BACKGROUND
saprophytic soil bacteria from the genus streptomyces produce a large number of secondary metabolites and extracellular enzymes .
streptomycetes are ecologically important in carbon recycling and constitute the largest genus of actinobacteria, a phylum including more than  <dig> species  <cit> .
in general, free-living bacteria must adapt to constantly changing environmental conditions.
therefore, they have developed mechanisms for finely modulate metabolism and growth.
one of the most remarkable control mechanisms is carbon catabolite repression .
this mechanism guarantees the sequential utilization of carbon sources when more than one is simultaneously present in the culture media.
even when the outcome effect of ccr in different bacteria is the same, their essential mechanisms can be quite different.
in gram-negative bacteria, ccr relies on the carbohydrate translocation phosphoenolpyruvate-dependent phosphotransferase system .
for escherichia coli and other gram-negative bacteria, the main control system is camp-receptor protein , whose activity is related to the levels of the phosphorylated pts enzyme eiiglc.
on the other hand, in some low guanine-cytosine gram-positive bacteria like bacillus subtilis, the control is exerted by the catabolite control protein , which is related to the phosphorylation levels of the pts protein hpr-his  <cit> .
in the streptomyces genus , glucose also exerts an inhibitory effect on the expression of genes involved in the uptake and utilization of alternative carbon sources .
but, in contrast with other bacteria in streptomyces glucose is not transported by the pts system, but via the major facilitator system , glcp  <cit> .
besides, orthologous crp  does not seem to play a significant role in ccr in these genera  <cit> .
after the observation that mutants of streptomyces coelicolor and other streptomycetes resistant to the non-utilizable glucose analog, 2-deoxyglucose , lack glucose kinase  activity and lose sensitivity to ccr, a key role was ascribed to glk in the ccr mechanism  <cit> .
furthermore, when glk activity and consequently, the glycolytic flux is restored by transformation in the s. coelicolor mutant with a heterologous non-related glk from zymomonas mobilis, its growth in glucose is recovered, but not its sensitivity to ccr  <cit> , suggesting a dual function of glk.
however, attempts to separate the phosphorylation activity from the regulatory function have failed  <cit> .
based on their primary structure, microbial glks are classified into three families  <cit> .
s. coelicolor glk belongs to the family iii, which contains a rok signature .
however, in contrast to the transcriptional repressors of this family, these kinases lack dna binding domains.
the only presumed interaction reported for glk is with the glucose transporter glcp  <cit> .
using a high-density microarray approach, in the present work the ccr paradigmatic model proposed by angell et al.
<cit>  for s. coelicolor was explored.
for this purpose, the transcriptomic profile of a glk null mutant, complemented with a heterologous glk gene was analyzed and compared to the wild-type scom <dig> strain.
in addition, this transcriptomic study was extended to evaluate the glucose effect under repressive and non-repressive conditions.
RESULTS
deletion of glk, growth and glucose utilization
for several years, studies to understand ccr and carbon regulation in streptomyces have depended on the isolation of spontaneous mutants resistant to the glucose analog, 2-deoxyglucose  <cit> .
although some of these mutations relied on the glk gene, the full effect of the mutagenic treatment remains unknown.
therefore, to isolate the “glk” effect, the glk gene  was deleted in the s. coelicolor m <dig> wild-type generating the sco∆glk strain.
in addition, the z. mobilis glk gen was cloned into the plasmid pij <dig>  generating the plasmid punamzm.
the plasmids pij <dig> and punamzm were independently transformed into the sco∆glk mutant generating the sco <dig> and scozm strains.
a qualitative in vivo visualization of glk activity using bpg agar medium displayed a clear glk activity in strains scom <dig> and scozm while sco <dig> was unable to grow in this medium .
the capacity of z. mobilis glk to reestablish growth of the Δglk mutant was quantitatively confirmed measuring the specific growth rate, μ =  <dig>  h− <dig> for scom <dig> and μ =  <dig>  h− <dig> for scozm.
next, the complemented mutant scozm was used in the transcriptomic assays.
global transcriptomic analysis
as introduced above, even when ccr has been largely studied in s. coelicolor, neither the glk protein role nor its precise regulatory mechanism has been elucidated.
therefore, to evaluate the effect of scglk in ccr, the gene expression of scom <dig> and the scozm, were compared under repressed conditions at the exponential growth phase, .
additionally, to evaluate the effect of glucose on gene expression, a global transcriptomic analysis   <cit>  was performed using a classical glucose-repressive condition over agar utilization, in exponential growing phase cultures using a classical glucose repressive condition over agar utilization  <cit> .
thus, the transcriptomic profile of scom <dig> was compared between repressive    .
additionally, to evaluate the scglk participation in ccr, the gene expression of scom <dig> and the scozm, were compared under repressive conditions at the exponential growth phase .
gta was applied to four independent biological replicates using the 104 k microarray from oxford gene technology ltd .fig.
