BACKGROUND
streptomycetes are gram-positive soil bacteria that display a complex morphological and metabolic differentiation.
streptomyces develop branched hyphae that expand by tip extension to form a vegetative mycelium meshwork.
in response to as yet unidentified signals and to nutritient depletion, aerial branches emerge from the surface of colonies and may produce spores.
as the aerial mycelium develops, streptomyces colonies produce diverse secondary metabolites and synthesise antibiotics  <cit> .
this differentiation cycle can be reproduced in laboratory conditions by growing streptomyces cells on solid media.
most streptomyces species do not form aerial mycelium or spores when in liquid media , and antibiotic production occurs in submerged cultures  <cit> .
adpa, also known as bldh, has been identified as a conserved major transcriptional regulator involved in the formation of aerial mycelia in various streptomyces species  <cit> .
adpa is a member of the family of arac/xyls regulator proteins that contain a c-terminal domain with two helix-turn-helix dna-binding motifs; these features are strictly conserved in all streptomyces adpas in the strepdb database  <cit> .
the n-terminal domain of adpa is responsible for its dimerization and regulation  <cit> .
protein/dna interaction experiments identified the following consensus adpa-binding site in s. griseus: 5′-tggcsngwwy-3′   <cit> .
adpa was discovered and has mostly been studied in s. griseus, in which it was first shown to activate expression of about thirty genes directly.
they include genes encoding secreted proteins , a sigma factor , a subtilisin inhibitor , ssga which is essential for spore septum formation and the amfr transcriptional regulator involved in production of amfs , a small hydrophobic peptide involved in the emergence of aerial hyphae  <cit> .
adpa also plays a role in secondary metabolism and directly activates streptomycin biosynthesis  <cit> .
proteomic, transcriptomic and chip-sequencing analyses revealed that, in fact, several hundred genes are under the control of s. griseus adpa and that adpa acts as transcriptional activator as well as repressor  <cit> .
in s. coelicolor, few genes have been identified as being directly regulated by adpa: sti <dig> , ramr , clpp <dig>   <cit>  and wbla   <cit> .
the regulation of adpa gene expression is complex and various mechanisms have been described  <cit> .
adpa represses its own gene expression in s. griseus <cit>  whereas it activates its own transcription in s. coelicolor <cit> .
in several streptomyces species, the binding of γ-butyrolactones to a γ-butyrolactone receptor represses the adpa promoter  <cit> .
in s. coelicolor, bldd represses adpa expression  <cit> .
at the translational level, a feedback-control loop regulates levels of adpa and absb  in s. coelicolor <cit> .
a positive feedback loop between adpa and blda, the only trna able to read the uua codon present in all adpa mrna, has been demonstrated in s. griseus <cit> .
in s. coelicolor, adpa expression is constant during growth in liquid media  <cit>  whereas on solid media, adpa is strongly expressed before aerial hyphae formation and adpa is most abundant during the early aerial mycelium stage  <cit> .
even though adpa plays a major role in development of streptomyces spp., little is known about the pathways it controls in s. lividans, a species closely related to s. coelicolor and whose genome has recently been sequenced  <cit> .
we have recently shown that in s. lividans adpa directly controls sti <dig> and the clpp1clpp <dig> operon, encoding important factors for streptomyces differentiation; we also found interplay between adpa and clpp <dig>  <cit> .
here, we report microarray experiments, quantitative real-time pcr , in silico analysis and protein/dna interaction studies that identify other genes directly regulated by adpa in s. lividans.
finally, in silico genome analysis allowed the identification of over hundred genes that are probably directly activated or repressed by adpa in s. lividans.
these findings and observations reveal new adpa-dependent pathways in s. lividans.
methods
bacterial strains, growth conditions and media
s. lividans  <dig> was obtained from the john innes culture collection.
in this s. lividans background, we constructed an adpa mutant in which adpa was replaced with an apramycin-resistance cassette  <cit> .
streptomyces was grown on ne plates  <cit>  and in yeme liquid medium  <cit>  in baffled flasks.
ms medium was used for sporulation experiments  <cit> .
apramycin was added to final concentrations of 25 μg ml- <dig> to solid media and 20 μg ml- <dig> to liquid media as appropriate.
microarray experiments
s. lividans microarrays were not available, so s. coelicolor oligonucleotide arrays covering most open reading frames  of the genome  were used.
aliquots of 60 ml of liquid yeme medium were inoculated with about  <dig> spores and incubated at 30°c with shaking at 200 rpm until early stationary phase .
samples of 12 ml of culture  were then collected and rna extracted as previously described  <cit> .
rna quality was assessed with an agilent  <dig> bioanalyser .
rna indirect labelling and array hybridization were performed as described  <cit>  and hybridized microarrays were scanned with a genepix 4000a scanner .
statistical analysis of array data
r software  <cit>  was used for normalization and differential analysis.
a loess normalization  <cit>  was performed on a slide-by-slide basis .
a paired t-test was used for differential analysis.
variance estimates for each gene were computed under the hypothesis of homoscedasticity, together with the benjamini and yekutieli p-value adjustment method  <cit> .
only genes with a significant  fold change  were taken into consideration.
empty and flagged spots were excluded, and only genes with no missing values were analysed.
a few genes which displayed excessive variation were analysed using the vmixt method from the varmixt package  <cit> .
we defined our cut-off for microarray data acquisition as fc < <dig>  or fc >  <dig>  with p-value <  <dig> .
the genome of s. lividans  <dig> was sequenced only recently  <cit> , so we used the strepdb database  <cit> , and in some cases a basic local alignment search tool , to identify s. lividans orthologs  of s. coelicolor genes.
we also used the protein classification scheme for the s. coelicolor genome available on the welcome trust sanger institute database  <cit> .
qrt-pcr analysis
oligonucleotide pairs specific for ccha , cchb , sli <dig>  sli <dig>  ramr , hyas  and hrdb   were designed using the beacon designer software .
rna samples were extracted from cultures in yeme liquid medium at od450nm values of about  <dig> ,  <dig> ,  <dig> ,  <dig>  and  <dig>  .
aliquots of 20 μg of rna were treated twice with  <dig> units of dnase i with the turbo dna-free reagent  for 30 min at 37°c.
reverse transcription and quantitative real-time pcr were performed as previously described  <cit> .
pcrs involved a hybridization step of 55°c, except for ramr, sli <dig> and cchb where a temperature of 58°c was used.
each assay was performed in triplicate and repeated with at least two independent rna samples.
the critical threshold cycle  was defined for each sample.
the relative amounts of cdna for the tested genes were normalized to that of the hrdb gene transcript which did not vary under our experimental conditions .
the change  in a transcript level was calculated using the following equations: Δct = ct - ct, ΔΔct = Δct - Δct, and ratio = 2-ΔΔct <cit> .
student’s t test was used to evaluate the significance of differences between the expression level of tested genes and that of a reference gene.
a p-value <  <dig>  was considered significant.
in silico analysis and electrophoretic mobility shift assays
several adpa-binding site sequences, identified in s. griseus by dnase i footprinting experiments  <cit> , were used with the predetector software   <cit>  to generate a s. griseus matrix  <cit> .
this matrix was used with the s. coelicolor genome sequence  to identify putative adpa-binding sites upstream from s. lividans adpa-dependent genes .
the strepdb database  <cit>  and blast were used to identify s. lividans, s. coelicolor and s. griseus ortholog gene names.
radioactively labelled dna fragments  corresponding to promoter regions of putative s. lividans adpa-regulated genes were obtained by pcr.
primers  were used to amplify the promoter regions of ccha , sli <dig>  sli <dig> , ramr and hyas as described elsewhere  <cit> .
purified radiolabelled fragments  were then used with purified adpa histidine-tagged protein  in emsa as previously described  <cit> .
