BACKGROUND
genome-wide surveys of transcript abundances in different conditions have become a major strategy toward delineating transcriptional regulation in many organisms.
while there is an infinite number of conditions and their combinations, certain conditions can be viewed as essential and our understanding of transcriptional regulation in such conditions is critical for associating macroscopic phenotypes with regulatory pathways.
such essential conditions include, but are not limited to, variations in nutrient composition of the growth environment.
indeed, bacterial growth depends on the availability of macronutrients that can serve as sources of carbon , nitrogen  phosphorus , and sulfur .
in the absence of any one of these macronutrients in the environment, cells cease to grow and divide.
while it is not surprising that biomass increase, which is prerequisite for normal growth, cannot happen without sufficient continued supply of nutrients, the regulatory consequences of nutritional challenges are not trivial.
understanding such effects on the molecular level, which would involve characterizing both condition-specific and condition-independent regulatory patterns, is especially important when dealing with reversible growth arrest or with secondary metabolic processes triggered by nutrient limitation or starvation.
one such secondary process is hydrogen production by cyanobacteria.
the fresh-water, unicellular non-nitrogen-fixing cyanobacterial strain synechocystis sp.
pcc  <dig>  is emerging as a model for photobiological production of hydrogen, a clean and sustainable energy carrier  <cit> .
under certain conditions, this oxygenic photosynthetic organism can use reductant from water-splitting photosynthetic processes to generate molecular hydrogen  <cit> .
the derived protons and electrons can be redirected to drive h <dig> production via the bidirectional hydrogenase, typically characterized by its sensitivity to oxygen  <cit> .
cyanobacterial hydrogen production is known to increase substantially under conditions of nutrient limitation, including n and s limitation  <cit> .
it has been suggested that limitation of macronutrients could result in accumulation of carbon that can then be used for h <dig> production.
however, the details of the bacterial h <dig> response to nutrient limitations and its regulation are yet to be determined.
in principle, it would be impossible to optimize h <dig> production without systematic understanding of the interactions between hydrogen metabolism, respiration, photosynthesis and bacterial nutritional status.
additionally, an understanding of the details of bacterial metabolism under various macro- and micronutrient limitations will help elucidate the effect of such interactions on the ecology of bacteria under natural conditions.
availability of the complete genome sequence of synechocystis sp.
pcc  <dig>  <cit>  offers an opportunity to develop technology to study and optimize metabolic and regulatory interactions in this bacterium.
the genome is approximately  <dig>  m bp long.
it contains more than  <dig> chromosomal protein coding genes and slightly over  <dig> genes encoding structural rnas  <cit> .
expression profiling by dna microarray technology  <cit>  has enabled tremendous advances in our ability to understand genome-wide regulatory and metabolic processes in a diversity of organisms.
this technology has provided insights into cyanobacterial transcriptional responses such as in acclimation to high light  <cit> , salt stress  <cit> , and nutrient limitations  <cit> .
here, we present a design and application of the genome-wide synechocystis microarray.
first, we developed and validated a new dna microarray platform, based on long oligonucleotide probes  spotted on a glass support.
we chose to build an oligo-based array because it allowed us to circumvent the labor-intensive and error-prone steps of probe amplification and purification.
additionally, the quality of cdna arrays may vary between pcr batches, which would affect researchers' ability to standardize data across the experimental sets.
the specificity of and hybridization consistency among the designed probes was assessed using a pilot array.
the validity of the genome-wide array was evaluated by successful identification of the well-characterized transcriptional responses to heat and salt stress.
next, the genomic transcriptional responses of synechocystis to s deprivation were investigated.
for photosynthetic organisms, including plants and microbes, s is an essential macronutrient present in proteins, lipids, electron transport components, and many cellular metabolites.
it is critical for the association of metal ions to form protein iron-sulfur  clusters, which perform indispensable functions including electron transfer, redox sensing, gene regulation, catalysis and maintenance of protein structure.
due to the limited intracellular storage of s, a continuous supply of this nutrient from the environment, mainly in the form of sulfate  anion, is necessary for cell growth and development.
following its acquisition, sulfate is either used for the direct sulfatation of compounds or it is reduced and converted to cysteine and methionine.
s limitation can occur in natural freshwater environments and strongly affect community composition.
in the laboratory, it was found that s deprivation was correlated with increase in h <dig> production in both green alga chlamydomonas reinhardtii  <cit>  and cyanobacterium synechocystis  <cit> .
the genomic transcriptional response of green alga chlamydomonas reinhardtii in response to s deprivation has been investigated using dna microarray analysis to obtain important regulatory insights underlying the physiological changes  <cit> .
the cyanobacterial genome-wide transcriptional responses to c, n and pi deficiencies have been investigated  <cit> , but no s acclimation study has been done.
in the present study, we used our microarray platform to explore the transcriptional response of the synechocystis genome to s deprivation.
our results demonstrated that the oligonucleotide microarray platform is applicable to systematic investigation of the transcriptional activities of the synechocystis genome under the conditions of ecological or biotechnological interest, such as nutrient limitation and h <dig> production.
the expression profiling indicated that regulation of gene transcription in the s acclimation conditions was critical for synechocystis to sense nutrient limitations and tune its metabolism for efficient utilization of available resources.
for example, repression of certain s-related genes was paralleled by activation of genes involved in internal and external s scavenging.
it remains to be determined if the two processes share a common regulatory mechanism.
RESULTS
experimental condition and microarray data processing
synechocystis transcriptional response to s deprivation was studied by two-color comparative hybridization using genome-wide oligonucleotide microarrays that were designed and validated as described in detail .
in short, a cost-effective two-stage design strategy was developed.
in the first stage, genomic dna was hybridized to the pilot array to evaluate the specificity of and hybridization consistency among the designed probes.
in the second stage, the genome-wide microarray  was developed and validated by successful identification of well-studied transcriptional responses including heat and salt stress.
synechocystis culture was grown in hepes -1-piperazineethanesulfonic acid) buffered bg- <dig> medium  <cit>  to mid-exponential phase, at which point  so42- was removed from the medium .
following s removal and a wash in s-free medium, time-point samples were taken over a short-term period  and a long-term period  in order to capture both "rapid" and prolonged transcriptional responses.
rna isolated from the time- <dig> sample was used as a common reference in time-course comparisons.
for each time-point,  <dig> or  <dig> biological samples were examined.
the scanned fluorescence intensity data were smoothed to remove undesirable local hybridization biases, and normalized using an anova model  <cit>  to remove systematic biases.
following the data reduction, differentially expressed genes were identified on the basis of passing a false discovery rate  cut-off applied to a two-class t-statistic.
in addition to individual gene analysis, the gene set analysis  <cit>  was used to identify gene sets which were strongly or moderately associated with the s deprivation response by assessing the significance of differential expression of pre-defined gene sets.
the  <dig> gene sets were assembled based on functional classification of synechocystis genes from cyanobase  <cit> , out of which  <dig> were first-level categories and  <dig> second-level categories.
the gene sets and annotation are available .
global features of temporal response
s deprivation had a slow-developing effect on cell growth and transcriptional activity of synechocystis, as shown in figure  <dig>  within  <dig> hr, s deprivation did not noticeably reduce synechocystis growth rate compared to the normally growing control .
consistent with a previous report  <cit> , the growth was arrested after long-term deprivation .
only a few dozen genes demonstrated high magnitude changes in transcription  at each time-point.
when panels b and c in figure  <dig> are compared, it could be noted that the number of genes whose transcript levels were affected after  <dig> hr of starvation was greater than after  <dig> hr.