<dig> scheme showing the number of differentially expressed genes in comparisons between strains and carbon sources.
orientation of the longer arrows shows the comparison between both conditions.
differential transcription values were obtained by rank product analysis at a pfp cutoff ≤  <dig>  value.
the small arrows up or down oriented indicate the number of genes up or down-expressed in each condition, respectively.
a
scom <dig> strain grown under repressive conditions.
b
scom <dig> strain grown under non-repressive conditions.
c
scozm mutant grown under repressive conditions
average expression data were compared, using the rank products analysis method .
for all expression data, the rpa method calculated a pfp value.
those genes with a pfp value ≈  <dig>  have the highest probability of biological relevance  <cit> .
differentially expressed  genes were identified as having a pfp value less than or equal to  <dig> , equal to a false discovery rate of approximately 15 %, as previously reported  <cit> .
this statistical analysis involving four biological replicates resulted in the identification of  <dig> up-regulated and  <dig> down-regulated genes in the glc/agar comparison and,  <dig> up-regulated and  <dig> down-regulated genes in the scom145/scozm comparison.
in total,  <dig> genes were differentially expressed between both comparisons .fig.
<dig>
a classification based on go biological processes for glk/agar and m145/scozm comparisons.
in all cases the most representatives go are those corresponding to metabolic processes, transport and regulation of biological processes.
b classification of genes coding for enzymes included in the go metabolic processes
the list of differentially expressed genes was submitted to the protein center  to group broadly them on the basis of its go-biological function term  .
half of the annotated genes were related to the go metabolic processes in both comparisons.
the go term “metabolic process”, includes anabolic and catabolic chemical reactions and pathways.
looking closer inside the go metabolic process, genes encoding enzymes were also classified.
a considerable number of oxidoreductases, hydrolases and transferases were repressed by glucose in glc/agar comparison .
comparison between scom <dig> and the scozm mutant revealed that the presence of a glk protein, but not its enzyme activity, stimulated or repressed mostly the expression of oxidoreductases and transferases .
considering their possible role in signal transduction, we have also looked for potential sensor kinases in this group.
for the glc/agar comparison, regarding to agar, the sensor kinase gene sco <dig>  was down-regulated by glucose .
in the scom14/scozm comparison, the putative sensor kinase gene sco <dig>  was up-regulated .
metabolic processes and catalysis
as previously mentioned glucose is mostly transported inside the cell by the glcp transporter, most probably bound to scglk  <cit> .
in this hypothesis, the glucose is transported and phosphorylated in a single step .
in the glc/agar comparison, the genes glcp <dig> , and glcp <dig>  showed the highest stimulation exerted by glucose  .
the transcriptional regulation of glcp was dependent on the presence of glucose but scglk independent since in the scom145/scozm comparison no changes were detected for glc transporters.
previous reports have demonstrated a constitutive expression of scglk  <cit> .
in agreement with this result, we did not observe differential expression of scglk in the glc/agar comparison.
as expected, this difference was detected when comparing m145/scozm due to scglk deletion.
no other glucokinases were found being regulated by glucose or scglk.
once glucose-6-phosphate is formed, it can enter to central carbon catabolic pathways .fig.
<dig> profile of glucose dependent genes involved in both, glycolysis  and pentose phosphate pathways .
a schematic representation of the central carbon metabolism pathways in s. coelicolor, in red are genes stimulated and in blue are genes repressed.
b the comparison between conditions is indicated as ratios of glc/agar.
results are given as log <dig> ratios, thus a positive number indicates up-regulation and a negative number down-regulation, respectively
fig.