RESULTS
deletion of adpa affects the expression of hundreds of genes during early stationary phase
we had previously inactivated adpa in s. lividans and found that this adpa mutant failed to produce aerial mycelium on rich media and that its growth was comparable to that of the parental strain  <dig> in liquid yeme medium at 30°c  <cit> .
expression studies with this s. lividans adpa mutant cultivated in liquid medium identified two differentiation-regulating factors  whose orfs were under the direct control of adpa  <cit> .
we used transcriptome analysis of this adpa mutant to identify other adpa-dependent pathways in s. lividans; however, this analysis was performed using s. coelicolor microarrays  <cit>  because the s. lividans genome sequence was not yet available  <cit>  and the two species are very closely related  <cit> .
total rna was isolated from s. lividans  <dig> and adpa cells during early stationary phase  because at this growth phase, s. coelicolor adpa is expressed  <cit> ; also the expression of genes involved in secondary metabolism in a s. coelicolor blda mutant  <cit> , a strain defective for adpa translation, starts to diverge from that in the wild-type.
global gene expression in the mutant was compared to that in the parental strain.
the expression of more than  <dig> genes was affected in the adpa mutant at early stationary phase :  <dig> genes were significantly down-regulated , and  <dig> were up-regulated  with a p-value <  <dig>  .
theses genes encode proteins of several different classes according to the welcome trust sanger institute s. coelicolor genome database  <cit> :  <dig> of the genes are involved in metabolism of small molecules, including seven playing a role in electron transport  ;  <dig> encode proteins involved in secondary metabolism, for example the ccha-cchf gene cluster  involved in coelichelin biosynthesis  <cit>  and the sli0339- <dig> cluster encoding the putative deoxysugar synthase/glycosyltransferase.
deletion of adpa in s. lividans also affected the expression of  <dig> genes involved in regulation including ramr , wbla , bldn , hrdd  and cutrs   <cit> .
sixty-two genes involved in the cell envelope  <cit>  were differentially expressed in the adpa mutant; they include hyas   <cit> , chpe, chph <cit> , sli <dig> and sli <dig> which were strongly down-regulated in the adpa mutant .
thirty-nine genes encoding proteins involved in various cellular processes   <cit>  were also deregulated in the absence of adpa .
the expression of  <dig> genes coding for proteins with unidentified or unclassified function was altered in the adpa mutant.
thus, deletion of adpa influenced the expression of a large number of genes involved in a broad range of metabolic pathways, and indeed other functions, in s. lividans.
s. lividans adpa
mutant at early stationary phase in yeme medium
a
s. coelicolor
gene
b
s. lividans
gene
c
d
b
e
f
agene expression in the s. lividans adpa mutant was compared to that in the wild-type, using s. coelicolor microarrays.
table  <dig> shows a selected subset of the genes .
the genes presented here were further studied or are discussed in the text because of their role in streptomyces primary or secondary metabolism  <cit> .
bgene names for s. coelicolor  and s. lividans  and annotated function are from the strepdb database  <cit> .
cs.
coelicolor microarrays were used for transcriptome analysis of the s. lividans adpa mutant .
the s. lividans genome sequence was recently made available  <cit>  and sli ortholog gene numbers were identified as sco gene orthologs with strepdb database  <cit> .
the expression of genes shown in bold was analysed by qrt-pcr.
intergenic dna regions between genes labelled with asterisks were analyzed by emsa .
a sco7658-orthologous sequence  was detected in s. lividans, downstream from hyas, but it was not annotated as a s. lividans coding dna sequence .
however our microarray data suggest that this sequence is indeed a cds or alternatively that the s. lividans hyas cds is longer than annotated.
dsco genes and their s. griseus orthologs studied and described under another name found on strepdb database  <cit>  or see “references”.
efold change  in gene expression in the s. lividans adpa mutant with respect to the parental strain with p-value <  <dig> , as calculated by student’s t-test applying the benjamini and hochberg multiple testing correction.
± indicates average fc of some gene operons .
ffrom a protein classification scheme for the s. coelicolor genome available from the welcome trust sanger institute database  <cit> : macromolecule metabolism , small molecule metabolism .
identification of new adpa-controlled genes
to confirm that s. lividans adpa controls the expression of genes identified as differentially expressed in microarray experiments, six genes were studied in more detail by qrt-pcr.
the six genes were selected as having biological functions related to streptomyces development or the cell envelope  or primary or secondary metabolism , and for having very large fold-change values .
the genes in s. coelicolor and griseus orthologous to sli <dig> and sli <dig> encode secreted proteins  <cit> .
the expression levels of these genes in s. lividans wild-type and adpa strains were measured after various times of growth in liquid yeme media , as shown in figure 1a.
the s. lividans hyas gene was strongly down-regulated in the adpa mutant compared to the wild-type   as previously observed for the sco <dig> homolog also known as sti <dig> <cit> .
this suggests that hyas expression is strongly dependent on s. lividans adpa or an adpa-dependent regulator.
sli <dig>  sli <dig> and ramr, were also expressed at a lower level in the adpa mutant than wild-type, particularly after mid-exponential phase ; cchb seemed to be mostly affected by adpa during stationary phase .
the expression of ccha was strongly down-regulated by the absence of adpa at times d and t : note that despite repeated efforts, ccha expression could not be detected in samples corresponding to times a to c for unknown reasons.
the findings for gene expression as determined by microarrays and by qrt-pcr were consistent, with the exception of those for ramr.
the expression of ramr observed by qrt-pcr at time t differed from that determined in microarray experiments , suggesting that some of our microarray data are flattened.
nevertheless, these qrt-pcr experiments confirmed that the expression of the six selected genes is indeed adpa-dependent in s. lividans at every growth time studied.
direct binding of adpa to the promoter regions of s. lividans adpa regulon members
to determine whether s. lividans adpa directly controls these genes, we searched for potential adpa-binding sites in their promoter regions in silico.
a consensus adpa-binding sequence  has been established in s. griseus, and adpa can bind up to five sites between positions -260 bp and +60 bp with respect to the transcriptional start point of the target gene  <cit> .
blast analysis revealed that the s. griseus adpa dna-binding domain is conserved in s. coelicolor and s. lividans adpas  suggesting that all three species share the same adpa-binding consensus sequence.
the dna sequences upstream from the s. coelicolor ramr and hyas genes and the intergenic region between the divergently transcribed genes ccha/cchb, sco0774/sco <dig> and sco6197/sco <dig> were analyzed using predetector software  <cit>  and a matrix was generated with identified s. griseus adpa-binding sequences  <cit> .
