s. flexneri m90t.
therefore, we examined the transcriptional profile of the shi- <dig> pathogenicity island that includes the aerobactin, iron-uptake system  <cit> .
as previously reported, we found that genes encoding the aerobactin system  were down-regulated under anaerobic conditions, as was shif, a gene which is also involved in iron acquisition  <cit> .
in contrast, shia, a shi- <dig> gene involved in attenuating host inflammatory responses, was over-expressed under anaerobic conditions when compared to aerobic conditions  <cit> .
of note, no shi- <dig> gene is subject to fnr regulation .
csrb and csrc srnas are induced in the absence of oxygen in s. flexnerim90t
little is known about the small rnas  in shigella or their expression under anaerobic conditions.
we analysed the srnas already described in shigella as well as potential srnas homologues to those described in s. enterica serovar typhimurium and found that anaerobic growth conditions induce the expression of csrb and csrc in an fnr-independent manner  .table  <dig>
srnas differentially expressed in response to anaerobic conditions
srna
a
b
b
2
/o
2
fnr
/wt no o
2
360
<dig>
yihi/yiha
245
<dig>
asrnas are arranged in descending order in relation to log <dig> of fold change values of wt no o2/wt o <dig> comparison.
blog <dig> of fold change values of wt no o2/wt o <dig> and Δfnr no o2/wt no o <dig> comparisons are presented.
only values considered differentially expressed are shown .
verification of srnas results by northern blot.
northern blot analysis of csrb and crsc srnas expression under aerobic/anaerobic conditions.
10 μg of total rna obtained from s. flexneri m90t wild‒type strain and its isogenic Δfnr mutant grown under aerobic and anaerobic conditions until od600 =  <dig>  were separated in  <dig> % mops‒agarose gels, transferred to membranes and detected using probes specific for the sense strand.
discussion
in vitro studies have several limitations in relation to in vivo studies; e.g., they cannot mimic the amount and type of carbon sources available for bacteria and lack the signals derived from the interaction with intestinal epithelium, human immune system or other bacteria present in the gut.
however, if conducted accurately can provide valuable information.
in the current study we have, for the first time, employed rna-sequencing to identify oxygen regulated genes in an enteric pathogen.
our findings confirm previous results, but as this method is more sensitive than array based approaches, we identified an extended repertoire of genes modulated by oxygen in an fnr-dependent or -independent manner.
for instance, little is known about the role of cra, a transcriptional regulator of carbon flux  here shown to be induced under anaerobic conditions  <cit> .
interestingly, mutation of cra increases both epithelial cell attachment and invasion by shigella in aerobic conditions  <cit> .
however, cra has an entirely distinct role in the virulence of enterohemorrhagic e. coli , a close relative of shigella, when investigated under conditions mimicking the anaerobic environment of the intestinal tract.
under these circumstances, loss of cra reduces attachment of bacteria to enterocytes  <cit> .
additionally, salmonella cra mutants are avirulent when administered orally, indicating that cra may have key roles in enteric pathogens in anaerobic conditions  <cit> .
while there is an increasing recognition that carbon metabolism affects microbial virulence, it is still not clear whether distinct carbon energy sources are important or preferable for different members of the enterobacteriaceae.
for example, our results show that the expression pattern under anaerobic conditions of ptsg, manxyc and frubka involved in the transport of sugars is opposite in shigella to that observed in e. coli <cit> .
this could be simply due to the different growth medium used in the experiments or to distinct metabolic strategies between shigella and other enterobacteriaceae.
in favour of the latter and its relationship with virulence it has been shown that mutation of ptsg induces the adherence and invasive capacity of enteroinvasive e. coli  strains but not in salmonella <cit> .
further differences between shigella and other enterobacteriaceae include adiy, an arac-like regulator, which activates expression of adia and adic, encoding the arginine-dependent acid resistance system .
in salmonella adiy expression is elevated under aerobic conditions, whereas in shigella and in e. coli, increased expression of adiy occurs in anaerobiosis  <cit> .
these differences could be due to the strikingly different acid survival strategies that these bacteria seem to develop in spite of being close relatives  <cit> .
deletion of cad locus, a typical pathoadaptive mutation in shigella spp., also induces the ar <dig> system suggesting that this system contributes to the survival of shigella in its particular niche in the intestinal tract  <cit> .
interestingly, we observed an fnr-dependent elevated expression under anaerobiosis of hns and overall of stpa and ygip that encode nucleoid-associated proteins responsible for dna compaction and global gene regulation, indicating that lack of oxygen profoundly modifies dna topology in shigella.
recently, it has been shown that fnr function is strongly inhibited by this kind of nucleoid-associated proteins, which block fnr access to many binding sites  <cit> .