<dig> selected genes encoding transporters and their expression profiles depending upon glucose and glk presence.
a glc/agar comparison reflects the glucose effect  and the m145/scozm comparison shows glk dependence .
the comparison between conditions is indicated as ratio of glc/agar and scom145/scozm.
results are given as log <dig> ratio, thus a positive number indicates up-regulation and a negative down-regulation, respectively.
b putative products of regulated genes are shown.
genes that showed the same pattern of repression/stimulation across glc/agar and m145/scozm comparisons, are indicated in bold.
genes that exhibit an opposite pattern of stimulation or repression between comparisons are underlined
glycolysis and gluconeogenesis
glycolysis or the embden-meyerhof-parnas pathway  usually consists of ten reactions in which glucose is degraded to pyruvate .
this pathway is present in all domains of life and hence is considered one of the most ancient pathways for sugar degradation.
the expression of eight glycolytic steps involved in glucose degradation was up-regulated by glucose .
this differential expression was not observed in the scom145/scozm comparison, suggesting that glycolysis proceeded more actively when glucose was present , but it was not affected by the absence of endogenous scglk .
in detail, two of the three isoforms of the glyceraldehyde-3-phosphate dehydrogenase genes  showed induction by glucose while sco <dig>  was glucose repressed.
expression of the sco <dig> gene encoding the gluconeogenic phosphoenolpyruvate carboxykinasewas glucose repressed  .
the genes with greater changes were those encoding a pyruvate kinase isoform  <dig>   and phosphoenolpyruvate carboxykinase  , which were strongly induced and repressed by this carbohydrate, respectively .
the large changes in the expression of these genes reflected their importance as regulatory points for the glucose/gluconeogenesis pathways.
it is generally known that enzymes of metabolic pathways catalyzing irreversible steps are potential sites for regulatory controls.
pyk <dig> catalyzes the transfer of phosphate from phosphoenolpyruvate to adp, producing pyruvate and atp.
this reaction has a large negative free energy change and represents the third regulated and irreversible enzyme of glycolysis.
the strong repression of glucose over the pepck gene whose product is involved in the conversion of oxalacetate to phosphoenolpyruvate, may reflect the strict control exerted by this carbohydrate, as this enzyme catalyzes the rate-controlling step of gluconeogenesis, and likely is the first committed step in all organisms  <cit> .
in b. subtilis, pepck is also reported to operate in reverse direction  <cit>  and might function in the anaplerotic node linking tca cycle and glycolysis/gluconeogenesis.
the tricarboxylic acid cycle
the tricarboxylic acid cycle  is the final common pathway for molecules from which energy is generated.
molecules enter tca as acetyl-coa and leave as co <dig>  in the glc/agar comparison, just the putative succinate dehydrogenase complex encoded by sco0922-sco <dig> was induced, while in the tested conditions, the expression of other genes coding or enzymes of the tca, remained unchanged, suggesting that the tca cycle may be regulated mostly by the metabolic intermediates rather than by transcriptional regulation .
proteomic studies have previously suggested that the tca enzymes are not regulated by protein expression  <cit>  .
pentose phosphate pathway
this pathway, also known as the phosphogluconate pathway or hexose monophosphate shunt, is another way to metabolize carbon sources like glucose, generating reducing power in the form of nadph and c <dig> carbohydrates .
the ribulose-5-phosphate is an important precursor of atp, coa, nad+, fad+, dna and rna production.
in s. coelicolor, there are two major clusters encoding enzymes involved in the pentose phosphate pathway .
in the first cluster, sco1935-sco <dig>  neither the presence of glucose nor the scglk have an effect on its expression level.
the second cluster, sco6658-sco <dig>  was entirely stimulated by glucose , and of these, the most noticeable change observed was in the sco <dig> gene, encoding a transaldolase with a log2 =  <dig>  .
it has been proposed that this cluster may be involved in providing nadph for secondary metabolite production  <cit> .
in concordance with the reported capacity of glk from z. mobilis to reestablish the glycolytic flux  <cit> , no changes were observed in the transcriptomic data of glycolysis, ppp, tca cycle or gluconeogenesis in the scom145/scozm comparison.
therefore, regarding central carbon metabolism the glycolysis and ppp were highly activated only by glucose.
enzymes in the tca cycle practically did not change, while gluconeogenesis was inhibited by glucose, supporting a preferential channeling of glucose to form essential building blocks.
in this regard, a good correlation between our transcriptomic data and previously reported proteomics  <cit>  data was observed.
additionally, a putative gluconokinase  coding gene  was also stimulated by glucose in the glc/agar comparison, while no changes were detected in the scom145/scozm comparison.
the finding of up-regulation in the glnk encoding gene sco <dig> , correlated well with a glucose positive regulation of sco <dig> , encoding a gluconate transporter .
in streptomyces lividans, the consumption of gluconate as a sole carbon source has been reported by  <cit> .
however, to our knowledge, no studies on the regulation of the gluconate catabolic genes in s. coelicolor have been previously performed.
in several genera of bacteria such as pseudomonas, gluconobacter, acetobacter, and various fungi, gluconate is produced from glucose through a simple dehydrogenation reaction catalyzed by a glucose oxidase  <cit> .