between three and nine putative adpa-binding sites were detected within the promoter region of the s. coelicolor genes and by analogy in orthologous s. lividans adpa-dependent genes .
during the course of this study, the s. lividans  <dig> genome sequence became available  <cit>    <cit> ) and its analysis suggested that the position and composition of adpa-binding sites were different from those predicted.
the putative adpa-binding sites of s. lividans ccha/cchb at -101 nt and -86 nt are gggccggttc and tggctggaac, respectively.
the adpa-binding sites located upstream of sli <dig>  sli <dig>  and hyas differ from their s. coelicolor orthologs .
in silico
in the promoter regions of
s. lividans
adpa-dependent genes
a
s. coelicolor
gene
b
c
c
d
e
f
ain silico analysis of the s. coelicolor genome using predetector software   <cit>  to analyse orthologs of s. lividans adpa-dependent genes.
the s. coelicolor adpa-binding sites identified were checked for their conservation and location using the s. lividans genome strepdb database  <cit>  .
bgenes are named according to the strepdb database  <cit> .
*binding sites located between s. coelicolor genes transcribed in the opposite orientation.
cputative s. coelicolor adpa-binding sites were found in silico with predetector  <cit> ; #putative site located in the upstream from the cds of cchb.
the site location given corresponds to the position of first nucleotide most distant from the translation start point of the first gene named.
the positions of some sites are not the same for the s. lividans orthologs as indicated in brackets .
~ putative sites are in the cds of sli <dig>  predicted cds diverge between sli <dig> and sli <dig> locus and their orthologs sco <dig> and sco <dig>  resulting in a smaller intergenic region in s. lividans.
dcs, coding strand; ncs, non coding strand with reference to the first gene named in the s. coelicolor gene column.
escores given by predetector software for s. coelicolor genes  <cit> .
fsites present  or absent  in the s. lividans dna probes used in emsa experiments.
we used emsa to test whether s. lividans adpa binds to predicted s. lividans adpa-binding sequence.
recombinant purified adpa-his <dig> bound to the promoter region of s. lividans sti <dig> , an adpa-dependent gene, whereas an excess of adpa-his <dig>  did not bind to the promoter of sli <dig> , a gene that is not controlled by s. lividans adpa.
this suggests that the binding of adpa with the promoter of genes tested in our previous study was specific  <cit> .
adpa-his <dig> was able to bind to the promoter regions of all s. lividans adpa-dependent genes tested , although with different affinities.
for sli6586/sli <dig>  ramr and hyas, displacement of the dna fragment to the slower migrating protein-dna complex was nearly complete with amounts of adpa of less than  <dig> pmoles .
for ccha/cchb and sli0755/sli <dig>  larger amounts of adpa were necessary for near complete displacement of the dna probe to a protein-dna complex.
in a competition emsa performed on sli6586/ <dig> with an excess of the corresponding unlabelled probe, adpa-binding to the labelled probe decreased .
we also tested a hyas promoter in which one  of the three putative adpa-binding sites was mutated : the affinity of adpa for this promoter region was reduced and one protein-dna complex disappeared .
these results suggest that one dimer of adpa binds the adjacent sites - <dig> and - <dig> of s. lividans hyas promoter and another dimer binds the - <dig> site resulting in the formation of the two dna-adpa complexes depicted in figure  <dig>
these emsa experiments demonstrated that s. lividans adpa directly binds to five intergenic regions and confirmed the in silico prediction presented in table  <dig>  s. lividans adpa directly regulates at least the six adpa-dependent genes listed above and identified by microarrays and qrt-pcr analysis.
these newly identified targets highlight the pleiotropic role of s. lividans adpa: it is involved in primary  and secondary  metabolisms, in regulation , and in cell development .
discussion
adpa, a transcriptional regulator of the arac/xyls family, is involved in the development and differentiation of various streptomyces <cit> .
we report here the first identification of several pathways directly regulated by adpa in s. lividans cultivated in liquid rich medium.
inactivation of adpa in s. lividans affected the expression of approximately  <dig> genes.
this large number was expected in the light of the size of the s. griseus adpa regulon  <cit> .
although adpa mutant growth was comparable to that of the parental strain in yeme liquid medium, the expression of around  <dig> genes involved in primary metabolism was influenced by adpa deletion.
these genes encode proteins involved in the major biosynthesis pathways for amino acids   <cit> , and in energy metabolism  including glycolysis, pentose phosphate, pyruvate dehydrogenase pathways, as well as in electron transport .
other s. lividans adpa-regulated genes influence streptomyces development on solid media    <cit> .
s. lividans adpa also influences the expression of  <dig> genes involved in secondary metabolism such as coelichelin biosynthesis   <cit>  and also genes described to affect metabolic differentiation    <cit> .
consistently with transcriptomic studies in s. griseus, these observations suggest that adpa is a pleiotropic transcriptional regulator in s. lividans.
we demonstrate that s. lividans adpa directly activates cchb, sli <dig> and hyas.
as a result of their co-transcription with these genes, the expression of cchcd, sli <dig> and sco7658-ortholog genes is adpa-dependent in s. lividans .
sli <dig> is probably a directly adpa-regulated gene because its promoter dna region is shared with sli0755-sli <dig> operon, which is transcribed in the opposite direction and directly regulated by adpa .
adpa directly regulates the genes ramr and sti <dig> in s. lividans   <cit>  and in the closely related species s. coelicolor <cit> .
in an s. coelicolor adpa mutant, levels of sti <dig> and ramr expression were lower than in the wild-type strain following growth for 48 h in a minimal agar medium  <cit> .
in vitro experiments showed a high affinity of adpa with a s. coelicolor sti <dig> probe  <cit> , consistent with our results with s. lividans sti <dig> <cit> .
however, adpa had a lower affinity to s. coelicolor ramr  than s. lividans ramr .
when we used a s. lividans ramr probe carrying the promoter region from -201 nt to +66 nt, we observed that less than half the probe was shifted .
therefore, the predicted sites for ramr promoter at positions - <dig> and - <dig>  may have the greatest affinity for adpa .
of the genes analysed by qrt-pcr, the ramr gene was that for which the observed expression was the least consistent with the microarray findings, even through the same sample was used for these analyses.
this suggests that the expression of genes close to the cut-off we applied to the microarray data will need further investigation by qrt-pcr.
among the  <dig> genes identified as direct targets of adpa in s. griseus,  <dig> have no orthologous gene in s. lividans and the orthologous genes of six are not under the control of s. lividans adpa in our conditions.
in addition to ramr  and sti <dig> , hyas  is also a directly adpa-regulated gene that is conserved in the s. lividans and s. griseus adpa regulons  <cit> .
in s. lividans, hyas affects hypha aggregation and the amount of mycelium-associated undecylprodigiosin  <cit> ; its function in s. griseus is unknown.
the expression of all of bldn, sli <dig>  sli <dig> and the sco <dig> ortholog  is influenced by adpa deletion in s. lividans.
it remains to be determined whether adpa directly controls s. lividans adpa and blda as described in s. coelicolor and griseus <cit> .