our findings suggest that fnr is involved in this inhibition, probably indirectly, due to the absence of putative fnr binding-boxes in the promoter region of these genes  <cit> .
to distinguish between direct and indirect effects of fnr, in vivo approaches based in chromatin immunoprecipitation followed by micro-array hybridization  or high-throughput sequencing  have been performed in e. coli <cit> .
correlation of fnr chip-seq peaks with transcriptomic data showed that less than half of the fnr-regulated operons could be attributed to direct fnr binding.
of note, fnr occupancy does not always correlate with the presence of a consensus fnr binding site or a change in expression  <cit> .
a total of  <dig> of e. coli chip-seq peaks are located in promoter regions of genes identified in table  <dig> ; only one of these, dbpa, has a canonical fnr binding sequence in its promoter region.
consistent with previous findings, only six of these  <dig> genes  were influenced by fnr in our transcriptomic analysis.
this result suggests that many fnr effects in table  <dig> are likely to be indirect.
however, we cannot rule out differences in regulation between e. coli and shigella that could affect fnr function.
of note, this is the first time that mend, slyb, yjea and yhhx have been identified as fnr regulated by transcriptome analysis, corroborating previous chip findings performed in e. coli.
srnas are widespread in bacteria and play critical roles in regulating physiological processes  <cit> .
in shigella, putative srnas have been identified by bioinformatics  <cit> .
however, the expression of these srnas has not been confirmed in all cases and little is known about their function or the physiological conditions that induce their expression.
here, we found that anaerobic growth induces expression of two srnas, csrb and csrc, independently of fnr.
in e. coli csrb and csrc regulate the activity of csra, the carbon storage regulator although their function in shigella has not been characterised so far  <cit> .
for genes directly involved in host:pathogen interactions, we found that oxygen influences the expression of almost all genes in the mxi-spa operon.
these t3ss-related genes were down-regulated in the absence of oxygen in an fnr-dependent manner.
this is likely to be mediated by virb as this transcription factor controls many genes in this operon, is influenced by h-ns dependent dna supercoiling and our findings demonstrate that virb gene is repressed in anaerobiosis  <cit> .
the effect of oxygen on the shigella t3ss is opposite to salmonella in which fnr induces expression of invasion genes, and probably reflects the different sites occupied in the host by these two related intestinal pathogens  <cit> .
the results further emphasise that the shigella t3ss is inactive in anaerobic environments as we previously reported  <cit> .
inflammation at the site of invasive infection is a hallmark of intestinal shigellosis  <cit> .
of note, expression of shia is induced under anaerobiosis.
this gene in the shi- <dig> pathogenicity island encodes a factor that attenuates the intestinal inflammatory response in shigellosis by decreasing the recruitment of polymorphonuclear leukocytes and t-cells  <cit> .
similarly ospi is the only t3ss-effector protein that was overexpressed in anaerobiosis; it also serves to dampen inflammatory responses by deaminating a glutamine in host ubiquitin-conjugating enzyme   <cit> .
thus, expression of both shia and ospi under low oxygen tension might dampen the extent of inflammatory responses to shigella while it is in the anoxic environment of the intestinal lumen, impairing immune responses.
only one operon on the virulence plasmid, shf-rfbu-virk-msbb, was induced under anaerobiosis in an fnr-dependent manner.
interestingly, all these genes are implicated in modification of shigella lipopolysaccharide , an important pro-inflammatory mediator .
the transcription of several genes encoding omps was induced under anaerobic growth.
both ompa and ompc have been implicated in shigella virulence, while our results suggest that tsx, slp, nmpc, slyb and ycid  could also contribute to pathogenesis and be considered as potential vaccine targets  <cit> .
indeed, salmonella ompw, tsx and nmpc have already been demonstrated to be immunogenic  <cit> .
in addition to omps, transcription of gapa, which encodes glyceraldehyde-3-phosphate dehydrogenase, was induced under anaerobic conditions.
interestingly, this enzyme is exported by ehec and enteropathogenic e. coli  strains but not by non-pathogenic strains.
due to its ability to interact with plasminogen, fibrinogen and intestinal epithelial cells, it has been suggested that gapa might contribute in vivo to the interaction of ehec and epec with the gut epithelium  <cit> .
CONCLUSIONS
overall, our rna-seq based analysis revealed that in the anaerobic lumen of the intestine shigella is predicted to prompt both survival and anti-host immune-modulatory activities of the bacterium.
this occurs through a reprogramming of bacterial metabolism including altered transcription of genes encoding transport systems and metabolic pathways , likely reflecting the carbon energy sources available in the intestine.
modulation of lps, along with shia and ospi may enable shigella to subvert inflammatory responses prior to mucosal invasion.
our results highlight the central role of oxygen and fnr in these processes and how it governs bacterial interactions and entry into host cells  <cit> .figure  <dig>
summary of novel genes influenced by the absence of oxygen in
shigella
identified by rna-seq.