in general, bacterial growth on gluconate requires two specific enzymes: a gluconate permease  for extracellular gluconate incorporation and a gluconate kinase , which phosphorylates gluconate to 6-phosphogluconate  <cit> .
in streptomyces, gluconate can eventually be degraded by ppp to generate reducing power, needed for secondary metabolite biosynthesis  <cit> .
in agreement with this observation, glucose activation of this ppp branch  was in synchrony with the over-expression of both sco <dig> and sco <dig> genes  .
ccr at the operon-specific mechanisms
in many bacteria ccr may operate at operon-specific mechanisms by both a) inducer exclusion, i.e., prevention of the internalization of non-preferred carbon sources to thwart induction of their catabolic operons, and b) induction prevention, i.e., control of the activity of operon-specific transcription factors  <cit> .
a) in the case of induction exclusion, approximately 10 % of the down or up-regulated genes from each transcriptome were implicated in transport functions .
glucose repression was detected over the maltose transporter malg and malf genes  and the putative operon sco3482-sco <dig>  were repressed .
it is noteworthy mentioning that only a limited number of sugar transporter genes were detected in this study, probably due to the lack of specific inducers in the system.
interestingly, the xylose transporter,  showed 10-fold stimulation, just after the glcp transporter .
this oddly up-regulation of the xylose transporter was also accompanied by changes in its transcriptional regulator rok7b <dig>  .
recently, it has been shown that the protein rok7b <dig> is also involved in s. coelicolor ccr  <cit> , but its function in this phenomenon remains unclear.
the genes involved in glutamate incorporation , encoding glud, gluc, glub and glua, respectively), were strongly stimulated  by glucose .
it was not surprising to find that glucose did not exert a repressive effect over the glutamate transporter since this amino acid is a preferred carbon source, even over glucose  <cit> .
besides, in the scom145/scozm comparison, this transporter was also up-regulated in scozm when compared to scom <dig>  however, it is unclear why and how glucose stimulates the glutamate permease and how is the scglk effect mediated.
likely, this is the result of transcriptional changes in the arginine repressor, which will be discussed later.
for induction prevention, it is well known that glucose represses glycerol kinase  production  <cit>  by negatively influencing transcription of the glycerol-inducible glpfkdx operon  <cit> .
this operon comprises genes for a putative glycerol transporter, a glycerol kinase, a glycerol-3-phosphate dehydrogenase, and a gene of unknown function.
in the glc/agar comparison, the glycerol kinase encoded by sco <dig>  was repressed while the glycerol operon repressor protein  was up-regulated by glucose .
in addition, a transcriptional repression of glpk was observed, confirming the findings of hindle and smith  <cit>  and supporting that the gylr effect is due to an operon specific mechanism rather than a pleiotropic one.
interestingly, no transcriptional changes were detected in this operon when comparing the scom <dig> with scozm, suggesting a scglk-independent repression of glpk or induction of gylr.
since many operons, specific ccr mechanisms require the presence of inductor we choose a classical and widely model to evaluate the effect of glucose and scglk over the utilization of an alternative carbon source looking for transcriptional changes in agar metabolism.
utilization of an alternative carbon source
agar metabolism
agar utilization is one of the most widely used tests to assess for ccr.
agar is constituted by a family of complex polysaccharides found in the cell wall of some red algae cells.
primarily, this polymer consists of agarobiose  and agaropectin.
agarobiose is formed of linear chains of alternating residues of 3-o-linked β-d-galactopyranose and 4-o-linked  <dig>  6-anhydro-α-l-galactose  while agaropectin seems to be formed by sulfate esters, pyruvate acetal and methyl esters, but its exact structure remains obscure  <cit> .
the genomic context of a cluster involved in agar degradation is presented in fig.
5a  <cit> .
complete agar hydrolysis produces monomeric sugars such as d-galactose , la, and l-galactose-6-sulfate   <cit> .
although used as a model to measure ccr, the s. coelicolor agarolytic pathway has not been widely studied.
the first enzyme described in this pathway was an endo-type agarase that degrades agar into neoagarotetraose and neoagarohexaose, β-agarase a, daga .
its transcription, controlled by four different promoters, is induced by agar but repressed by glucose  <cit> .
a second agarase , was recently identified.
this enzyme  is an exo and endo-type β-agarase that degrades agarose, neoagarotetraose and neoagarohexaose into neoagarobiose  <cit> .
in the proposed agarolytic pathway daga and dagb can hydrolyze agarose into neoagarobiose in a cooperative way  <cit> .
likely, neoagarobiose is transported into the cell by an unknown transporter and once inside the cell, a cytosol glycoside hydrolase  hydrolyze neoagarobiose into gal and la  <cit> .
the reconstructed agarolytic pathway proposed by chi et al.