at each time-point, the number of significantly down-regulated genes was roughly twice that of the up-regulated ones.
the predominance of low-magnitude effects cannot be entirely attributed to the microarray platform and/or data processing, as we were able to detect more than 10-fold changes and hundreds of highly responsive genes in the salt stress treatment .
transcriptional dynamics of significantly affected genes and gene sets
the transcriptional dynamics of significantly affected genes were examined in time courses following s withdrawal.
the statistically significant genes were selected based on fdr cutoff of <1% for each time-point experiment .
from the list of genes satisfying the fdr criterion, average transcript abundance of  <dig> genes changed more than  <dig> -fold in at least one time point were selected for subsequent analysis .
these genes were hierarchically clustered using pearson correlation as a distance metric and displayed as a heat-map, as shown in figure 2a.
twenty clusters could be delineated based on a correlation coefficient  cut-off of  <dig> .
most of the clusters contained more than five genes.
figure 2b shows the transcriptional profile of each cluster, plotted as a mean ± s.e.m.
a partial list of the differentially expressed genes encoding proteins with known functions, along with their transcriptional profile classifications, is given in table  <dig>
the differentially expressed genes in synechocystis sulfate deprivation time course were identified and clustered according to figure  <dig>  the complete list, with annotations  and mean fold change for each time-point comparison, is available .
the cluster analysis revealed the structure and dynamics of the transcriptional data obtained in the study.
overall, the profiles of up-regulated genes  and down-regulated genes  were highly anti-correlated with a pearson correlation coefficient of - <dig> .
clusters  <dig> to  <dig> showed an early induction with average transcriptional changes peaking before the 24-hr time point.
cluster  <dig>  containing three genes encoding for proteins with hypothetical functions, showed a late repression.
genes belonging to cluster  <dig>  which included genes encoding for phycobilisome  degradation  and sulfate transport system , were consistently up-regulated until  <dig> hr after s withdrawal.
clusters  <dig> to  <dig> contained genes that were significantly induced at a late stage ;  <dig> out of a total of  <dig> genes encoding for proteins with hypothetical or unknown functions could be found in those clusters.
cluster  <dig> contained  <dig> genes encoding for transposon-related functions, showing an immediate up-regulation.
clusters  <dig> to  <dig> showed overall down-regulation in transcript abundances.
the lowest transcript levels for clusters  <dig> and  <dig>  enriched for genes encoding atp synthase, co <dig> fixation and cytochrome b6/f complex, was reached at 3-hr time point.
however, for clusters  <dig> and  <dig>  which contained all of the genes responsible for light harvesting and photosynthesis  and most of the genes encoding for transcription and translation machineries, the largest repression occurred  <dig> hr following s removal.
cluster  <dig>  which contained  <dig> genes encoding components of the translational machinery and  <dig> encoding hypothetical and unknown protein, showed a single-peak transitional profile .
cluster  <dig> exhibited initial up-regulation followed by late down-regulation.
clusters  <dig> to  <dig> showed a dynamic profile reciprocal to that of cluster 17: an initial down-regulation followed by steady up-regulation in later time points.
<dig> out of the  <dig> genes contained in clusters  <dig> and  <dig> encoded proteins involved in protein synthesis and folding  as well as components of the protein/peptide secretion machinery.
in sum, the clustered transcriptional profiles indicated a three-stage temporal dynamic of genome-wide transcriptional regulation: early , transitional  and late  stages.
the genome-wide transcriptional activity may underpin the observed growth phenotype, which exhibited at least a two-stage response to s starvation: 1– <dig> normal doublings followed by the growth arrest.
the cluster analysis also revealed co-expression, and, potentially, co-regulation.
first, genes in the same operon were found to be co-regulated , sbpa-cystwa , hypothetical , atpihgfdacbe , psaab , psbflj , apcabc ).
most of the genes encoding for ribosomal protein subunits and sharing the same cluster profile were distributed continuously on the synechocystis chromosome.
second, genes encoding components of multi-protein complexes, which are scattered along the synechocystis chromosome, were also co-expressed.
for example, psbd  and psbd <dig>  were clustered together , both encoding for photosystems  ii reaction center d <dig> protein; rpoa  shared the same cluster with rpob  and rpoc <dig> , all encoding for core subunits for rna polymerase.
however, several duplicated genes indicated disparate transcriptional profiles.
the psba <dig>  and psba <dig>  genes, encoding form ii and iii of ps ii reaction center d <dig> polypeptide, were found to be significantly down-regulated, while psba <dig> , which is thought to be a cryptic gene  <cit> , showed no change in its transcript abundances during the time course.
this pattern of regulation was also noted for high light  <cit>  and salt stresses  <cit> .
other disparately active genes included those encoding the proteins sensing co <dig> concentration ccmk , ferredoxin petf , atp-dependent protease clpp , and a group  <dig> sigma factor rpoe .
the distinct transcriptional activities of those genes may indicate their complementary roles in mediating cellular stress responses.
in addition to individual genes, we also identified groups of genes that were significantly affected in the experiment.
the top-ranking significantly up-regulated or down-regulated sets of genes are listed in table s <dig> .
consistent with the analysis of differentially expressed genes, the sets of photosynthetic and respiration genes  as well as protein synthesis genes  were significantly down-regulated.
the sets of genes whose products are involved in protein and peptide secretion, phosphorus and energy metabolism  were also among the most down-regulated groups.
the sets of genes involved in rna metabolism, including transcription and degradation, were down-regulated by  <dig> hr of s starvation.
however by  <dig> hr, at least some genes involved in rna degradation appeared to be significantly up-regulated, including the ribonuclease e encoding gene rne, in cluster  <dig>  up-regulated gene sets included respiratory terminal oxidases, transport and binding proteins, cell envelope, membranes, lipoproteins, and porins, fatty acid, phospholipid and sterol metabolism.
of note, many sets of genes encoding for biosynthesis of cofactors, prosthetic groups and carriers  were also significantly up-regulated.
this might be relevant since these cofactors participate in important primary as well as secondary metabolic pathways, e.g., pigment synthesis, metal homestasis, and electron transfer.
verification of microarray data by quantitative rt-pcr
the microarray data were partly verified by quantitative real-time pcr following reverse transcription of total rna isolated from independent biological replicas of time point samples.
while 16s rrna is routinely used as an endogenous control for cell number, it may not be an ideal internal standard under conditions of variable growth  <cit> .
therefore in addition to 16s rrna, we used trpa and lysc as an internal reference.
the trpa gene was previously shown to be non-responsive to light or to chemical treatments  <cit> .
more importantly, both trpa and lysc were not differentially affected in our experimental conditions .
we confirmed that during early s deprivation , the abundance of cpca  and psba <dig>  transcripts was significantly decreased, while that of nbla <dig>  and cysw  was consistently increased .
the three internal references produced qualitatively similar results.
we observed that the quantitative pcr technique yielded relatively larger differences in transcript abundances than those estimated from microarray ratios.
it has also been observed in other studies that the quantitative pcr values are, in general, much higher than microarray data, whether based on oligonucleotide or cdna microarray platform  <cit> .
the somewhat larger disparity between microarray signals and the quantitative pcr observed in this study was likely due to the fact that, in the reported microarray experiment, fluorescent intensities were not corrected for background noise: surprisingly, application of available background models, suitable for microarrays with double stranded dna probes, resulted in a dramatic deterioration of the statistical properties of the data.
at the same time, quantitative pcr can have close-to-100% amplification efficiency at optimal conditions.
though the magnitudes of microarray data are smaller, it can sensitively discriminate the direction of transcriptional changes  for a large number of genes simultaneously.
a. transcript abundances of significantly induced nbla <dig>  cysw and repressed cpca, psba <dig> genes, relative to stable rna, trpa, or lysc, as endogenous references.
b. measurements of transcript abundances of hydrogenase-related genes using lysc as an endogenous reference.
mid-exponential cells were harvested and fixed in phenol-ethanol stop solution before sulfate withdrawal  and  <dig>   <dig>   <dig> hr after sulfate withdrawal.
total rna was isolated and reverse transcribed.
the relative abundances of each transcript were measured by sybr green real-time pcr and analyzed according to the comparative ct method.
the data are presented as transcript abundances, normalized to a reference mrna, in a time-point sample relative to control  on the log <dig> basis.
errors are the pooled s.e.m.