 shigella flexneri is a gram-negative bacterium that causes dysentery, an acute human rectocolitis that usually results in destruction of the intestinal mucosa and bloody diarrhoea. the ability of this pathogen to invade epithelial cells at the colonic and rectal mucosal surface is a key determinant in the establishment of the disease. this is mediated by a type iii secretion system encoded on the large shigella virulence plasmid. the t3ss acts like a molecular syringe that delivers molecules directly from the bacterial cytoplasm into host cells via a needle-like structure. however, before the bacterium reaches the large intestine and invades mucosal epithelial cells, shigella must successfully survive the hostile conditions found in the gastrointestinal tract. therefore the capacity of the bacterium to adapt to anaerobiosis, changes in ph, resist antimicrobial peptides, and acquire nutrients is essential for its pathogenesis. anaerobiosis is known to influence the virulence of several enteric pathogens including shigella, escherichia coli, salmonella spp., vibrio cholerae and yersinia enterocolitica. in particular, s. flexneri has been shown to be primed for invasion in anaerobic conditions, in which it expresses longer t3ss needles while reducing ipa effector secretion; this results from fnr-mediated repression of the virulence plasmid genes, spa and spa. fnr is a major regulator of anaerobic metabolism that is inactivated by the presence of oxygen. its function depends on the integrity of its o2-sensitive cluster, which is required for fnr dimerization and thence site-specific dna binding and transcriptional regulation. one rna deep sequencing and several microarray studies have been performed to characterise the extent of the fnr regulon in e. coli and other gram negative pathogens such as salmonella enterica and neisseria gonorrhoeae. in e. coli, there were significant discrepancies between studies even when the same strain was examined. however some differences could be attributed to the use of media containing high levels of glucose, which represses expression from some fnr-activated promoters, and the delayed growth rate of mutants lacking fnr compared with wild-type strains under anaerobic conditions. here we define the regulatory role of oxygen and fnr in s. flexneri. we have applied two powerful whole-transcriptome approaches, rna-seq complemented with flow cell reverse transcription sequencing, in which there is no amplification during library preparation, to quantify differences in gene expression induced by anaerobiosis and to define the contribution of fnr in this process. we found that shigella grown anaerobically exhibits global transcriptional changes compared to when grown aerobically, with marked changes in metabolic and transport genes, as well as those involved in regulatory and virulence functions. importantly, transcription from the shigella virulence plasmid is extensively modified in anaerobiosis, with most of t3ss-related genes being down regulated in the absence of oxygen in an fnr-dependent manner, demonstrating that this highly conserved regulator of metabolism also controls the horizontally-acquired virulence genes on the plasmid, but not on the chromosome, in this important human pathogen.  growth conditions and rna sequencing strategies to determine the response of shigella to anaerobiosis and the role of fnr in this process, we employed rna-seq to compare the transcriptional profiles of wild type s. flexneri m90t and its Δfnr mutant grown in luria-bertani medium in the presence and absence of oxygen. constantinidou et al. designed a supplemented, minimal salts medium in which an e. coli fnr mutant exhibited similar growth as the parental strain in the absence of oxygen. however, this medium did not support the growth of s. flexneri m90t. on the other hand, enriched-glucose media have been shown to repress some fnr-activated promoters. therefore, we chose lb with no added glucose for our experiments. particular attention was paid to ensure that the culture volume, agitation, temperature and the growth stage of bacteria did not differ in aerobic and anaerobic conditions. cultures were grown to an optical density at 600 nm of to avoid a reduction in the concentration of dissolved oxygen tension and total depletion of sugars that occurs during exponential growth. furthermore until reach od of under anaerobiosis, there was no obvious delay in growth rate of the Δfnr mutant in relation to the wild-type strain. three biological replicates were performed per strain in each condition, and differential expression between conditions was analysed with the deseq r statistical package. to assess the reproducibility of results obtained with rna-seq data and to further characterise the role of fnr, the shigella fnr regulon under anaerobiosis was also examined using frt-seq, an alternative sequencing approach in which cdna synthesis is performed on the sequencing flowcell thereby avoiding the possible pcr biases generated during library preparation using standard rna-seq methods. frt-seq confirmed 77% of the genes found differentially expressed by rna-seq, showing a robust concordance between the two techniques. due to its higher sensitivity, frt-seq detected more genes whose transcription was significantly influenced by the absence of fnr than rna-seq. a complete catalogue of significant differences is shown in additional