<cit>  is summarized in fig.
5b.fig.
<dig>
a genomic context of a cluster possible involved in agar degradation.
b the agar metabolism.
c average expression changes observed among experimental conditions.
glc/agar comparison reflects the glucose effect and m145/scozm comparison shows the glk dependence .
the comparison between these conditions is indicated as ratios of glc/agar  and scom145/scozm .
results are given as the log <dig> ratio, thus a positive number indicates up-regulation and a negative number down-regulation, respectively
as expected, the genes encoding for agarases , were repressed by glucose in glc/agar comparison.
this effect was observed in a large genomic region , except for the sco <dig> that encodes for a laci-transcriptional regulator .
this region  contains nine possible transcription units , although the function of only three of them has been described .
previous attempts to find the transcriptional regulator of daga have failed  <cit> .
however, in our tested conditions, no changes were detected in the only gene encoding a putative transcriptional regulator  present in this agar degradation cluster.
the functions of this putative regulator are unknown, but apparently it is not transcriptionally regulated by glucose.
in the scom145/scozm comparison, genes from the agar degradation cluster showed higher repression in the scom <dig> strain compared to scozm , particularly that of sco <dig> which codes for dagb  implying, as previously reported, a repressive effect dependent on scglk per se.
it is hard to establish the mechanism by which the scglk protein itself could exert a regulatory function.
as mentioned before, at the central carbon metabolism , and hence the metabolites produced, operates in a similar way when endogenous scglk is present and when is substituted by a non-related glk.
so the repression, at least that of agar metabolism, most be not dependent on the glycolytic flux, as previously proposed  <cit> , but dependent on the expression of dna binding proteins and its interactions with scglk.
it is becoming evident that besides the specific operon regulators, the presence of other pleiotropic or general regulators such as the recently described rok7b <dig>  <cit>  may be important in understanding the ccr in streptomyces and also in establishing the scglk effect.
in the next section, the transcriptional changes in genes coding for putative transcriptional regulators responsive to glucose and scglk are described.
interestingly, in this study, a possible transporter associated with neoagarobiose incorporation , was found .
since this chromosomal region is implicated in agar metabolism, genes sco3482- <dig> showed the same expression pattern observed for agarases and genes in this region seemed to respond to a common regulation.
the gene products of sco <dig> and sco <dig> have  <dig> transmembrane domains  and are therefore predicted to be membrane proteins.
on the other hand, sco <dig> does not have any tm and, therefore, it is predicted to be the sugar binding protein .
likely sco <dig> is the transporter associated with neoagarabiose internalization.
regarding to agar utilization, this is the first report describing the genes and transcriptomic changes in a cluster involved in agar metabolism.fig.
<dig> genes and its predicted products involved in cpk synthesis  .
on the right, transcriptomic expression of the cpk genes.
glc/agar comparison  reflects the glucose effect on cpk production and the m145/scozm comparison  shows its glk dependence.
comparisons between these conditions are indicated as ratios of glc/agar  and scom145/scozm .
results are given as log <dig> ratio, thus positive numbers indicate up-regulation and negative numbers down-regulation, respectively
ccr and transcription regulators
prokaryotic cells can adapt and respond to environmental conditions by modifying their genome expression pattern; therefore, as previously proposed, describing the transcriptional dynamics mediated by regulatory proteins could be an important clue to unraveling ccr mechanisms.
in model organisms, transcriptional regulators operate both global and operon-specific mechanisms.
in our study, around 15 % of all differentially expressed genes in glc/agar and scom145/scozm comparisons, comprised go regulation of metabolic processes and stimuli responses .
transcriptional factors stimulated by glucose were found among the merr, laci, tetr, gntr, marr and asnc families, as well as sigma factors .
on the other hand, transcriptional factors repressed by glucose included members of the gntr, marr, and deor families, but also sigma and anti-sigma factors .
down-regulated transcriptional regulators with known functions included: the rna polymerase sigma factor sigu , the anti-sigma factor rsua, the sporulation transcription factor whih, as well as the rna polymerase sigma factor bldn.