.
"n.d." – not determined.
control experiments indicated that sulfate deprivation induced specific transcriptional changes
because the medium ph may affect h <dig> production  <cit> , the s deprivation experiment was also performed with hepes buffering.
both the growth curve  and transcriptional profile   were comparable with the hepes-free condition.
out of the top  <dig> significantly affected genes,  <dig> were common between the two s deprivation conditions.
another control experiment was done to verify that the transcriptional regulation was indeed due to s deprivation and not a growth stage-dependent artifact.
as shown in figure s <dig> , the growth stage time-course profile was significantly different from the profile of s deprivation conditions.
out of the top  <dig> significant genes, about half of down-regulated genes in s deprivation conditions were up-regulated in normal growth stage transition, including many psi genes.
however, both psi and psii genes were significantly down-regulated in late stationary phase .
typically the sulfate transporter sbpa-cystwa operon was not up-regulated during normal growth control.
many differentially expressed individual genes  and gene sets  , including up-regulation of detoxification genes and simultaneous down-regulation of genes for n  and s  assimilation, represented known transcriptional features of cells transitioning from exponential to stationary phase of growth  <cit> .
these results suggested that the transcriptional regulation following s deprivation was indeed attributable in large part to sulfate availability and not a general response to slowed growth.
s starvation induced general and specific responses to macronutrient deficiencies
the s deprivation microarray data identified both the general and specific responses to macronutrient deficiencies  <cit> .
the specific responses result from a particular nutrient limitation, whereas the general, or common, responses occur under any starvation condition such as c, n or pi deprivation.
upon s deprivation, the high-affinity periplasmic sulfate transport system, encoded by sbpa-cystwa operon in synechocystis, was rapidly and significantly induced , and stayed induced until 72-hr after s withdrawal .
the physiological roles of the proteins encoded by these genes in sulfate acquisition have been elucidated in synechococcus sp.
strain pcc <dig>  <cit> , another extensively studied freshwater cyanobacterium.
their homologs in synechocystis may have a similar function.
sbpa  is a polypeptide that binds to sulfate.
the binding causes a conformational change in the protein which results in trapping of a sulfate substrate molecule.
the protein ligand complex then interacts with two hydrophobic proteins  which are thought to span the cytoplasmic membrane and form a pore.
this interaction results in the release of the substrate.
it then traverses the pore and enters the cell against a concentration gradient using energy derived from atp hydrolysis which is a function of the nucleotide binding protein encoded by cysa.
though sulfate acquisition genes were significantly induced, some s assimilation pathway genes, including sulfate adenylytransferase , adenylylsulfate kinase , phosphoadenosine phosphosulfate reductase , d-3-phosphoglycerate dehydrogenase , serine hydroxymethyltransferase  and cystein synthase , were not identified in our analysis as being significantly affected.
the responses mentioned above are specifically related to s limitation/starvation.
the common response of cyanobacteria upon nutrient deficiencies typically includes shutting down photosynthesis and degrading their pbs, the specific light harvesting complexes for ps ii.
as analyzed above, the photosynthesis and respiration genes, including those encoding the five major macromolecular complexes localized on the thylakoid membrane, pbs, ps ii, cytochrom b6/f complex, ps i and atp synthase, were consistently down-regulated during the experimental time course.
pbs structural genes, including genes encoding core , rod  and linker  polypeptides, were significantly down-regulated.
pbs, which can constitute up to half of the total cellular protein  <cit> , is known to be rapidly and progressively degraded during nutrient-limited growth.
the pbs degradation was thought to minimize the absorption of excess excitation energy under stressful conditions  <cit> .
however, another possibility is that it can be scavenged as a source of s-containing amino acids, although pbs is thought to be a poor s-source  <cit> .
the pbs degradation is triggered by a small polypeptide encoded by nbla  genes , which were significantly up-regulated in our microarray experiments  and confirmed by quantitative reverse-transcription pcr .
the opposite transcriptional regulation of the pbs structural genes and pbs degradation protein genes suggests that synechocystis down-regulates pbs abundance and/or activity for light harvest and energy transfer at the transcriptional level, though this down-regulation may not be causing dramatic bleaching in the process   <cit> .
the declined pbs transcription was correlated with the down-regulation of the downstream energy transfer components: ps ii, cytochrom b6/f complex, ps i and atp synthase .
furthermore, key co <dig> fixation genes, corresponding to the ribulose bisphosphate carboxylase  and the co <dig> concentrating mechanism protein ccmk , were down-regulated; soluble electron carrier genes , except cytm, which encodes cytochrome cm, were all consistently repressed upon s removal.
notably, no genes in respiratory terminal oxidase and nadh dehydrogenase sub-categories were identified as down-regulated, from both the individual gene analysis  and gene set analysis  .
it may indicate that cytochrome oxidase-mediated respiration activity was not significantly repressed on a transcriptional level during s acclimation.
s deprivation elicited transcriptional responses associated with general growth arrest and stress
other components of the transcriptional response were associated with general growth arrest and stress.
first, down-regulation of both transcription and translation machineries was observed.
rna polymerase core subunits genes  were significantly repressed.
the ribosomal protein subunit genes and genes encoding the initiation factor if- <dig> , and two elongation factors  were significantly down-regulated.
many of these significantly repressed ribosomal protein subunits genes are clustered on the synechocystis chromosome .
the transcription of the principle sigma factor siga, encoded by rpod , was significantly induced at 24-hr time-point, and the ecf-type  sigma-e factor, encoded by rpoe , showed pronounced late induction.
they may be involved in the transcriptional modulation during the transition stage .
sige is also involved in the response to n deprivation as well as in positive regulation of sugar catabolism  <cit> .
second, transcriptional adjustment of genes involved in macromolecular biosynthesis and energy metabolism was observed.
transcriptional regulation of several genes involved in n assimilation, including glna , sll <dig> and sll <dig> , may indicate the slowing of ammonium assimilation under s limitation.
similarly, ppa , a key pi starvation induced gene  <cit> , was down-regulated.
the repression of pyrophosphatase activity, however, may suggest accumulation of polyphosphate during s deprivation.
s deprivation induced a decline in photosynthetic electron transfer, concurrent with down-regulation of pbs and photosynthesis, which were exemplified by reduced abundances of trxa  and grxc .
in addition, three energy metabolism genes, cbba , cfxe  and tkta , were repressed.
all three gene activities lead to production of glyceraldehydes 3-phosphate, through glycolysis  or non-oxidative branch of pentose phosphate pathways .
the effect of this regulation may be to lower the sugar catabolism and preserve carbohydrate storage in the form of glycogen.