s. coelicolor adpa is one of  <dig> identified tta-containing genes; the production of the proteins encoded by these genes is dependent on blda, encoding the only trna for the rare leucine codon tta  <cit> .
our study has revealed that expression of  <dig> tta-containing genes and of  <dig> genes regulated by s. coelicolor blda <cit>  was affected by adpa deletion in s. lividans .
we show that ccha, cchb, sti <dig>  hyas, sli <dig> and sli <dig>  previously identified in s. coelicolor as blda-dependent genes, are direct targets of s. lividans adpa  <cit> .
of the  <dig> other blda-dependent genes,  <dig> are probable direct s. lividans adpa targets: in silico analysis indicated the presence of putative adpa-binding sites upstream from these genes .
by analogy, this suggests that the deregulation of certain genes observed in the s. coelicolor blda mutant may have been the consequence of s. coelicolor adpa down-regulation, as previously suggested  <cit> .
to predict probable direct targets of adpa in s. lividans and contribute to knowledge of the adpa regulon, we carried out in silico analysis of the entire s. coelicolor genome using predetector  <cit> , and also restricted to the s. lividans genes identified as being adpa-dependent .
we identified  <dig> genes probably directly activated by s. lividans adpa and  <dig> genes that could be directly repressed .
most of the putative adpa-binding sites identified by this analysis are coherent with the findings of yao et al., demonstrating the importance of g and c nucleotides at positions  <dig> and  <dig>  respectively  <cit> .
six genes have been identified as directly regulated by adpa in other species   <cit> , and  <dig> more in s. griseus are also probable adpa-direct targets   <cit> .
sixty-three of the  <dig> probable direct targets of adpa in s. lividans have no ortholog in the s. griseus genome .
s. lividans
adpa in liquid rich medium
a
gene
b
b
b
b
c
c
c
d
e
adbs3-orfa
glpq1
adpa bldh
bldn, adsa
aorthologs of s. lividans adpa-dependent genes  were analysed in silico using the s. coelicolor genome database .
adpa-binding sites upstream from s. coelicolor genes were identified and are presented in additional file 5: table s <dig>  table  <dig> presents a selected subset of this complete compilation.
bgene names for s. griseus  and annotated function are from the strepdb database  <cit> .
ortholog gene names were identified using strepdb.
genes identified in other streptomyces as being directly adpa-regulated are in bold, and those described as being adpa-dependent are italicized  <cit> .
* binding sites in the promoters of these genes were identified in silico <cit> .
the sco2921-ortholog was not annotated as a s. lividans cds; however, our microarray data suggest that this cds exists.
ccis-element, score, and binding site position as determined by analysing s. coelicolor genes with predetector  <cit> .
when more than one putative adpa-binding site was detected, only the one with the highest score was shown here.
other genes putatively directly regulated by s. lividans adpa are listed in additional file 5: table s <dig>  # site found in the sco <dig> cds at position  <dig> .
dfold change  in gene expression in s. lividans adpa mutant relative to the parental strain with p-value <  <dig> , as determined by student’s t-test applying the benjamini and hochberg multiple testing correction .
efrom a protein classification scheme for the s. coelicolor genome available on the welcome trust sanger institute database  <cit> : unknown function , cell process , macromolecule metabolism , small molecule metabolism , cell envelope , extrachromosomal , regulation  and not classified .
CONCLUSIONS
in conclusion, this study has extended our knowledge of the s. lividans adpa regulon.
we identified s. lividans adpa-regulated genes by transcriptomic analysis, and used in silico analysis to identify over a hundred probable direct targets of adpa in s. lividans.
most of them are absent from the current predicted s. griseus adpa regulon.
discovering new s. lividans genes directly regulated by adpa and that are involved in primary and secondary metabolism will provide valuable information about streptomyces development and differentiation in liquid culture.
availability of supporting data
microarray data are available in the arrayexpress database  <cit>  under accession number a-mexp- <dig>
abbreviations
qrt-pcr: quantitative real-time pcr; orf: open reading frame; fc: fold change; ct: critical threshold cycle; blast: basic local alignment search tool; emsa: electrophoretic mobility shift assay; adpa-his6: recombinant adpa protein with a six-histine tag at the c-terminus; cds: coding dna sequence; cs: coding strand; ncs: non coding strand; u. f.: unknown function; c. p.: cell process; m. m.: macromolecule metabolism; s. m.: small molecule metabolism; c. e.: cell envelope; e.: extrachromosomal; r.: regulation; n. c.: not classified.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
ag, nb and pm wrote and revised the manuscript.
cp and jyc have given final approval for this version to be published.
pm helped ag to design the project.
ag performed qrt-pcr, emsa and in silico analysis; and prepared figures, tables and additional files.
nb purified adpa-his <dig> protein.
cp carried out the microarray experiments.
jyc helped cp with the statistical analysis of microarray results and wrote the associated methods sections.
ag interpreted the microarrays data.
mg help with qrt-pcr experiments and provided technical support.
all authors read and approved the final manuscript.
authors’ information
ag performed qrt-pcr and emsa experiments while working at pasteur institute.
her current address is centre for bacterial cell biology, institute for cell and molecular biosciences, newcastle university, newcastle-upon-tyne ne <dig> 4hh, uk.
supplementary material
additional file 1: table s1
oligonucleotides used in this study.
click here for file
additional file 2: table s2
complete set of genes differentially expressed in the s. lividans adpa mutant.
s. coelicolor microarrays were used to test for genes differentially expressed in the s. lividans adpa mutant and wild-type  <dig>  at growth time point t, in liquid yeme medium.
annotated function, fc, p-values, and classification of the proteins are presented according to the microarray sco genes, by increasing sco gene number.
click here for file
additional file 3: figure s1
effect of the mutation of one adpa-binding site in the s. lividans hyas promoter on adpa-binding specificity.
mutation of an adpa-binding site in the s. lividans hyas promoter region prevents formation of an adpa-dna complex in vitro.
sequence of the mutated adpa-binding site  and emsa performed with the mutated hyas promoter region are shown.
click here for file
additional file 4: table s3
comparison of gene expression profiles between s. coelicolor blda-dependent and s. lividans adpa-dependent genes.
comparison of the gene expression profiles of some s. coelicolor blda-dependent genes whose s. lividans orthologs are adpa-dependent .
putative adpa-binding sites were identified in silico , suggesting that in the s. coelicolor blda mutant, the adpa translation defect leads to blda-dependence of the genes identified previously  <cit> .
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additional file 5: table s4
putative s. coelicolor adpa-binding sites upstream from the s. lividans adpa-dependent genes.
we identified putative adpa-binding sites in silico using the s. coelicolor genome and we analysed orthologs of s. lividans adpa-dependent genes ; the sequences and positions of the sites with the highest scores according to predetector are shown.
s. coelicolor, s. lividans and s. griseus ortholog genes are indicated and previously identified direct or probably direct s. griseus adpa-dependent genes are highlighted.