up-regulated transcriptional regulators were: the regulatory protein , the glycerol operon regulatory protein , and the regulatory protein rok7b <dig> previously discussed.table  <dig> transcriptional regulators differentially expressed in the glc/agar comparison
sco1541
sco0140
sco1699
sco2489
sco2209
sco2935
sco3134
sco3361
sco3933
sco3423
sco3986
sco3810
sco4122
sco3943
sco4412
sco4020
sco4677
sco4158
sco4920
sco4640
sco5785
sco4850
sco5811
sco5413
sco5819
sco5552
sco6162
sco5982
sco6992
sco7424
sco7727
sco1658
sco2954
sco2950
sco2953
sco4159
sco5819
sco4762
sco3323
sco6008
the transcriptional factors activated by glucose can be divided into three groups: a) pleiotropic regulators, b) regulators of specific operons and c) dna-associated proteins.
regarding pleiotropic regulators, the glnr regulator functions as the main nitrogen regulator  <cit> , but it is also involved in different metabolic processes such as carbon metabolism, synthesis of catabolic enzymes, and secondary metabolism  <cit> .
the recently described regulator rok7b <dig>  was activated by glucose in the scom <dig> background, following the same activation pattern as the xylfgh operon.
likely, rok7b <dig> may act as a positive regulator.
thus, in addition to regulating the xylose operon, rok7b <dig> activates morphological development and actinorhodine production  <cit> .
on the contrary, this regulator blocked the calcium dependent antibiotic and prodigiosin formation.
rok7b <dig> is also known to be involved in the ccr process  <cit> .
regarding regulators of specific operons, the only specific regulator gene observed with differential expression was the gylr gene .
this regulator was stimulated by glucose and in agreement with this effect, the glycerol kinase gene  was repressed.
on dna associated proteins, hupa expression was stimulated by glucose.
hupa is a protein found in vegetative mycelium growing in liquid culture  <cit>  and involved in gene expression, dna protection  and nucleoid structuring.
additionally, transcription of the chaperonine groel <dig> was also stimulated by glucose and was heat shock activated  <cit> .
an interesting regulator with an unknown function is sco <dig>  a probable member of the luxr family , which showed more than 20-fold down-regulation as well as its cognate kinase .
markedly, the divergent gene sco <dig> , exhibited the highest glucose repression .
in the scom145/scozm comparison, the four genes encoding regulatory proteins that showed stimulation belonged to the gntr , luxr families  and a transcriptional activator  .
the two regulators belonging to the gntr family, the sco <dig>  and sco <dig>  were highly overexpressed .
remarkably, none of these up-regulated transcriptional factors have been previously described.
the in silico prediction of the putative targets of sco <dig> has revealed a large set of sensitive genes involved in primary and secondary metabolism.
actually, in a s. coelicolor ∆sco <dig> mutant constructed in our lab, a premature and high-level production of secondary metabolites was observed .
on the other side, in the group of transcriptional down-expressed regulators, the arginine metabolism and its regulator argr , deserve special attention   <cit> .
in a recent transcriptomic and proteomic comparison between scom <dig> and its ∆argr mutant, the versatile functions of this regulator were demonstrated  <cit> .
beyond its regulatory role in the arginine and pyrimidine biosynthesis, argr was also able to regulate some different transcriptional factors from different families such as deor, arac and gntr  <cit> .
therefore, stimulation or repression of argr affected secondary metabolite production, morphology and synthesis of proteases and peptidases.
the versatile functions of this regulator and its higher expression in the absence of scglk suggest a complex regulatory network.
to make this panorama more complex, when scom145/scozm were compared, none of the differentially expressed genes of the putative transcriptional regulators seemed to be the transcriptional regulators found by pérez-redondo  <cit> .table  <dig> differentially expressed genes found in the scom145/scozm comparison
sco0016
sco0138
sco0555
sco0556
sco1570
sco1576
sco1577
sco1578
sco1579
sco1580
sco1815
sco2126
sco2296
sco2513
sco2930
sco2986
sco3138
sco3411
sco3413
sco3985
sco4173
sco4174
sco4175
sco4317
sco4903
sco4950
sco5025
sco5026
sco5367
sco5536
sco5839
sco5976
sco6266
sco6268
sco6288
sco7036
sco7262
sco7530
sco7586
sco7587
sco7698
role of scglk in carbon catabolite repression
as previously mentioned, the transcriptional response to glucose  included  <dig> differentially expressed genes while in the scglk response   <dig> differentially expressed genes were identified.
from the total of differentially expressed genes in scom145/scozm comparison,  <dig> were included in the glc/agar comparison .
this result conferred only a limited regulatory function to this enzyme in ccr, since specifically affected only  <dig> out of a total of  <dig> genes.
this contrasted with previous results, which proposed a significant role of glk in ccr  <cit> .
as seen in additional file 1: table s <dig> several of this shared genes were involved in transport functions  and agar metabolism .