however, genes for fatty acid and lipid biosynthesis, accd and fabh, were significantly up-regulated, which is interesting with respect to the carbon storage and h <dig> production hypothesis  <cit> .
third, stress protection responses were observed.
the stress response involved genes encoding chaperon and proteolysis functions , preprotein translocase , a pigment biosynthesis pathway gene , a stationary phase survival protein sure homolog , ribonuclease e , light-repressed protein a homolog , antioxidant protein , oxidative stress protection ahpc/tsa family protein , and two-component response regulator genes .
additionally, control of the cell cycle , nucleotide synthesis , dna protection , and modulation of cell envelop permeability  were affected by s stress.
of note, a large number of genes regulated by s availability encode hypothetical proteins or proteins of unknown function .
interestingly, about 30% of those genes were significantly up-regulated during the late stage of s starvation .
their involvement, if any, in s acclimation and/or utilization remains to be elucidated.
overall, our genome-wide analysis presented a broad view of transcriptional changes accompanying sulfur starvation in cyanobacteria.
some strategies unveiled here are also "employed" by cyanobacteria to cope with c, n and pi deficiencies  <cit> .
however, to fully understand how cells sense, transmit and integrate information about s status, it may be necessary to study various mutants affecting s acclimation, including:  a mutant of the sll <dig> gene, whose chlamydomonas reinhardtii homologue sac <dig>  is a putative sensor of s levels in the environment and is required for survival of s-deprived chlamydomonas cells in the light  <cit> ;  mutants of dspa   <cit>  and sll <dig>  whose synechococcus orthologues, nbls and nblr, respectively, encode the components of the histindine kinase-response regulator  two-component system nbls-nblr  <cit> , which was shown to be critical for acclimation of synechococcus to high-light and nutrient limitation conditions  <cit> .
it is possible, however, that other two-component signalling systems mediate s acclimation in synechocystis, including those that are regulated by sll <dig> of the ompr subfamily and slr <dig> of the pata subfamily, which were significantly up-regulated in our experimental condition;  mutants of global regulators of c, n and pi metabolism, such as ntca  <cit>  and ndhr  <cit> , which mediate a "cross-talk" between nutrient-specific responses in the cell  <cit> .
it might also be informative to compare the s acclimation response of cyanobacteria with eukaryotic oxygenic photosynthetic organisms and heterotrophic eubacteria.
in green alga chlamydomonas reinhardtii, s deprivation leads to increased accumulation of arylsulfatase, which releases sulfate anions from esterified organic sulfates, allowing cells to access new sulfur stores  <cit> .
in the opportunistic pathogen pseudomonas aeruginosa, s deprivation induces genes encoding sulfatases and sulfonatases for desulfurizing a range of organic sulfur compounds such as sulfate esters and sulfonates  <cit> .
in contrast, the sulfate starvation response in e. coli is dominated by increased expression of the s assimilation cysteine biosynthetic pathway, and propagated into methionine metabolism, synthesis of fe-s clusters, and oxidative stress  <cit> .
these differences, to some extent, may be associated with the special cellular component that only cyanobacteria have – pbs.
on one hand, pbs degradation may directly contribute to nutrient scavenging  because it constitutes up to 50% of the total cellular protein.
on the other hand, the transcriptional shut down of pbs biosynthesis, accompanied by reduced photosynthetic activity, allows nutrient limited cells to survive for relatively long periods of time under stress conditions via a "stand-by" mode of energy metabolism.
relevance to biohydrogen production
as a secondary metabolic process of significant biotechnological interest, photobiological h <dig> production from water by cyanobacteria depends on coordination between photosynthetic electron transport and the catalytic activity of the hydrogenase.
the optimal conditions for h <dig> production should direct more electrons to hydrogenase than other competing pathways   <cit> .
at the same time, anaerobic conditions are necessary to induce hydrogenase expression and/or activity  <cit> .
we observed that s deprivation significantly increased photoautotrophic h <dig> evolution by synechocystis, compared with +s controls  .
the microarray expression profiling suggested similar underlying principles for h <dig> production as observed in the green alga chlamydomonas reinhardtii  <cit> .
s deprivation leads to inactivation of ps ii which performs water splitting.
oxygen evolution ceases and it is depleted by respiration, as it was observed that ps ii genes were significantly repressed but genes encoding for nadh dehydrogenase and respiratory terminal oxidase were not.
this process leads to anaerobiosis, which in turn could induce hydrogenase activity.
s starvation inhibits co <dig> fixation and thus removes another important electron sink.
furthermore, this process likely leads to decreased sugar catabolism and the accumulation of carbohydrates, which is important for sustained h <dig> production in the long run.
previous work  <cit>  proposed that s limitation triggers h <dig> production by causing a lack of the d <dig> protein  which is essential for ps ii and needs to be constantly replaced.
the microarray data suggested that h <dig> evolution was accompanied by a complex and dynamic transcriptional process.
however, the question remains whether, and to what extent, the observed complexity of transcriptional regulation is essential for renewable, i.e., without the loss of viability, bioproduction of h <dig> by cyanobacteria.
the mrna levels of genes encoding activities directly involved in h <dig> evolution were specifically focused on by quantitative rt-pcr.
synechocystis uses heteropentameric  hydrogenases  and maturation accessory proteins  for h <dig> production.
none of those genes was identified as significantly differentially affected in microarray experiments.
moreover their known transcriptional activator lexa  <cit>  was down-regulated in the conditions we tested .
the qrt-pcr assay confirmed that the transcript levels of most of the hydrogenase genes were not significantly increased .
this indicates that hydrogenase levels were not limiting at the transcript level.
however, hoxe and hoxh were induced more than  <dig>  fold at 1-hr and 3-hr time points as measured by qrt-pcr.
there may be other factors involved in the regulation of the hox operon transcription besides lexa.
for instance, recently an abrb-like protein was identified as a positive regulator of expression of the bidirectional hydrogenase in synechocystis  <cit> .
however, cells only survive for a limited period of time in s-deprived medium and will eventually die.
such a shortcoming may be overcome by designing systems that mimic s deprivation for h <dig> production.
for example, an inducible nbla gene expression system could be constructed under the control of promoters whose activity can be modulated by nonessential micronutrient inducers, e.g., pete promoter by copper  <cit> , or by environmental parameters, e.g., groes-groel promoter by heat shock.
the cyclic induction and repression of nbla transcription may lead to repeated pbs degradation and synthesis, which in turn cyclically inactivates and activates ps ii.
an inducible ps ii gene expression system can potentially generate repeated cycles of carbon storage and h <dig> production, allowing for scalable and sustainable h <dig> production.
a similar idea has been explored in chlamydomonas reinhardtii for triggering h <dig> production by means other than macronutrient limitation  <cit> .
CONCLUSIONS
here we reported the study of transcriptional activity of the synechocystis genome in response to s deprivation by using a newly designed microarray of more than  <dig>  protein coding genes of synechocystis sp.
pcc  <dig>  the genome-wide expression profiling indicated that s deprivation induced relatively weak, but significant, transcriptional changes.
a complex transcriptional change during s acclimation was described in terms of general and specific responses characteristic of macronutrient deficiencies.
a large portion of significantly affected genes were the genes of unknown function, highlighting gaps in our understanding of the nature of nutrient adaptation.