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acknowledgements
we thank t. msadek, s. dubrac, e. johnson and j.-l. pernodet for helpful discussion and critical reading of the manuscript, and o. poupel for assistance with qrt-pcr analysis.
we are grateful to g. bucca for her advice and help with microarray handling.
we thank alex edelman & associates for correcting the manuscript.
this work was supported by research funds from the institut pasteur and centre national de recherche scientifique.
a. guyet was the recipient of fellowships from the ministère de l’education nationale, de la recherche et de la technologie, the pasteur-weizmann foundation and the era-ib european grant.
ag thanks bbsrc and r. daniel for his constant support during the preparation of this manuscript.

 streptomycetes are gram-positive soil bacteria that display a complex morphological and metabolic differentiation. streptomyces develop branched hyphae that expand by tip extension to form a vegetative mycelium meshwork. in response to as yet unidentified signals and to nutritient depletion, aerial branches emerge from the surface of colonies and may produce spores. as the aerial mycelium develops, streptomyces colonies produce diverse secondary metabolites and synthesise antibiotics. this differentiation cycle can be reproduced in laboratory conditions by growing streptomyces cells on solid media. most streptomyces species do not form aerial mycelium or spores when in liquid media, and antibiotic production occurs in submerged cultures. adpa, also known as bldh, has been identified as a conserved major transcriptional regulator involved in the formation of aerial mycelia in various streptomyces species. adpa is a member of the family of arac/xyls regulator proteins that contain a c-terminal domain with two helix-turn-helix dna-binding motifs; these features are strictly conserved in all streptomyces adpas in the strepdb database. the n-terminal domain of adpa is responsible for its dimerization and regulation. protein/dna interaction experiments identified the following consensus adpa-binding site in s. griseus: 5′-tggcsngwwy-3′. adpa was discovered and has mostly been studied in s. griseus, in which it was first shown to activate expression of about thirty genes directly. they include genes encoding secreted proteins, a sigma factor, a subtilisin inhibitor, ssga which is essential for spore septum formation and the amfr transcriptional regulator involved in production of amfs, a small hydrophobic peptide involved in the emergence of aerial hyphae. adpa also plays a role in secondary metabolism and directly activates streptomycin biosynthesis. proteomic, transcriptomic and chip-sequencing analyses revealed that, in fact, several hundred genes are under the control of s. griseus adpa and that adpa acts as transcriptional activator as well as repressor. in s. coelicolor, few genes have been identified as being directly regulated by adpa: sti, ramr, clpp and wbla. the regulation of adpa gene expression is complex and various mechanisms have been described. adpa represses its own gene expression in s. griseus whereas it activates its own transcription in s. coelicolor. in several streptomyces species, the binding of γ-butyrolactones to a γ-butyrolactone receptor represses the adpa promoter. in s. coelicolor, bldd represses adpa expression. at the translational level, a feedback-control loop regulates levels of adpa and absb in s. coelicolor. a positive feedback loop between adpa and blda, the only trna able to read the uua codon present in all adpa mrna, has been demonstrated in s. griseus. in s. coelicolor, adpa expression is constant during growth in liquid media whereas on solid media, adpa is strongly expressed before aerial hyphae formation and adpa is most abundant during the early aerial mycelium stage. even though adpa plays a major role in development of streptomyces spp., little is known about the pathways it controls in s. lividans, a species closely related to s. coelicolor and whose genome has recently been sequenced. we have recently shown that in s. lividans adpa directly controls sti and the clpp1clpp operon, encoding important factors for streptomyces differentiation; we also found interplay between adpa and clpp. here, we report microarray experiments, quantitative real-time pcr, in silico analysis and protein/dna interaction studies that identify other genes directly regulated by adpa in s. lividans. finally, in silico genome analysis allowed the identification of over hundred genes that are probably directly activated or repressed by adpa in s. lividans. these findings and observations reveal new adpa-dependent pathways in s. lividans. methods bacterial strains, growth conditions and media s. lividans was obtained from the john innes culture collection. in this s. lividans background, we constructed an adpa mutant in which adpa was replaced with an apramycin-resistance cassette. streptomyces was grown on ne plates and in yeme liquid medium in baffled flasks. ms medium was used for sporulation experiments. apramycin was added to final concentrations of 25 μg ml- to solid media and 20 μg ml- to liquid media as appropriate. microarray experiments s. lividans microarrays were not available, so s. coelicolor oligonucleotide arrays covering most open reading frames of the genome were used. aliquots of 60 ml of liquid yeme medium were inoculated with about spores and incubated at 30°c with shaking at 200 rpm until early stationary phase. samples of 12 ml of culture were then collected and rna extracted as previously described. rna quality was assessed with an agilent bioanalyser. rna indirect labelling and array hybridization were performed as described and hybridized microarrays were scanned with a genepix 4000a scanner. statistical analysis of array data r software was used for normalization and differential analysis. a loess normalization was performed on a slide-by-slide basis. a paired t-test was used for differential analysis. variance estimates for each gene were computed under the hypothesis of homoscedasticity, together with the benjamini and yekutieli p-value adjustment method. only genes with a significant fold change were taken into consideration. empty and flagged spots were excluded, and only genes with no missing values were analysed. a few genes which displayed excessive variation were analysed using the vmixt method from the varmixt package. we defined our cut-off for microarray data acquisition as fc < or fc >  with p-value < . the genome of s. lividans was sequenced only recently, so we used the strepdb database, and in some cases a basic local alignment search tool, to identify s. lividans orthologs of s. coelicolor genes. we also used the protein classification scheme for the s. coelicolor genome available on the welcome trust sanger institute database. qrt-pcr analysis oligonucleotide pairs specific for ccha, cchb, sli sli ramr, hyas and hrdb were designed using the beacon designer software. rna samples were extracted from cultures in yeme liquid medium at od450nm values of about,,, and. aliquots of 20 μg of rna were treated twice with units of dnase i with the turbo dna-free reagent for 30 min at 37°c. reverse transcription and quantitative real-time pcr were performed as previously described. pcrs involved a hybridization step of 55°c, except for ramr, sli and cchb where a temperature of 58°c was used. each assay was performed in triplicate and repeated with at least two independent rna samples. the critical threshold cycle was defined for each sample. the relative amounts of cdna for the tested genes were normalized to that of the hrdb gene transcript which did not vary under our experimental conditions. the change in a transcript level was calculated using the following equations: Δct = ct - ct, ΔΔct = Δct - Δct, and ratio = 2-ΔΔct. student’s t test was used to evaluate the significance of differences between the expression level of tested genes and that of a reference gene. a p-value <  was considered significant. in silico analysis and electrophoretic mobility shift assays several adpa-binding site sequences, identified in s. griseus by dnase i footprinting experiments, were used with the predetector software to generate a s. griseus matrix. this matrix was used with the s. coelicolor genome sequence to identify putative adpa-binding sites upstream from s. lividans adpa-dependent genes. the strepdb database and blast were used to identify s. lividans, s. coelicolor and s. griseus ortholog gene names. radioactively labelled dna fragments corresponding to promoter regions of putative s. lividans adpa-regulated genes were obtained by pcr. primers were used to amplify the promoter regions of ccha, sli sli, ramr and hyas as described elsewhere. purified radiolabelled fragments were then used with purified adpa histidine-tagged protein in emsa as previously described.  