concerning transport functions, these included genes encoding abc transporters , cation transporters like sco3717-sco <dig>  amino acid transporters as the glutamate permease  and also the transporter putatively associated with neoagarabiose internalization .
the genes encoding the abc transporter  were stimulated by glucose in the wild-type strain, but repressed in the scom145/scozm comparison.
as mentioned previously, the genes involved in agar metabolism were repressed by glucose and also were affected by scglk.
aside from primary metabolic pathways and transport systems, significant transcriptional changes in the yellow cryptic polyketide  cluster was also observed in both transcriptomes, and, therefore, deserved special mention.
the repressive effects exerted by glucose on differentiation and secondary metabolite production are well known .
however, in our experimental conditions, relative to agar, the cpk cluster was up-regulated by glucose.
previously, pawlick et al.
<cit>  reported that the presumptive product of the cpk polyketide synthase is a yellow compound secreted into the medium.
its production depended on the medium composition, inoculum density and required the absence of glucose.
in parallel, gottelt et al.
<cit>  reported in s. coelicolor that cpk production is enhanced by glutamate supplementation.
recently, in a s. coelicolor engineered strain, the product of this cluster has been named coelimycin p <dig> and suggested to be a glutamate adduct  <cit> .
in the glc/agar comparison, glucose exerted a stimulatory effect on the genes sco6272-sco <dig>  and sco6282-sco <dig>  .
additionally, in the scom145/scozm comparison, sco <dig>  and sco <dig> , also showed increased expression in the scom <dig> strain, relative to the scozm mutant and this effect was not observed in the glc/agar comparison.
although pawlick et al.
<cit>  previously reported that glucose exerts a negative effect on the synthesis of ycpk, in our experimental conditions this carbohydrate exerted an up-regulation on the cpk genes.
interestingly, in the absence of scglk a reduced expression of cpk genes was observed in scozm, relative to scom <dig>  in the model proposed by gubbens et al.
<cit> , cpk synthesis is activated by glucose + mannitol and repressed by glucose + fructose.
it would be interesting to test the scozm in glucose + fructose or any other carbon source combinations to evaluate their effect on the cpk cluster expression.
as gubbens et al.
<cit>  anticipated for the cpk secondary metabolite production, the influence of glk likely involves a complex regulatory mechanism, also dependent on nutritional signals.
from the proposed genes involved in the cpk synthesis, the only one that showed a down-regulation in scom <dig>  relative to scozm, was sco <dig>  which encodes a butenolide synthase .
additionally, in the scom145/scozm comparison, sco <dig>  and sco <dig> , also showed increased expression in the scom <dig> strain, relative to the scozm mutant and this effect was not observed in the glc/agar comparison.
depending on the used carbon source, proteomic studies have also reported the differential expression of proteins of this cluster  <cit> .
regarding the genes that were exclusively found in the scom145/scozm comparison , as previously discussed, the gene encoding the versatile regulator argr  was down-regulated in the scom <dig> strain , when compared to scozm.
it is interesting to note that the absence of scglk  somehow generates a higher transcription of the gene encoding the regulator argr in the scozm strain, relative to scom <dig>  in agreement with the up-regulation of argr genes, the arginine biosynthetic pathway showed down-regulation on the mutant or, in other words, the arginine biosynthetic genes were up-regulated in the scozm when compared scom <dig> .
rt-pcr validation of selected genes up- or down-regulated by glucose or scglk
microarray data were validated by rt-qpcr utilizing glucose-stimulated  and repressed  genes in glc/agar comparison.
to look for suitable reference genes, the sigma factor gene hrdb  <cit> , the beta chain from dna polymerase iii , the dna gyrase subunit a gene , the recombinase a gene  and the dna-directed rna polymerase alpha chain   <cit>  were utilized.
to select the most stable reference genes, the bestkeeper program was utilized  <cit> .
this program calculates a bestkeeper index, which is the geometric mean of all housekeeping raw ct values.
pearson correlations between individual genes and the bestkeeper index were calculated and reported as the bestkeeper correlation coefficient.
genes with the highest correlation coefficient were considered the most stably expressed.
in our conditions, reca, rpoa, and gyra were selected as reference genes due to their high bestkeeper index and low standard deviation .
the geometrically averaged reference genes were used to normalize the following target genes: daga, glcp, rok7b <dig>  sco <dig> and sco <dig>  the relative transcript profile  of the target genes corresponded well to the microarray analysis, confirming our results on glucose regulation.
also, glcp and rok7b <dig> were largely induced by glucose, while expression of daga, sco <dig> and sco <dig>  were largely repressed.
their neighboring genes  also were down-regulated by glucose.
the sco <dig> and its cognate kinase sco <dig> constitute a two-component system.
the response regulator  belongs to the luxr family of transcriptional regulators and is conserved in many streptomyces species .
its domain architecture is present in many activators.
the two component systems are important to sense and respond to the environment.
a great unexplored area in streptomyces ccr is the potential role of two-component systems.
in other soil living or environmental strains such pseudomonas, sinorhizobium and bacillus, two-component systems are important in ccr control .