 genome-wide surveys of transcript abundances in different conditions have become a major strategy toward delineating transcriptional regulation in many organisms. while there is an infinite number of conditions and their combinations, certain conditions can be viewed as essential and our understanding of transcriptional regulation in such conditions is critical for associating macroscopic phenotypes with regulatory pathways. such essential conditions include, but are not limited to, variations in nutrient composition of the growth environment. indeed, bacterial growth depends on the availability of macronutrients that can serve as sources of carbon, nitrogen phosphorus, and sulfur. in the absence of any one of these macronutrients in the environment, cells cease to grow and divide. while it is not surprising that biomass increase, which is prerequisite for normal growth, cannot happen without sufficient continued supply of nutrients, the regulatory consequences of nutritional challenges are not trivial. understanding such effects on the molecular level, which would involve characterizing both condition-specific and condition-independent regulatory patterns, is especially important when dealing with reversible growth arrest or with secondary metabolic processes triggered by nutrient limitation or starvation. one such secondary process is hydrogen production by cyanobacteria. the fresh-water, unicellular non-nitrogen-fixing cyanobacterial strain synechocystis sp. pcc is emerging as a model for photobiological production of hydrogen, a clean and sustainable energy carrier. under certain conditions, this oxygenic photosynthetic organism can use reductant from water-splitting photosynthetic processes to generate molecular hydrogen. the derived protons and electrons can be redirected to drive h production via the bidirectional hydrogenase, typically characterized by its sensitivity to oxygen. cyanobacterial hydrogen production is known to increase substantially under conditions of nutrient limitation, including n and s limitation. it has been suggested that limitation of macronutrients could result in accumulation of carbon that can then be used for h production. however, the details of the bacterial h response to nutrient limitations and its regulation are yet to be determined. in principle, it would be impossible to optimize h production without systematic understanding of the interactions between hydrogen metabolism, respiration, photosynthesis and bacterial nutritional status. additionally, an understanding of the details of bacterial metabolism under various macro- and micronutrient limitations will help elucidate the effect of such interactions on the ecology of bacteria under natural conditions. availability of the complete genome sequence of synechocystis sp. pcc offers an opportunity to develop technology to study and optimize metabolic and regulatory interactions in this bacterium. the genome is approximately m bp long. it contains more than chromosomal protein coding genes and slightly over genes encoding structural rnas. expression profiling by dna microarray technology has enabled tremendous advances in our ability to understand genome-wide regulatory and metabolic processes in a diversity of organisms. this technology has provided insights into cyanobacterial transcriptional responses such as in acclimation to high light, salt stress, and nutrient limitations. here, we present a design and application of the genome-wide synechocystis microarray. first, we developed and validated a new dna microarray platform, based on long oligonucleotide probes spotted on a glass support. we chose to build an oligo-based array because it allowed us to circumvent the labor-intensive and error-prone steps of probe amplification and purification. additionally, the quality of cdna arrays may vary between pcr batches, which would affect researchers' ability to standardize data across the experimental sets. the specificity of and hybridization consistency among the designed probes was assessed using a pilot array. the validity of the genome-wide array was evaluated by successful identification of the well-characterized transcriptional responses to heat and salt stress. next, the genomic transcriptional responses of synechocystis to s deprivation were investigated. for photosynthetic organisms, including plants and microbes, s is an essential macronutrient present in proteins, lipids, electron transport components, and many cellular metabolites. it is critical for the association of metal ions to form protein iron-sulfur clusters, which perform indispensable functions including electron transfer, redox sensing, gene regulation, catalysis and maintenance of protein structure. due to the limited intracellular storage of s, a continuous supply of this nutrient from the environment, mainly in the form of sulfate anion, is necessary for cell growth and development. following its acquisition, sulfate is either used for the direct sulfatation of compounds or it is reduced and converted to cysteine and methionine. s limitation can occur in natural freshwater environments and strongly affect community composition. in the laboratory, it was found that s deprivation was correlated with increase in h production in both green alga chlamydomonas reinhardtii and cyanobacterium synechocystis. the genomic transcriptional response of green alga chlamydomonas reinhardtii in response to s deprivation has been investigated using dna microarray analysis to obtain important regulatory insights underlying the physiological changes. the cyanobacterial genome-wide transcriptional responses to c, n and pi deficiencies have been investigated, but no s acclimation study has been done. in the present study, we used our microarray platform to explore the transcriptional response of the synechocystis genome to s deprivation. our results demonstrated that the oligonucleotide microarray platform is applicable to systematic investigation of the transcriptional activities of the synechocystis genome under the conditions of ecological or biotechnological interest, such as nutrient limitation and h production. the expression profiling indicated that regulation of gene transcription in the s acclimation conditions was critical for synechocystis to sense nutrient limitations and tune its metabolism for efficient utilization of available resources. for example, repression of certain s-related genes was paralleled by activation of genes involved in internal and external s scavenging. it remains to be determined if the two processes share a common regulatory mechanism.  experimental condition and microarray data processing synechocystis transcriptional response to s deprivation was studied by two-color comparative hybridization using genome-wide oligonucleotide microarrays that were designed and validated as described in detail. in short, a cost-effective two-stage design strategy was developed. in the first stage, genomic dna was hybridized to the pilot array to evaluate the specificity of and hybridization consistency among the designed probes. in the second stage, the genome-wide microarray was developed and validated by successful identification of well-studied transcriptional responses including heat and salt stress. synechocystis culture was grown in hepes -1-piperazineethanesulfonic acid) buffered bg- medium to mid-exponential phase, at which point so42- was removed from the medium. following s removal and a wash in s-free medium, time-point samples were taken over a short-term period and a long-term period in order to capture both "rapid" and prolonged transcriptional responses. rna isolated from the time- sample was used as a common reference in time-course comparisons. for each time-point, or biological samples were examined. the scanned fluorescence intensity data were smoothed to remove undesirable local hybridization biases, and normalized using an anova model to remove systematic biases. following the data reduction, differentially expressed genes were identified on the basis of passing a false discovery rate cut-off applied to a two-class t-statistic. in addition to individual gene analysis, the gene set analysis was used to identify gene sets which were strongly or moderately associated with the s deprivation response by assessing the significance of differential expression of pre-defined gene sets. the gene sets were assembled based on functional classification of synechocystis genes from cyanobase, out of which were first-level categories and second-level categories. the gene sets and annotation are available. global features of temporal response s deprivation had a slow-developing effect on cell growth and transcriptional activity of synechocystis, as shown in figure within hr, s deprivation did not noticeably reduce synechocystis growth rate compared to the normally growing control. consistent with a previous report, the growth was arrested after long-term deprivation. only a few dozen genes demonstrated high magnitude changes in transcription at each time-point. when panels b and c in figure are compared, it could be noted that the number of genes whose transcript levels were affected after hr of starvation was greater than after hr. at each time-point, the number of significantly down-regulated genes was roughly twice that of the up-regulated ones. the predominance of low-magnitude effects cannot be entirely attributed to the microarray platform and/or data processing, as we were able to detect more than 10-fold changes and hundreds of highly responsive genes in the salt stress treatment. transcriptional dynamics of significantly affected genes and gene sets the transcriptional dynamics of significantly affected genes were examined in time courses following s withdrawal. the statistically significant genes were