deletion of adpa affects the expression of hundreds of genes during early stationary phase we had previously inactivated adpa in s. lividans and found that this adpa mutant failed to produce aerial mycelium on rich media and that its growth was comparable to that of the parental strain in liquid yeme medium at 30°c. expression studies with this s. lividans adpa mutant cultivated in liquid medium identified two differentiation-regulating factors whose orfs were under the direct control of adpa. we used transcriptome analysis of this adpa mutant to identify other adpa-dependent pathways in s. lividans; however, this analysis was performed using s. coelicolor microarrays because the s. lividans genome sequence was not yet available and the two species are very closely related. total rna was isolated from s. lividans and adpa cells during early stationary phase because at this growth phase, s. coelicolor adpa is expressed; also the expression of genes involved in secondary metabolism in a s. coelicolor blda mutant, a strain defective for adpa translation, starts to diverge from that in the wild-type. global gene expression in the mutant was compared to that in the parental strain. the expression of more than genes was affected in the adpa mutant at early stationary phase: genes were significantly down-regulated, and were up-regulated with a p-value < . theses genes encode proteins of several different classes according to the welcome trust sanger institute s. coelicolor genome database: of the genes are involved in metabolism of small molecules, including seven playing a role in electron transport; encode proteins involved in secondary metabolism, for example the ccha-cchf gene cluster involved in coelichelin biosynthesis and the sli0339- cluster encoding the putative deoxysugar synthase/glycosyltransferase. deletion of adpa in s. lividans also affected the expression of genes involved in regulation including ramr, wbla, bldn, hrdd and cutrs. sixty-two genes involved in the cell envelope were differentially expressed in the adpa mutant; they include hyas, chpe, chph, sli and sli which were strongly down-regulated in the adpa mutant. thirty-nine genes encoding proteins involved in various cellular processes were also deregulated in the absence of adpa. the expression of genes coding for proteins with unidentified or unclassified function was altered in the adpa mutant. thus, deletion of adpa influenced the expression of a large number of genes involved in a broad range of metabolic pathways, and indeed other functions, in s. lividans. s. lividans adpa mutant at early stationary phase in yeme medium a s. coelicolor gene b s. lividans gene c d b e f agene expression in the s. lividans adpa mutant was compared to that in the wild-type, using s. coelicolor microarrays. table  shows a selected subset of the genes. the genes presented here were further studied or are discussed in the text because of their role in streptomyces primary or secondary metabolism. bgene names for s. coelicolor and s. lividans and annotated function are from the strepdb database. cs. coelicolor microarrays were used for transcriptome analysis of the s. lividans adpa mutant. the s. lividans genome sequence was recently made available and sli ortholog gene numbers were identified as sco gene orthologs with strepdb database. the expression of genes shown in bold was analysed by qrt-pcr. intergenic dna regions between genes labelled with asterisks were analyzed by emsa. a sco7658-orthologous sequence was detected in s. lividans, downstream from hyas, but it was not annotated as a s. lividans coding dna sequence. however our microarray data suggest that this sequence is indeed a cds or alternatively that the s. lividans hyas cds is longer than annotated. dsco genes and their s. griseus orthologs studied and described under another name found on strepdb database or see “references”. efold change in gene expression in the s. lividans adpa mutant with respect to the parental strain with p-value < , as calculated by student’s t-test applying the benjamini and hochberg multiple testing correction. ± indicates average fc of some gene operons. ffrom a protein classification scheme for the s. coelicolor genome available from the welcome trust sanger institute database: macromolecule metabolism, small molecule metabolism. identification of new adpa-controlled genes to confirm that s. lividans adpa controls the expression of genes identified as differentially expressed in microarray experiments, six genes were studied in more detail by qrt-pcr. the six genes were selected as having biological functions related to streptomyces development or the cell envelope or primary or secondary metabolism, and for having very large fold-change values. the genes in s. coelicolor and griseus orthologous to sli and sli encode secreted proteins. the expression levels of these genes in s. lividans wild-type and adpa strains were measured after various times of growth in liquid yeme media, as shown in figure 1a. the s. lividans hyas gene was strongly down-regulated in the adpa mutant compared to the wild-type as previously observed for the sco homolog also known as sti. this suggests that hyas expression is strongly dependent on s. lividans adpa or an adpa-dependent regulator. sli sli and ramr, were also expressed at a lower level in the adpa mutant than wild-type, particularly after mid-exponential phase; cchb seemed to be mostly affected by adpa during stationary phase. the expression of ccha was strongly down-regulated by the absence of adpa at times d and t: note that despite repeated efforts, ccha expression could not be detected in samples corresponding to times a to c for unknown reasons. the findings for gene expression as determined by microarrays and by qrt-pcr were consistent, with the exception of those for ramr. the expression of ramr observed by qrt-pcr at time t differed from that determined in microarray experiments, suggesting that some of our microarray data are flattened. nevertheless, these qrt-pcr experiments confirmed that the expression of the six selected genes is indeed adpa-dependent in s. lividans at every growth time studied. direct binding of adpa to the promoter regions of s. lividans adpa regulon members to determine whether s. lividans adpa directly controls these genes, we searched for potential adpa-binding sites in their promoter regions in silico. a consensus adpa-binding sequence has been established in s. griseus, and adpa can bind up to five sites between positions -260 bp and +60 bp with respect to the transcriptional start point of the target gene. blast analysis revealed that the s. griseus adpa dna-binding domain is conserved in s. coelicolor and s. lividans adpas suggesting that all three species share the same adpa-binding consensus sequence. the dna sequences upstream from the s. coelicolor ramr and hyas genes and the intergenic region between the divergently transcribed genes ccha/cchb, sco0774/sco and sco6197/sco were analyzed using predetector software and a matrix was generated with identified s. griseus adpa-binding sequences. between three and nine putative adpa-binding sites were detected within the promoter region of the s. coelicolor genes and by analogy in orthologous s. lividans adpa-dependent genes. during the course of this study, the s. lividans genome sequence became available ) and its analysis suggested that the position and composition of adpa-binding sites were different from those predicted. the putative adpa-binding sites of s. lividans ccha/cchb at -101 nt and -86 nt are gggccggttc and tggctggaac, respectively. the adpa-binding sites located upstream of sli sli and hyas differ from their s. coelicolor orthologs. in silico in the promoter regions of s. lividans adpa-dependent genes a s. coelicolor gene b c c d e f ain silico analysis of the s. coelicolor genome using predetector software to analyse orthologs of s. lividans adpa-dependent genes. the s. coelicolor adpa-binding sites identified were checked for their conservation and location using the s. lividans genome strepdb database. bgenes are named according to the strepdb database. *binding sites located between s. coelicolor genes transcribed in the opposite orientation. cputative s. coelicolor adpa-binding sites were found in silico with predetector; #putative site located in the upstream from the cds of cchb. the site location given corresponds to the position of first nucleotide most distant from the translation start point of the first gene named. the positions