material as well as a summary of the mapping statistics. to confirm the results obtained by global analysis of the transcriptional profile, we performed strand-specific qrt-pcr to analyse mrna levels of several genes found to be differentially expressed under anaerobic and aerobic growth conditions. identification of novel chromosomal genes influenced by the absence of oxygen in s. flexneri analysis of the rna-seq data revealed that chromosomal genes were differentially expressed by wild-type s. flexneri m90t grown under anaerobic conditions compared with aerobic conditions, with genes being up-regulated, and genes down-regulated. additional file 1: table s shows these genes classified into functional categories based on the database of clusters of orthologous groups. as expected, most of the genes differentially expressed were related to energy production and metabolism. the remaining genes were involved in cellular processes and signalling, information storage and processing or were poorly characterized. rna-seq data also showed that from the above differentially expressed genes, genes were influenced by the absence of fnr under anaerobic conditions. importantly the majority of genes that we found to be anaerobically induced/repressed have been identified in previous microarray studies with other enteric pathogens examining the effect of oxygen on the transcriptome and/or the two main anaerobic regulators, fnr and arca. consistent with previous work, we found increased expression of genes involved in anoxic carbon metabolism, respiratory pathways, production of hydrogenases, fermentation and acid response under anaerobiosis. our analysis also identified several anaerobically repressed genes that have been previously characterised. these genes encode enzymes of the tricarboxylic acid cycle, aerobic dehydrogenases, transhydrogenases and iron acquisition systems, and others. the sensitivity of the direct sequencing approaches, rna-seq and frt-seq, compared with array-based methods enabled us to extend the repertoire of shigella genes modulated by ambient oxygen. table  shows all genes influenced by the presence of oxygen and not detected in previous microarray studies on e. coli and s. flexneri. the effect of fnr mutation on the transcription of previous genes under anaerobiosis is also shown in table  several members of the phosphoenolpyruvate–carbohydrate phosphotransferase system, involved in the transport and phosphorylation of sugars, were up-regulated under anaerobic conditions. examples include ptshi, which encode the general pts components phosphohistidine carrier protein and enzyme i respectively, and sugar-specific pts components like ptsg and manxyz, trebc, mtla and murqp that contribute to the uptake and catabolism of n-acetylmuramic acid. of note, the murqp operon, which is also involved in peptidoglycan recycling, showed an fnr-dependent expression pattern.table chromosomal genes differentially expressed in response to anaerobic conditions not previously published in e. coli and s. flexneri microarray analysis orf id ab c c c 2 /o 2 fnr /wt no o 2 fnr /wt no o 2 metabolism - - yiay - - - pcka yiak ugpq hmpa caib atpf atpe ppc galt ydja - ygaf - gltp - rnfb - fldb - rnfa - ruta - treb trec manx many manz rbsd ptsg tref murp - pfka agp rpia pgk gapa yegt fba ptsh mtla ydhc tpia ptsi fuci bgla malp ycfo talb gnd galm eda galk tkta frub - fruk - ptso - - frua - arga fliy ybgh - - pepd gcvt proa potd - cysd - - dcp prob - glya gsp edd - hisj ybik potc - - pepp gcvp asna - - lysc - trpe - cyca - yeda - yifk - livj - lysa - prop - ytff - udp nupc nupg ybek adk - purc cpdb gpt - add pure - menb - - mend - - menc - - ispb pdxh visc ribb - yigc - ygfa - bira - - thim - acpp - mene - - ucpa - sbma - ycbo cysp - - sodb copa yjce cysn - - ybal - - mnth - tauc - shif - ygjt - fhue - ydie - - - mdti - taud - cellular processes and signalling ycib - ampc - emrd - - shia ampd ybhf ybhs ybhr mdla - yehu uspa yfea - - crec - nmpc - - slyb - - ompa tsx gtrb - rfbc - slt muri - rfad tonb - murb - - fliq sece - - tatc - yiio - mopb slyd ybbn clpp ibpa - yegd - - information storage and processing yfia gltx frr glns glyq - yjea - - glys ygip - - murr rhar mali yeht cspc - malt ompr yiag gatr cra yjdc rfah - - yjeb - csph - enda - - ygjf hupb rhle - dead - dbpa - poorly characterized stpa - - muq - - yfbt ybhg hns - yfbb - - yrda hfq - gph yeir yede - - yhfc - - yejk yegh yhgh yram ybip - yejm - yhbe - - ycca - - yqab - bax - - yhhx - aslb - - - yead ybjo - dsrb - yfbu ybak - - - yaaa yiiu - yjgd - - yhbn ybbf - rtn - yban - - - - ymcd - yjba - ycfj - - yoho - - - - - ybaa - relf - - agenomes used as reference are: s. flexneri 5a str. m90t, s. flexneri 2a str. s. flexneri shigella sp. d and s. flexneri 2457 t with genbank accession numbers agnm nc_ nc_ nz_gg and nc_ respectively. bgenes are classified in functional categories based on the database of clusters of orthologous groups. http://www.ncbi.nlm.nih.gov/cog/. inside each subgroup, genes are arranged in descending order in relation to log of fold change values of wt no o2/wt o comparison. clog of fold change values of wt no o2/wt o and Δfnr no o2/wt no o comparisons are presented. only values considered differentially expressed are shown. qrt-pcr verification of s. flexneri chromosomal genes induced under anaerobic growth conditions and the role of fnr in the process. strand specific qrt-pcr