selected based on fdr cutoff of <1% for each time-point experiment. from the list of genes satisfying the fdr criterion, average transcript abundance of genes changed more than -fold in at least one time point were selected for subsequent analysis. these genes were hierarchically clustered using pearson correlation as a distance metric and displayed as a heat-map, as shown in figure 2a. twenty clusters could be delineated based on a correlation coefficient cut-off of. most of the clusters contained more than five genes. figure 2b shows the transcriptional profile of each cluster, plotted as a mean ± s.e.m. a partial list of the differentially expressed genes encoding proteins with known functions, along with their transcriptional profile classifications, is given in table the differentially expressed genes in synechocystis sulfate deprivation time course were identified and clustered according to figure the complete list, with annotations and mean fold change for each time-point comparison, is available. the cluster analysis revealed the structure and dynamics of the transcriptional data obtained in the study. overall, the profiles of up-regulated genes and down-regulated genes were highly anti-correlated with a pearson correlation coefficient of -. clusters to showed an early induction with average transcriptional changes peaking before the 24-hr time point. cluster containing three genes encoding for proteins with hypothetical functions, showed a late repression. genes belonging to cluster which included genes encoding for phycobilisome degradation and sulfate transport system, were consistently up-regulated until hr after s withdrawal. clusters to contained genes that were significantly induced at a late stage; out of a total of genes encoding for proteins with hypothetical or unknown functions could be found in those clusters. cluster contained genes encoding for transposon-related functions, showing an immediate up-regulation. clusters to showed overall down-regulation in transcript abundances. the lowest transcript levels for clusters and enriched for genes encoding atp synthase, co fixation and cytochrome b6/f complex, was reached at 3-hr time point. however, for clusters and which contained all of the genes responsible for light harvesting and photosynthesis and most of the genes encoding for transcription and translation machineries, the largest repression occurred hr following s removal. cluster which contained genes encoding components of the translational machinery and encoding hypothetical and unknown protein, showed a single-peak transitional profile. cluster exhibited initial up-regulation followed by late down-regulation. clusters to showed a dynamic profile reciprocal to that of cluster 17: an initial down-regulation followed by steady up-regulation in later time points. out of the genes contained in clusters and encoded proteins involved in protein synthesis and folding as well as components of the protein/peptide secretion machinery. in sum, the clustered transcriptional profiles indicated a three-stage temporal dynamic of genome-wide transcriptional regulation: early, transitional and late stages. the genome-wide transcriptional activity may underpin the observed growth phenotype, which exhibited at least a two-stage response to s starvation: 1– normal doublings followed by the growth arrest. the cluster analysis also revealed co-expression, and, potentially, co-regulation. first, genes in the same operon were found to be co-regulated, sbpa-cystwa, hypothetical, atpihgfdacbe, psaab, psbflj, apcabc ). most of the genes encoding for ribosomal protein subunits and sharing the same cluster profile were distributed continuously on the synechocystis chromosome. second, genes encoding components of multi-protein complexes, which are scattered along the synechocystis chromosome, were also co-expressed. for example, psbd and psbd were clustered together, both encoding for photosystems ii reaction center d protein; rpoa shared the same cluster with rpob and rpoc, all encoding for core subunits for rna polymerase. however, several duplicated genes indicated disparate transcriptional profiles. the psba and psba genes, encoding form ii and iii of ps ii reaction center d polypeptide, were found to be significantly down-regulated, while psba, which is thought to be a cryptic gene, showed no change in its transcript abundances during the time course. this pattern of regulation was also noted for high light and salt stresses. other disparately active genes included those encoding the proteins sensing co concentration ccmk, ferredoxin petf, atp-dependent protease clpp, and a group sigma factor rpoe. the distinct transcriptional activities of those genes may indicate their complementary roles in mediating cellular stress responses. in addition to individual genes, we also identified groups of genes that were significantly affected in the experiment. the top-ranking significantly up-regulated or down-regulated sets of genes are listed in table s. consistent with the analysis of differentially expressed genes, the sets of photosynthetic and respiration genes as well as protein synthesis genes were significantly down-regulated. the sets of genes whose products are involved in protein and peptide secretion, phosphorus and energy metabolism were also among the most down-regulated groups. the sets of genes involved in rna metabolism, including transcription and degradation, were down-regulated by hr of s starvation. however by hr, at least some genes involved in rna degradation appeared to be significantly up-regulated, including the ribonuclease e encoding gene rne, in cluster up-regulated gene sets included respiratory terminal oxidases, transport and binding proteins, cell envelope, membranes, lipoproteins, and porins, fatty acid, phospholipid and sterol metabolism. of note, many sets of genes encoding for biosynthesis of cofactors, prosthetic groups and carriers were also significantly up-regulated. this might be relevant since these cofactors participate in important primary as well as secondary metabolic pathways, e.g., pigment synthesis, metal homestasis, and electron transfer. verification of microarray data by quantitative rt-pcr the microarray data were partly verified by quantitative real-time pcr following reverse transcription of total rna isolated from independent biological replicas of time point samples. while 16s rrna is routinely used as an endogenous control for cell number, it may not be an ideal internal standard under conditions of variable growth. therefore in addition to 16s rrna, we used trpa and lysc as an internal reference. the trpa gene was previously shown to be non-responsive to light or to chemical treatments. more importantly, both trpa and lysc were not differentially affected in our experimental conditions. we confirmed that during early s deprivation, the abundance of cpca and psba transcripts was significantly decreased, while that of nbla and cysw was consistently increased. the three internal references produced qualitatively similar results. we observed that the quantitative pcr technique yielded relatively larger differences in transcript abundances than those estimated from microarray ratios. it has also been observed in other studies that the quantitative pcr values are, in general, much higher than microarray data, whether based on oligonucleotide or cdna microarray platform. the somewhat larger disparity between microarray signals and the quantitative pcr observed in this study was likely due to the fact that, in the reported microarray experiment, fluorescent intensities were not corrected for background noise: surprisingly, application of available background models, suitable for microarrays with double stranded dna probes, resulted in a dramatic deterioration of the statistical properties of the data. at the same time, quantitative pcr can have close-to-100% amplification efficiency at optimal conditions. though the magnitudes of microarray data are smaller, it can sensitively discriminate the direction of transcriptional changes for a large number of genes simultaneously. a. transcript abundances of significantly induced nbla cysw and repressed cpca, psba genes, relative to stable rna, trpa, or lysc, as endogenous references. b. measurements of transcript abundances of hydrogenase-related genes using lysc as an endogenous reference. mid-exponential cells were harvested and fixed in phenol-ethanol stop solution before sulfate withdrawal and hr after sulfate withdrawal. total rna was isolated and reverse transcribed. the relative abundances of each transcript were measured by sybr green real-time pcr and analyzed according to the comparative ct method. the data are presented as transcript abundances, normalized to a reference mrna, in a time-point sample relative to control on the log basis. errors are the pooled s.e.m.. "n.d." – not determined. control experiments indicated that sulfate deprivation induced specific transcriptional changes because the medium ph may affect h production, the s deprivation experiment was also performed with hepes buffering. both the growth curve and transcriptional profile were comparable with the hepes-free condition. out of the top significantly affected genes, were common between the two s deprivation conditions. another control experiment was done to verify that the transcriptional regulation was indeed due to s deprivation and not a growth stage-dependent artifact. as shown in figure s, the growth stage time-course profile was significantly different from the profile of s deprivation conditions. out of the top significant genes, about half of down-regulated genes in s deprivation conditions were up-regulated in normal growth stage transition, including many psi genes. however, both psi and psii genes were