of some sites are not the same for the s. lividans orthologs as indicated in brackets. ~ putative sites are in the cds of sli predicted cds diverge between sli and sli locus and their orthologs sco and sco resulting in a smaller intergenic region in s. lividans. dcs, coding strand; ncs, non coding strand with reference to the first gene named in the s. coelicolor gene column. escores given by predetector software for s. coelicolor genes. fsites present or absent in the s. lividans dna probes used in emsa experiments. we used emsa to test whether s. lividans adpa binds to predicted s. lividans adpa-binding sequence. recombinant purified adpa-his bound to the promoter region of s. lividans sti, an adpa-dependent gene, whereas an excess of adpa-his did not bind to the promoter of sli, a gene that is not controlled by s. lividans adpa. this suggests that the binding of adpa with the promoter of genes tested in our previous study was specific. adpa-his was able to bind to the promoter regions of all s. lividans adpa-dependent genes tested, although with different affinities. for sli6586/sli ramr and hyas, displacement of the dna fragment to the slower migrating protein-dna complex was nearly complete with amounts of adpa of less than pmoles. for ccha/cchb and sli0755/sli larger amounts of adpa were necessary for near complete displacement of the dna probe to a protein-dna complex. in a competition emsa performed on sli6586/ with an excess of the corresponding unlabelled probe, adpa-binding to the labelled probe decreased. we also tested a hyas promoter in which one of the three putative adpa-binding sites was mutated: the affinity of adpa for this promoter region was reduced and one protein-dna complex disappeared. these results suggest that one dimer of adpa binds the adjacent sites - and - of s. lividans hyas promoter and another dimer binds the - site resulting in the formation of the two dna-adpa complexes depicted in figure  these emsa experiments demonstrated that s. lividans adpa directly binds to five intergenic regions and confirmed the in silico prediction presented in table  s. lividans adpa directly regulates at least the six adpa-dependent genes listed above and identified by microarrays and qrt-pcr analysis. these newly identified targets highlight the pleiotropic role of s. lividans adpa: it is involved in primary and secondary metabolisms, in regulation, and in cell development. discussion adpa, a transcriptional regulator of the arac/xyls family, is involved in the development and differentiation of various streptomyces. we report here the first identification of several pathways directly regulated by adpa in s. lividans cultivated in liquid rich medium. inactivation of adpa in s. lividans affected the expression of approximately genes. this large number was expected in the light of the size of the s. griseus adpa regulon. although adpa mutant growth was comparable to that of the parental strain in yeme liquid medium, the expression of around genes involved in primary metabolism was influenced by adpa deletion. these genes encode proteins involved in the major biosynthesis pathways for amino acids, and in energy metabolism including glycolysis, pentose phosphate, pyruvate dehydrogenase pathways, as well as in electron transport. other s. lividans adpa-regulated genes influence streptomyces development on solid media. s. lividans adpa also influences the expression of genes involved in secondary metabolism such as coelichelin biosynthesis and also genes described to affect metabolic differentiation. consistently with transcriptomic studies in s. griseus, these observations suggest that adpa is a pleiotropic transcriptional regulator in s. lividans. we demonstrate that s. lividans adpa directly activates cchb, sli and hyas. as a result of their co-transcription with these genes, the expression of cchcd, sli and sco7658-ortholog genes is adpa-dependent in s. lividans. sli is probably a directly adpa-regulated gene because its promoter dna region is shared with sli0755-sli operon, which is transcribed in the opposite direction and directly regulated by adpa. adpa directly regulates the genes ramr and sti in s. lividans and in the closely related species s. coelicolor. in an s. coelicolor adpa mutant, levels of sti and ramr expression were lower than in the wild-type strain following growth for 48 h in a minimal agar medium. in vitro experiments showed a high affinity of adpa with a s. coelicolor sti probe, consistent with our results with s. lividans sti. however, adpa had a lower affinity to s. coelicolor ramr than s. lividans ramr. when we used a s. lividans ramr probe carrying the promoter region from -201 nt to +66 nt, we observed that less than half the probe was shifted. therefore, the predicted sites for ramr promoter at positions - and - may have the greatest affinity for adpa. of the genes analysed by qrt-pcr, the ramr gene was that for which the observed expression was the least consistent with the microarray findings, even through the same sample was used for these analyses. this suggests that the expression of genes close to the cut-off we applied to the microarray data will need further investigation by qrt-pcr. among the genes identified as direct targets of adpa in s. griseus, have no orthologous gene in s. lividans and the orthologous genes of six are not under the control of s. lividans adpa in our conditions. in addition to ramr and sti, hyas is also a directly adpa-regulated gene that is conserved in the s. lividans and s. griseus adpa regulons. in s. lividans, hyas affects hypha aggregation and the amount of mycelium-associated undecylprodigiosin; its function in s. griseus is unknown. the expression of all of bldn, sli sli and the sco ortholog is influenced by adpa deletion in s. lividans. it remains to be determined whether adpa directly controls s. lividans adpa and blda as described in s. coelicolor and griseus. s. coelicolor adpa is one of identified tta-containing genes; the production of the proteins encoded by these genes is dependent on blda, encoding the only trna for the rare leucine codon tta. our study has revealed that expression of tta-containing genes and of genes regulated by s. coelicolor blda was affected by adpa deletion in s. lividans. we show that ccha, cchb, sti hyas, sli and sli previously identified in s. coelicolor as blda-dependent genes, are direct targets of s. lividans adpa. of the other blda-dependent genes, are probable direct s. lividans adpa targets: in silico analysis indicated the presence of putative adpa-binding sites upstream from these genes. by analogy, this suggests that the deregulation of certain genes observed in the s. coelicolor blda mutant may have been the consequence of s. coelicolor adpa down-regulation, as previously suggested. to predict probable direct targets of adpa in s. lividans and contribute to knowledge of the adpa regulon, we carried out in silico analysis of the entire s. coelicolor genome using predetector, and also restricted to the s. lividans genes identified as being adpa-dependent. we identified genes probably directly activated by s. lividans adpa and genes that could be directly repressed. most of the putative adpa-binding sites identified by this analysis are coherent with the findings of yao et al., demonstrating the importance of g and c nucleotides at positions and respectively. six genes have been identified as directly regulated by adpa in other species, and more in s. griseus are also probable adpa-direct targets. sixty-three of the probable direct targets of adpa in s. lividans have no ortholog in the s. griseus genome. s. lividans adpa in liquid rich medium a gene b b b b c c c d e adbs3-orfa glpq1 adpa bldh bldn, adsa aorthologs of s. lividans adpa-dependent genes were analysed in silico using the s. coelicolor genome database. adpa-binding sites upstream from s. coelicolor genes were identified and are presented in additional file 5: table s table  presents a selected subset of this complete compilation. bgene names for s. griseus and annotated function are from the strepdb database. ortholog gene names were identified using strepdb. genes identified in other streptomyces as being directly adpa-regulated are in bold, and those described as being adpa-dependent are italicized. * binding sites in the promoters of these genes were identified in silico. the sco2921-ortholog was not annotated as a s. lividans cds; however, our microarray data suggest that this cds exists. ccis-element, score, and binding site position as determined by analysing s. coelicolor genes with predetector. when more than one putative adpa-binding site was detected, only the one with the highest score was shown here. other genes putatively directly regulated by s. lividans adpa are listed in additional file 5: table s # site found in the sco cds at position. dfold change in gene expression in s. lividans adpa mutant relative to the parental strain with p-value < , as determined by student’s t-test applying the benjamini and hochberg multiple testing correction. efrom a protein classification scheme for the s. coelicolor genome available on the welcome trust sanger institute database: unknown function, cell process, macromolecule metabolism, small molecule metabolism, cell envelope, extrachromosomal, regulation and not classified.  