analysis of mrna levels of s. flexneri m90t chromosomal genes shown to be induced under anaerobiosis in rna-seq analysis. panel a shows transport and metabolic genes, and panel b acid resistance, omp and regulatory genes. data were calculated as the n-fold difference relative to pola. results are shown in relation to the wild-type strain 2-Δct levels under aerobic conditions, here referred to as thus, values greater than indicate increased transcription under anaerobiosis, and lower than indicate the opposite. significant differences were detected when wild-type 2-Δct levels under aerobic and anaerobic conditions, or wild-type vs. Δfnr 2-Δct levels under anaerobiosis were compared. ns = non-significant, p < , *; p <, **; n = 4; mann–whitney test. error bars show standard deviation. the expression of other genes involved in transport displayed altered expression in anaerobiosis. for instance, emrd, coding for a drug transporter, cysp, involved in the binding and uptake of sulfate and thiosulfate, yjce, coding for a na+/h+ exchanger, ybgh, which encodes a peptide transporter and genes involved in nucleoside transport and catabolism are induced in anaerobiosis. we found several metabolic genes induced under anaerobic growth such as cra, coding for the catabolite repressor/activator protein, cra, tpia, encoding a key enzyme of the gluconeogenic and glycolytic pathways, gapa, involved in glycolysis, yehu/yeht, coding for a two component system involved in responses to carbon starvation, malt, the transcriptional activator of the genes responsible for uptake and metabolism of maltodextrins and proa, which encodes an enzyme in proline biosynthesis. the expression of these genes was not fnr-dependent. in addition to metabolism, we observed anaerobic up-regulation of: genes involved in stress response such as cspc; genes coding for outer membrane proteins such as nmpc, ompa and slyb; genes with global regulatory functions such as yjgd that codes for rrab, which interacts with the endonuclease rnase e; yfia, encoding a ribosome-associated protein that inhibits protein translation; and yejk, hns and its paralogue stpa coding for nucleoid-associated proteins responsible for chromosomal dna compaction and global gene regulation. interestingly, anaerobic induction of cspc, nmpc, slyb, yjgd, hns and stpa was dependent, at least in part, on fnr. anaerobiosis can also down-regulate transcription. this is the case for frubka, encoding the fructose pts.figure qrt-pcr verification of s. flexneri chromosomal genes repressed under anaerobic growth conditions and the role of fnr in the repression. strand specific qrt-pcr analysis of mrna levels of s. flexneri m90t chromosomal genes shown to be repressed in rna-seq analysis. data were calculated as the n-fold difference relative to pola. results are shown in relation to wild-type 2-Δct levels under aerobic conditions, here referred to as thus, values greater than indicate increased transcription under anaerobiosis and lower than indicates the opposite. significant differences were detected when wild-type 2-Δct levels under aerobic and anaerobic conditions or wild-type vs. Δfnr 2-Δct levels under anaerobiosis were compared. p <, **; n = 4; mann–whitney test. error bars show standard deviation. the analysis of genes known to be influenced by anaerobiosis revealed further functions of fnr. this is the case for ygip, encoding a nucleoid-associated protein induced under anaerobic growth conditions, which we found is fnr-dependent. furthermore, we observed that mendbce, genes required for the biosynthesis of quinones with essential roles in anaerobic electron transport systems, are affected by the presence of fnr in contrast to e. coli. our study revealed extended regulatory roles for fnr, such as in the biosynthesis of l-cysteine. previous work has demonstrated that cysk, which encodes an enzyme in l-cysteine biosynthesis, is subject to fnr regulation and identified an fnr-like domain in cysj, which encodes a component of the sulfite reductase. here, we found that loss of fnr affects the entire l-cysteine biosynthetic pathway including genes involved in the uptake and transport of sulfate, sulfate activation, reduction to sulfide and transformation into l-cysteine. reprogramming of t3ss related genes under anaerobic conditions analysis of genes involved in shigella virulence revealed that multiple genes on the shigella virulence plasmid, including ipa-mxi-spa genes, were repressed under anaerobic growth in an fnr-dependent manner. in contrast, only seven genes on the plasmid were up-regulated in the absence of oxygen; all of these are regulated by fnr except para and yigb. figure  shows effect of oxygen on expression of genes on the virulence plasmid genes. these findings were confirmed by strand specific qrt-pcr for several genes. since excess para levels compared with parb can affect plasmid partitioning, we also examined the transcription profile of parb. similar to para, mrna levels of parb are elevated during anaerobic growth. consistent with this finding, there was no significant difference in loss of the virulence plasmid from bacteria grown in aerobic and anaerobic conditions.table virulence plasmid genes differentially expressed in response to anaerobic conditions orf id ab c c c 2 /o 2 fnr /wt no o 2 fnr /wt no o 2 yigb ospi - shf - - rfbu - - virk - - para msbb - - sepa - - ipah - - - - - - - virg - ipgf - virf - - ipge - mxih - - - vira - mxii - mkad - mxig - mxij - - - sopa - - ipgb - spa15 - shet2-2 - mxia - spa32 - - phon2 - mxil - acp - spa-orf10 - spa47 - icsb - spa24 - spa13 - spa33 - mxim - mxic - - - spa9 - ipgc - ipac - spa40 - ipga - mxid - mxie - mxid - ipad - spa-orf11 - ipab - ipaa - - spa29 - virb - a s. flexneri 5a str. m90t pwr virulence plasmid sequence was used as reference genbank accession numbers af bgenes are arranged in descending order in relation to log of fold change values of wt no o2/wt o comparison. clog of fold change values of wt no o2/wt o and Δfnr no o2/wt no o comparisons are presented. only values considered differentially expressed are shown. circular map of genes differentially expressed in the virulence plasmid under anaerobiosis. outer ring shows orfs and their orientations. genes differentially repressed and induced in the wild type m90t strain under anaerobiosis in relation to aerobic conditions were marked in deep blue and red respectively. scale is in base pairs. the figure was generated with dnaplotter. qrt-pcr verification of s. flexneri virulence plasmid genes repressed under anaerobic growth conditions and the role of fnr in the repression. strand specific qrt-pcr analysis of s. flexneri m90t virulence plasmid genes mrna levels shown to be repressed in rna-seq analysis. data were calculated as the n-fold difference relative to pola. results are shown in relation to the wild-type 2-Δct levels under aerobic conditions. values greater than indicate increased transcription under anaerobiosis, while lower than indicate the opposite. significant differences were detected with the wild-type strain 2-Δct levels under aerobic and anaerobic conditions, or wild-type vs. Δfnr 2-Δct levels under anaerobiosis were compared. p <, **; n = 4; mann–whitney test. error bars show standard deviation. qrt-pcr verification of s. flexneri virulence plasmid genes induced under anaerobic growth conditions and the role of fnr in the induction. strand specific qrt-pcr analysis of s. flexneri m90t virulence genes mrna levels shown to be induced in rna-seq analysis. data were calculated as the n-fold difference relative to pola. results are shown in relation to the wild-type 2-Δct levels under aerobic conditions. values greater than indicate increased transcription under anaerobiosis, while lower than indicate the opposite. significant differences were detected with the wild-type strain 2-Δct levels under aerobic and anaerobic conditions, or wild-type vs. Δfnr 2-Δct levels under anaerobiosis were compared. p < , *; p <, **; n = 4; mann–whitney test. error bars show standard deviation. the shigella pathogenicity island shi- is not present in s. flexneri m90t. therefore, we examined the transcriptional profile of the shi- pathogenicity island that includes the aerobactin, iron-uptake system. as previously reported, we found that genes encoding the aerobactin system were down-regulated under anaerobic conditions, as was shif, a gene which is also involved in iron acquisition. in contrast, shia, a shi- gene involved in attenuating host inflammatory responses, was over-expressed under anaerobic conditions when compared to aerobic conditions. of note, no shi- gene is subject to fnr regulation. csrb and csrc srnas are induced in the absence of oxygen in s. flexnerim90t little is known about the small rnas in shigella or their expression under anaerobic conditions. we analysed the srnas already described in shigella as well as potential srnas homologues to those described in s. enterica serovar typhimurium and found that anaerobic growth conditions induce the expression of csrb and csrc in an fnr-independent manner.table srnas differentially expressed in response to anaerobic conditions srna a b b 2 /o 2 fnr /wt no o 2 360 yihi/yiha 245 asrnas are arranged in descending order in relation to log of fold change values of wt no o2/wt o comparison. blog of fold change values of wt no o2/wt o and Δfnr no o2/wt no o comparisons are presented. only values considered differentially expressed are shown. verification of srnas results by northern blot. northern blot analysis of csrb and crsc srnas expression under aerobic/anaerobic conditions. 10 μg of total rna obtained from s. flexneri m90t wild‒type strain and its isogenic Δfnr mutant grown under aerobic and anaerobic conditions until od600 =  were separated in % mops‒agarose gels, transferred to membranes and detected using probes specific for the sense strand. discussion in vitro studies have several limitations in relation to in vivo studies; e.g., they cannot mimic the amount and type of carbon sources available for bacteria and lack the signals derived from the interaction with intestinal epithelium, human immune system or other bacteria present in the gut. however, if conducted accurately can provide valuable information. in the current study we have, for the first time, employed rna-sequencing to identify oxygen regulated genes in an enteric pathogen. our findings confirm previous results, but as this method is more sensitive than array based approaches, we identified an extended repertoire of genes modulated by oxygen in an fnr-dependent or -independent manner. for instance, little is known about the role of cra, a transcriptional regulator of carbon flux here shown to be induced under anaerobic conditions. interestingly, mutation of cra increases both epithelial cell attachment and