significantly down-regulated in late stationary phase. typically the sulfate transporter sbpa-cystwa operon was not up-regulated during normal growth control. many differentially expressed individual genes and gene sets, including up-regulation of detoxification genes and simultaneous down-regulation of genes for n and s assimilation, represented known transcriptional features of cells transitioning from exponential to stationary phase of growth. these results suggested that the transcriptional regulation following s deprivation was indeed attributable in large part to sulfate availability and not a general response to slowed growth. s starvation induced general and specific responses to macronutrient deficiencies the s deprivation microarray data identified both the general and specific responses to macronutrient deficiencies. the specific responses result from a particular nutrient limitation, whereas the general, or common, responses occur under any starvation condition such as c, n or pi deprivation. upon s deprivation, the high-affinity periplasmic sulfate transport system, encoded by sbpa-cystwa operon in synechocystis, was rapidly and significantly induced, and stayed induced until 72-hr after s withdrawal. the physiological roles of the proteins encoded by these genes in sulfate acquisition have been elucidated in synechococcus sp. strain pcc, another extensively studied freshwater cyanobacterium. their homologs in synechocystis may have a similar function. sbpa is a polypeptide that binds to sulfate. the binding causes a conformational change in the protein which results in trapping of a sulfate substrate molecule. the protein ligand complex then interacts with two hydrophobic proteins which are thought to span the cytoplasmic membrane and form a pore. this interaction results in the release of the substrate. it then traverses the pore and enters the cell against a concentration gradient using energy derived from atp hydrolysis which is a function of the nucleotide binding protein encoded by cysa. though sulfate acquisition genes were significantly induced, some s assimilation pathway genes, including sulfate adenylytransferase, adenylylsulfate kinase, phosphoadenosine phosphosulfate reductase, d-3-phosphoglycerate dehydrogenase, serine hydroxymethyltransferase and cystein synthase, were not identified in our analysis as being significantly affected. the responses mentioned above are specifically related to s limitation/starvation. the common response of cyanobacteria upon nutrient deficiencies typically includes shutting down photosynthesis and degrading their pbs, the specific light harvesting complexes for ps ii. as analyzed above, the photosynthesis and respiration genes, including those encoding the five major macromolecular complexes localized on the thylakoid membrane, pbs, ps ii, cytochrom b6/f complex, ps i and atp synthase, were consistently down-regulated during the experimental time course. pbs structural genes, including genes encoding core, rod and linker polypeptides, were significantly down-regulated. pbs, which can constitute up to half of the total cellular protein, is known to be rapidly and progressively degraded during nutrient-limited growth. the pbs degradation was thought to minimize the absorption of excess excitation energy under stressful conditions. however, another possibility is that it can be scavenged as a source of s-containing amino acids, although pbs is thought to be a poor s-source. the pbs degradation is triggered by a small polypeptide encoded by nbla genes, which were significantly up-regulated in our microarray experiments and confirmed by quantitative reverse-transcription pcr. the opposite transcriptional regulation of the pbs structural genes and pbs degradation protein genes suggests that synechocystis down-regulates pbs abundance and/or activity for light harvest and energy transfer at the transcriptional level, though this down-regulation may not be causing dramatic bleaching in the process. the declined pbs transcription was correlated with the down-regulation of the downstream energy transfer components: ps ii, cytochrom b6/f complex, ps i and atp synthase. furthermore, key co fixation genes, corresponding to the ribulose bisphosphate carboxylase and the co concentrating mechanism protein ccmk, were down-regulated; soluble electron carrier genes, except cytm, which encodes cytochrome cm, were all consistently repressed upon s removal. notably, no genes in respiratory terminal oxidase and nadh dehydrogenase sub-categories were identified as down-regulated, from both the individual gene analysis and gene set analysis. it may indicate that cytochrome oxidase-mediated respiration activity was not significantly repressed on a transcriptional level during s acclimation. s deprivation elicited transcriptional responses associated with general growth arrest and stress other components of the transcriptional response were associated with general growth arrest and stress. first, down-regulation of both transcription and translation machineries was observed. rna polymerase core subunits genes were significantly repressed. the ribosomal protein subunit genes and genes encoding the initiation factor if-, and two elongation factors were significantly down-regulated. many of these significantly repressed ribosomal protein subunits genes are clustered on the synechocystis chromosome. the transcription of the principle sigma factor siga, encoded by rpod, was significantly induced at 24-hr time-point, and the ecf-type sigma-e factor, encoded by rpoe, showed pronounced late induction. they may be involved in the transcriptional modulation during the transition stage. sige is also involved in the response to n deprivation as well as in positive regulation of sugar catabolism. second, transcriptional adjustment of genes involved in macromolecular biosynthesis and energy metabolism was observed. transcriptional regulation of several genes involved in n assimilation, including glna, sll and sll, may indicate the slowing of ammonium assimilation under s limitation. similarly, ppa, a key pi starvation induced gene, was down-regulated. the repression of pyrophosphatase activity, however, may suggest accumulation of polyphosphate during s deprivation. s deprivation induced a decline in photosynthetic electron transfer, concurrent with down-regulation of pbs and photosynthesis, which were exemplified by reduced abundances of trxa and grxc. in addition, three energy metabolism genes, cbba, cfxe and tkta, were repressed. all three gene activities lead to production of glyceraldehydes 3-phosphate, through glycolysis or non-oxidative branch of pentose phosphate pathways. the effect of this regulation may be to lower the sugar catabolism and preserve carbohydrate storage in the form of glycogen. however, genes for fatty acid and lipid biosynthesis, accd and fabh, were significantly up-regulated, which is interesting with respect to the carbon storage and h production hypothesis. third, stress protection responses were observed. the stress response involved genes encoding chaperon and proteolysis functions, preprotein translocase, a pigment biosynthesis pathway gene, a stationary phase survival protein sure homolog, ribonuclease e, light-repressed protein a homolog, antioxidant protein, oxidative stress protection ahpc/tsa family protein, and two-component response regulator genes. additionally, control of the cell cycle, nucleotide synthesis, dna protection, and modulation of cell envelop permeability were affected by s stress. of note, a large number of genes regulated by s availability encode hypothetical proteins or proteins of unknown function. interestingly, about 30% of those genes were significantly up-regulated during the late stage of s starvation. their involvement, if any, in s acclimation and/or utilization remains to be elucidated. overall, our genome-wide analysis presented a broad view of transcriptional changes accompanying sulfur starvation in cyanobacteria. some strategies unveiled here are also "employed" by cyanobacteria to cope with c, n and pi deficiencies. however, to fully understand how cells sense, transmit and integrate information about s status, it may be necessary to study various mutants affecting s acclimation, including: a mutant of the sll gene, whose chlamydomonas reinhardtii homologue sac is a putative sensor of s levels in the environment and is required for survival of s-deprived chlamydomonas cells in the light; mutants of dspa and sll whose synechococcus orthologues, nbls and nblr, respectively, encode the components of the histindine kinase-response regulator two-component system nbls-nblr, which was shown to be critical for acclimation of synechococcus to high-light and nutrient limitation conditions. it is possible, however, that other two-component signalling systems mediate s acclimation in synechocystis, including those that are regulated by sll of the ompr subfamily and slr of the pata subfamily, which were significantly up-regulated in our experimental condition; mutants of global regulators of c, n and pi metabolism, such as ntca and ndhr, which mediate a "cross-talk" between nutrient-specific responses in the cell. it might also be informative to compare the s acclimation response of cyanobacteria with eukaryotic oxygenic photosynthetic organisms and heterotrophic eubacteria. in green alga chlamydomonas reinhardtii, s deprivation leads to increased accumulation of arylsulfatase, which releases sulfate anions from esterified organic sulfates, allowing cells to access new sulfur stores. in the opportunistic pathogen pseudomonas aeruginosa, s deprivation induces genes encoding sulfatases and sulfonatases for desulfurizing a range of organic sulfur compounds such as sulfate esters and sulfonates. in contrast, the