in conclusion, this study has extended our knowledge of the s. lividans adpa regulon. we identified s. lividans adpa-regulated genes by transcriptomic analysis, and used in silico analysis to identify over a hundred probable direct targets of adpa in s. lividans. most of them are absent from the current predicted s. griseus adpa regulon. discovering new s. lividans genes directly regulated by adpa and that are involved in primary and secondary metabolism will provide valuable information about streptomyces development and differentiation in liquid culture. availability of supporting data microarray data are available in the arrayexpress database under accession number a-mexp- abbreviations qrt-pcr: quantitative real-time pcr; orf: open reading frame; fc: fold change; ct: critical threshold cycle; blast: basic local alignment search tool; emsa: electrophoretic mobility shift assay; adpa-his6: recombinant adpa protein with a six-histine tag at the c-terminus; cds: coding dna sequence; cs: coding strand; ncs: non coding strand; u. f.: unknown function; c. p.: cell process; m. m.: macromolecule metabolism; s. m.: small molecule metabolism; c. e.: cell envelope; e.: extrachromosomal; r.: regulation; n. c.: not classified. competing interests the authors declare that they have no competing interests. authors’ contributions ag, nb and pm wrote and revised the manuscript. cp and jyc have given final approval for this version to be published. pm helped ag to design the project. ag performed qrt-pcr, emsa and in silico analysis; and prepared figures, tables and additional files. nb purified adpa-his protein. cp carried out the microarray experiments. jyc helped cp with the statistical analysis of microarray results and wrote the associated methods sections. ag interpreted the microarrays data. mg help with qrt-pcr experiments and provided technical support. all authors read and approved the final manuscript. authors’ information ag performed qrt-pcr and emsa experiments while working at pasteur institute. her current address is centre for bacterial cell biology, institute for cell and molecular biosciences, newcastle university, newcastle-upon-tyne ne 4hh, uk. supplementary material additional file 1: table s1 oligonucleotides used in this study. click here for file additional file 2: table s2 complete set of genes differentially expressed in the s. lividans adpa mutant. s. coelicolor microarrays were used to test for genes differentially expressed in the s. lividans adpa mutant and wild-type at growth time point t, in liquid yeme medium. annotated function, fc, p-values, and classification of the proteins are presented according to the microarray sco genes, by increasing sco gene number. click here for file additional file 3: figure s1 effect of the mutation of one adpa-binding site in the s. lividans hyas promoter on adpa-binding specificity. mutation of an adpa-binding site in the s. lividans hyas promoter region prevents formation of an adpa-dna complex in vitro. sequence of the mutated adpa-binding site and emsa performed with the mutated hyas promoter region are shown. click here for file additional file 4: table s3 comparison of gene expression profiles between s. coelicolor blda-dependent and s. lividans adpa-dependent genes. comparison of the gene expression profiles of some s. coelicolor blda-dependent genes whose s. lividans orthologs are adpa-dependent. putative adpa-binding sites were identified in silico, suggesting that in the s. coelicolor blda mutant, the adpa translation defect leads to blda-dependence of the genes identified previously. click here for file additional file 5: table s4 putative s. coelicolor adpa-binding sites upstream from the s. lividans adpa-dependent genes. we identified putative adpa-binding sites in silico using the s. coelicolor genome and we analysed orthologs of s. lividans adpa-dependent genes; the sequences and positions of the sites with the highest scores according to predetector are shown. s. coelicolor, s. lividans and s. griseus ortholog genes are indicated and previously identified direct or probably direct s. griseus adpa-dependent genes are highlighted. click here for file acknowledgements we thank t. msadek, s. dubrac, e. johnson and j.-l. pernodet for helpful discussion and critical reading of the manuscript, and o. poupel for assistance with qrt-pcr analysis. we are grateful to g. bucca for her advice and help with microarray handling. we thank alex edelman & associates for correcting the manuscript. this work was supported by research funds from the institut pasteur and centre national de recherche scientifique. a. guyet was the recipient of fellowships from the ministère de l’education nationale, de la recherche et de la technologie, the pasteur-weizmann foundation and the era-ib european grant. ag thanks bbsrc and r. daniel for his constant support during the preparation of this manuscript.
in silico analysis revealed the presence of potential adpa-binding sites upstream from these genes; electrophoretic mobility shift assays indicated that adpa binds directly to their promoter regions. furthermore, in silico analysis suggests that over hundred genes may be directly activated or repressed by s. lividans adpa, although few have been described as being part of any streptomyces adpa regulons. several of these genes are orthologous to blda-dependent genes in s. coelicolor. microarray experiments revealed that the expression of hundreds of genes was affected in a s. lividans adpa mutant during early stationary phase cultures in yeme liquid medium. we recently discovered interplay between clpp peptidases and adpa in s. lividans. here, we report the identification of genes directly regulated by adpa in s. lividans. we studied the expression of the s. lividans adpa-regulated genes by quantitative real-time pcr analysis after various times of growth. this work identifies new pathways directly controlled by adpa and that are involved in s. lividans development, and primary or secondary metabolism. we characterised six s. lividans adpa-dependent genes whose expression is directly activated by this pleiotropic regulator. most work on adpa has been in s. griseus, and little is known about the pathways it controls in other streptomyces spp. streptomyces are gram-positive bacteria well-known for their production of secondary metabolites and antibiotics.
 adpa is a key transcriptional regulator involved in the complex growth cycle of streptomyces. streptomyces are gram-positive bacteria well-known for their production of secondary metabolites and antibiotics. most work on adpa has been in s. griseus, and little is known about the pathways it controls in other streptomyces spp. we recently discovered interplay between clpp peptidases and adpa in s. lividans. here, we report the identification of genes directly regulated by adpa in s. lividans.  microarray experiments revealed that the expression of hundreds of genes was affected in a s. lividans adpa mutant during early stationary phase cultures in yeme liquid medium. we studied the expression of the s. lividans adpa-regulated genes by quantitative real-time pcr analysis after various times of growth. in silico analysis revealed the presence of potential adpa-binding sites upstream from these genes; electrophoretic mobility shift assays indicated that adpa binds directly to their promoter regions. this work identifies new pathways directly controlled by adpa and that are involved in s. lividans development, and primary or secondary metabolism.  we characterised six s. lividans adpa-dependent genes whose expression is directly activated by this pleiotropic regulator. several of these genes are orthologous to blda-dependent genes in s. coelicolor. furthermore, in silico analysis suggests that over hundred genes may be directly activated or repressed by s. lividans adpa, although few have been described as being part of any streptomyces adpa regulons. this study increases the number of known adpa-regulated pathways in streptomyces spp. streptomyceslividansmicroarraysadpabldaramrhyascyp105d5cchbcoelichelin 