invasion by shigella in aerobic conditions. however, cra has an entirely distinct role in the virulence of enterohemorrhagic e. coli, a close relative of shigella, when investigated under conditions mimicking the anaerobic environment of the intestinal tract. under these circumstances, loss of cra reduces attachment of bacteria to enterocytes. additionally, salmonella cra mutants are avirulent when administered orally, indicating that cra may have key roles in enteric pathogens in anaerobic conditions. while there is an increasing recognition that carbon metabolism affects microbial virulence, it is still not clear whether distinct carbon energy sources are important or preferable for different members of the enterobacteriaceae. for example, our results show that the expression pattern under anaerobic conditions of ptsg, manxyc and frubka involved in the transport of sugars is opposite in shigella to that observed in e. coli. this could be simply due to the different growth medium used in the experiments or to distinct metabolic strategies between shigella and other enterobacteriaceae. in favour of the latter and its relationship with virulence it has been shown that mutation of ptsg induces the adherence and invasive capacity of enteroinvasive e. coli strains but not in salmonella. further differences between shigella and other enterobacteriaceae include adiy, an arac-like regulator, which activates expression of adia and adic, encoding the arginine-dependent acid resistance system. in salmonella adiy expression is elevated under aerobic conditions, whereas in shigella and in e. coli, increased expression of adiy occurs in anaerobiosis. these differences could be due to the strikingly different acid survival strategies that these bacteria seem to develop in spite of being close relatives. deletion of cad locus, a typical pathoadaptive mutation in shigella spp., also induces the ar system suggesting that this system contributes to the survival of shigella in its particular niche in the intestinal tract. interestingly, we observed an fnr-dependent elevated expression under anaerobiosis of hns and overall of stpa and ygip that encode nucleoid-associated proteins responsible for dna compaction and global gene regulation, indicating that lack of oxygen profoundly modifies dna topology in shigella. recently, it has been shown that fnr function is strongly inhibited by this kind of nucleoid-associated proteins, which block fnr access to many binding sites. our findings suggest that fnr is involved in this inhibition, probably indirectly, due to the absence of putative fnr binding-boxes in the promoter region of these genes. to distinguish between direct and indirect effects of fnr, in vivo approaches based in chromatin immunoprecipitation followed by micro-array hybridization or high-throughput sequencing have been performed in e. coli. correlation of fnr chip-seq peaks with transcriptomic data showed that less than half of the fnr-regulated operons could be attributed to direct fnr binding. of note, fnr occupancy does not always correlate with the presence of a consensus fnr binding site or a change in expression. a total of of e. coli chip-seq peaks are located in promoter regions of genes identified in table ; only one of these, dbpa, has a canonical fnr binding sequence in its promoter region. consistent with previous findings, only six of these genes were influenced by fnr in our transcriptomic analysis. this result suggests that many fnr effects in table  are likely to be indirect. however, we cannot rule out differences in regulation between e. coli and shigella that could affect fnr function. of note, this is the first time that mend, slyb, yjea and yhhx have been identified as fnr regulated by transcriptome analysis, corroborating previous chip findings performed in e. coli. srnas are widespread in bacteria and play critical roles in regulating physiological processes. in shigella, putative srnas have been identified by bioinformatics. however, the expression of these srnas has not been confirmed in all cases and little is known about their function or the physiological conditions that induce their expression. here, we found that anaerobic growth induces expression of two srnas, csrb and csrc, independently of fnr. in e. coli csrb and csrc regulate the activity of csra, the carbon storage regulator although their function in shigella has not been characterised so far. for genes directly involved in host:pathogen interactions, we found that oxygen influences the expression of almost all genes in the mxi-spa operon. these t3ss-related genes were down-regulated in the absence of oxygen in an fnr-dependent manner. this is likely to be mediated by virb as this transcription factor controls many genes in this operon, is influenced by h-ns dependent dna supercoiling and our findings demonstrate that virb gene is repressed in anaerobiosis. the effect of oxygen on the shigella t3ss is opposite to salmonella in which fnr induces expression of invasion genes, and probably reflects the different sites occupied in the host by these two related intestinal pathogens. the results further emphasise that the shigella t3ss is inactive in anaerobic environments as we previously reported. inflammation at the site of invasive infection is a hallmark of intestinal shigellosis. of note, expression of shia is induced under anaerobiosis. this gene in the