sulfate starvation response in e. coli is dominated by increased expression of the s assimilation cysteine biosynthetic pathway, and propagated into methionine metabolism, synthesis of fe-s clusters, and oxidative stress. these differences, to some extent, may be associated with the special cellular component that only cyanobacteria have – pbs. on one hand, pbs degradation may directly contribute to nutrient scavenging because it constitutes up to 50% of the total cellular protein. on the other hand, the transcriptional shut down of pbs biosynthesis, accompanied by reduced photosynthetic activity, allows nutrient limited cells to survive for relatively long periods of time under stress conditions via a "stand-by" mode of energy metabolism. relevance to biohydrogen production as a secondary metabolic process of significant biotechnological interest, photobiological h production from water by cyanobacteria depends on coordination between photosynthetic electron transport and the catalytic activity of the hydrogenase. the optimal conditions for h production should direct more electrons to hydrogenase than other competing pathways. at the same time, anaerobic conditions are necessary to induce hydrogenase expression and/or activity. we observed that s deprivation significantly increased photoautotrophic h evolution by synechocystis, compared with +s controls. the microarray expression profiling suggested similar underlying principles for h production as observed in the green alga chlamydomonas reinhardtii. s deprivation leads to inactivation of ps ii which performs water splitting. oxygen evolution ceases and it is depleted by respiration, as it was observed that ps ii genes were significantly repressed but genes encoding for nadh dehydrogenase and respiratory terminal oxidase were not. this process leads to anaerobiosis, which in turn could induce hydrogenase activity. s starvation inhibits co fixation and thus removes another important electron sink. furthermore, this process likely leads to decreased sugar catabolism and the accumulation of carbohydrates, which is important for sustained h production in the long run. previous work proposed that s limitation triggers h production by causing a lack of the d protein which is essential for ps ii and needs to be constantly replaced. the microarray data suggested that h evolution was accompanied by a complex and dynamic transcriptional process. however, the question remains whether, and to what extent, the observed complexity of transcriptional regulation is essential for renewable, i.e., without the loss of viability, bioproduction of h by cyanobacteria. the mrna levels of genes encoding activities directly involved in h evolution were specifically focused on by quantitative rt-pcr. synechocystis uses heteropentameric hydrogenases and maturation accessory proteins for h production. none of those genes was identified as significantly differentially affected in microarray experiments. moreover their known transcriptional activator lexa was down-regulated in the conditions we tested. the qrt-pcr assay confirmed that the transcript levels of most of the hydrogenase genes were not significantly increased. this indicates that hydrogenase levels were not limiting at the transcript level. however, hoxe and hoxh were induced more than fold at 1-hr and 3-hr time points as measured by qrt-pcr. there may be other factors involved in the regulation of the hox operon transcription besides lexa. for instance, recently an abrb-like protein was identified as a positive regulator of expression of the bidirectional hydrogenase in synechocystis. however, cells only survive for a limited period of time in s-deprived medium and will eventually die. such a shortcoming may be overcome by designing systems that mimic s deprivation for h production. for example, an inducible nbla gene expression system could be constructed under the control of promoters whose activity can be modulated by nonessential micronutrient inducers, e.g., pete promoter by copper, or by environmental parameters, e.g., groes-groel promoter by heat shock. the cyclic induction and repression of nbla transcription may lead to repeated pbs degradation and synthesis, which in turn cyclically inactivates and activates ps ii. an inducible ps ii gene expression system can potentially generate repeated cycles of carbon storage and h production, allowing for scalable and sustainable h production. a similar idea has been explored in chlamydomonas reinhardtii for triggering h production by means other than macronutrient limitation.  here we reported the study of transcriptional activity of the synechocystis genome in response to s deprivation by using a newly designed microarray of more than protein coding genes of synechocystis sp. pcc the genome-wide expression profiling indicated that s deprivation induced relatively weak, but significant, transcriptional changes. a complex transcriptional change during s acclimation was described in terms of general and specific responses characteristic of macronutrient deficiencies. a large portion of significantly affected genes were the genes of unknown function, highlighting gaps in our understanding of the nature of nutrient adaptation. 
we designed, validated and used synechocystis genome-wide oligonucleotide microarray to study transcriptional activity of the cyanobacterial genome in response to sulfur starvation. we made five main observations: 1) transcriptional changes upon sulfate starvation were relatively moderate, but significant and consistent with growth kinetics; 2) s acquisition genes encoding for a high-affinity sulfate transporter were significantly induced, while decreased transcription of genes for phycobilisome, photosystems i and ii, cytochrome b6/f, and atp synthase indicated reduced light-harvesting and photosynthetic activity; 3) s starvation elicited transcriptional responses associated with general growth arrest and stress; 4) a large number of genes regulated by s availability encode hypothetical proteins or proteins of unknown function; 5) hydrogenase structural and maturation accessory genes were not identified as differentially expressed, even though increased hydrogen evolution was observed. the expression profiles recorded by using this oligonucleotide-based microarray platform revealed that during transition from the condition of plentiful s to s starvation, synechocystis undergoes coordinated transcriptional changes, including changes in gene expression whose products are involved in sensing nutrient limitations and tuning bacterial metabolism. the microarray data were verified by quantitative rt-pcr. characteristic transcriptional responses to changes in the growth environment are expected to provide a scaffold for describing the synechocystis transcriptional regulatory network as well as efficient means for functional annotation of genes in the genome. however, these changes were unlikely due to the across-the-board, non-specific shut down of transcription in a condition of growth arrest. the repression of certain s-related genes was paralleled by activation of genes involved in internal and external s scavenging. the transcriptional profile of the nutrient starvation was dominated by a decrease in abundances of many transcripts.
 the unicellular cyanobacterium synechocystis sp. pcc is a model microbe for studying biochemistry, genetics and molecular biology of photobiological processes. importance of this bacterium in basic and applied research calls for a systematic, genome-wide description of its transcriptional regulatory capacity. characteristic transcriptional responses to changes in the growth environment are expected to provide a scaffold for describing the synechocystis transcriptional regulatory network as well as efficient means for functional annotation of genes in the genome.  we designed, validated and used synechocystis genome-wide oligonucleotide microarray to study transcriptional activity of the cyanobacterial genome in response to sulfur starvation. the microarray data were verified by quantitative rt-pcr. we made five main observations: 1) transcriptional changes upon sulfate starvation were relatively moderate, but significant and consistent with growth kinetics; 2) s acquisition genes encoding for a high-affinity sulfate transporter were significantly induced, while decreased transcription of genes for phycobilisome, photosystems i and ii, cytochrome b6/f, and atp synthase indicated reduced light-harvesting and photosynthetic activity; 3) s starvation elicited transcriptional responses associated with general growth arrest and stress; 4) a large number of genes regulated by s availability encode hypothetical proteins or proteins of unknown function; 5) hydrogenase structural and maturation accessory genes were not identified as differentially expressed, even though increased hydrogen evolution was observed.  the expression profiles recorded by using this oligonucleotide-based microarray platform revealed that during transition from the condition of plentiful s to s starvation, synechocystis undergoes coordinated transcriptional changes, including changes in gene expression whose products are involved in sensing nutrient limitations and tuning bacterial metabolism. the transcriptional profile of the nutrient starvation was dominated by a decrease in abundances of many transcripts. however, these changes were unlikely due to the across-the-board, non-specific shut down of transcription in a condition of growth arrest. down-regulation of transcripts encoding proteins whose function depends on a cellular s status indicated that the observed repression has a specific regulatory component. the repression of certain s-related genes was paralleled by activation of genes involved in internal and external s scavenging. 