shi- pathogenicity island encodes a factor that attenuates the intestinal inflammatory response in shigellosis by decreasing the recruitment of polymorphonuclear leukocytes and t-cells. similarly ospi is the only t3ss-effector protein that was overexpressed in anaerobiosis; it also serves to dampen inflammatory responses by deaminating a glutamine in host ubiquitin-conjugating enzyme. thus, expression of both shia and ospi under low oxygen tension might dampen the extent of inflammatory responses to shigella while it is in the anoxic environment of the intestinal lumen, impairing immune responses. only one operon on the virulence plasmid, shf-rfbu-virk-msbb, was induced under anaerobiosis in an fnr-dependent manner. interestingly, all these genes are implicated in modification of shigella lipopolysaccharide, an important pro-inflammatory mediator. the transcription of several genes encoding omps was induced under anaerobic growth. both ompa and ompc have been implicated in shigella virulence, while our results suggest that tsx, slp, nmpc, slyb and ycid could also contribute to pathogenesis and be considered as potential vaccine targets. indeed, salmonella ompw, tsx and nmpc have already been demonstrated to be immunogenic. in addition to omps, transcription of gapa, which encodes glyceraldehyde-3-phosphate dehydrogenase, was induced under anaerobic conditions. interestingly, this enzyme is exported by ehec and enteropathogenic e. coli strains but not by non-pathogenic strains. due to its ability to interact with plasminogen, fibrinogen and intestinal epithelial cells, it has been suggested that gapa might contribute in vivo to the interaction of ehec and epec with the gut epithelium.  overall, our rna-seq based analysis revealed that in the anaerobic lumen of the intestine shigella is predicted to prompt both survival and anti-host immune-modulatory activities of the bacterium. this occurs through a reprogramming of bacterial metabolism including altered transcription of genes encoding transport systems and metabolic pathways, likely reflecting the carbon energy sources available in the intestine. modulation of lps, along with shia and ospi may enable shigella to subvert inflammatory responses prior to mucosal invasion. our results highlight the central role of oxygen and fnr in these processes and how it governs bacterial interactions and entry into host cells.figure summary of novel genes influenced by the absence of oxygen in shigella identified by rna-seq. 
interestingly, there was a marked effect of available oxygen on genes involved in type iii secretion system, which is required for host cell invasion and pathogenesis. anaerobiosis promotes survival and adaption strategies of shigella, while modulating virulence plasmid genes involved in t3ss-mediated host cell invasion. we also confirmed anaerobic induction of csrb and csrc small rnas in an fnr-independent manner. we found that chromosomal genes were differentially expressed in response to anaerobic conditions; of these, genes were also influenced by fnr. genes that were up-regulated in anaerobic conditions are involved in carbon transport and metabolism, dna topology and regulation, host interactions and survival within the gastrointestinal tract. shigella flexneri is an important human pathogen that has to adapt to the anaerobic environment in the gastrointestinal tract to cause dysentery. these genes, located on the large shigella virulence plasmid, were down regulated in anaerobiosis in an fnr-dependent manner. the influence of fnr on this process is more extensive than previously appreciated, although aside from the virulence plasmid, this transcriptional regulator does not govern expression of genes on other horizontally acquired sequences on the chromosome such as pathogenicity islands.
 shigella flexneri is an important human pathogen that has to adapt to the anaerobic environment in the gastrointestinal tract to cause dysentery. to define the influence of anaerobiosis on the virulence of shigella, we performed deep rna sequencing to identify transcriptomic differences that are induced by anaerobiosis and modulated by the anaerobic fumarate and nitrate reduction regulator, fnr.  we found that chromosomal genes were differentially expressed in response to anaerobic conditions; of these, genes were also influenced by fnr. genes that were up-regulated in anaerobic conditions are involved in carbon transport and metabolism, dna topology and regulation, host interactions and survival within the gastrointestinal tract. interestingly, there was a marked effect of available oxygen on genes involved in type iii secretion system, which is required for host cell invasion and pathogenesis. these genes, located on the large shigella virulence plasmid, were down regulated in anaerobiosis in an fnr-dependent manner. we also confirmed anaerobic induction of csrb and csrc small rnas in an fnr-independent manner.  anaerobiosis promotes survival and adaption strategies of shigella, while modulating virulence plasmid genes involved in t3ss-mediated host cell invasion. the influence of fnr on this process is more extensive than previously appreciated, although aside from the virulence plasmid, this transcriptional regulator does not govern expression of genes on other horizontally acquired sequences on the chromosome such as pathogenicity islands. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2014 