CONCLUSIONS
glucose can elicit significant metabolic and transcriptional changes in s. coelicolor.
whereas glucose influx and its flow through glycolysis and pentose phosphate pathway were stimulated, gluconeogenesis was inhibited.
glucose also triggered carbon catabolic repression by repressing a number of transporter systems and repressing transcription of enzymes needed for the utilization of secondary carbon sources.
additionally,  <dig> dna binding proteins, including  <dig> transcriptional regulators and four two component systems were regulated by glucose and probably were involved in the signals and the effects elicited by this carbon source.
the scglk enzyme per se was required for the glucose repression, at least in the agar model proposed by angel et al.
<cit> .
in this regard, our results confirm and update the agar model of ccr in streptomyces and its dependence on scglk per se.
nevertheless, the expected regulatory function was not as global as thought before, but likely may be accompanied or coordinated by other transcriptional regulators.
the remaining transcription factors, whose expression was affected by glucose or scglk with unknown functions, are an interesting area to describe in the s. coelicolor ccr.
currently, we are constructing regulatory networks  to find out the possible regulatory relations between genes regulated by glucose and by scglk.
understanding the functions of the glucose-targeted transcriptional regulators and their transcriptional network may lead to unraveling one of the most fundamental regulatory mechanisms in the streptomyces metabolism.

 saprophytic soil bacteria from the genus streptomyces produce a large number of secondary metabolites and extracellular enzymes. streptomycetes are ecologically important in carbon recycling and constitute the largest genus of actinobacteria, a phylum including more than species. in general, free-living bacteria must adapt to constantly changing environmental conditions. therefore, they have developed mechanisms for finely modulate metabolism and growth. one of the most remarkable control mechanisms is carbon catabolite repression. this mechanism guarantees the sequential utilization of carbon sources when more than one is simultaneously present in the culture media. even when the outcome effect of ccr in different bacteria is the same, their essential mechanisms can be quite different. in gram-negative bacteria, ccr relies on the carbohydrate translocation phosphoenolpyruvate-dependent phosphotransferase system. for escherichia coli and other gram-negative bacteria, the main control system is camp-receptor protein, whose activity is related to the levels of the phosphorylated pts enzyme eiiglc. on the other hand, in some low guanine-cytosine gram-positive bacteria like bacillus subtilis, the control is exerted by the catabolite control protein, which is related to the phosphorylation levels of the pts protein hpr-his. in the streptomyces genus, glucose also exerts an inhibitory effect on the expression of genes involved in the uptake and utilization of alternative carbon sources. but, in contrast with other bacteria in streptomyces glucose is not transported by the pts system, but via the major facilitator system, glcp. besides, orthologous crp does not seem to play a significant role in ccr in these genera. after the observation that mutants of streptomyces coelicolor and other streptomycetes resistant to the non-utilizable glucose analog, 2-deoxyglucose, lack glucose kinase activity and lose sensitivity to ccr, a key role was ascribed to glk in the ccr mechanism. furthermore, when glk activity and consequently, the glycolytic flux is restored by transformation in the s. coelicolor mutant with a heterologous non-related glk from zymomonas mobilis, its growth in glucose is recovered, but not its sensitivity to ccr, suggesting a dual function of glk. however, attempts to separate the phosphorylation activity from the regulatory function have failed. based on their primary structure, microbial glks are classified into three families. s. coelicolor glk belongs to the family iii, which contains a rok signature. however, in contrast to the transcriptional repressors of this family, these kinases lack dna binding domains. the only presumed interaction reported for glk is with the glucose transporter glcp. using a high-density microarray approach, in the present work the ccr paradigmatic model proposed by angell et al. for s. coelicolor was explored. for this purpose, the transcriptomic profile of a glk null mutant, complemented with a heterologous glk gene was analyzed and compared to the wild-type scom strain. in addition, this transcriptomic study was extended to evaluate the glucose effect under repressive and non-repressive conditions.  deletion of glk, growth and glucose utilization for several years, studies to understand ccr and carbon regulation in streptomyces have depended on the isolation of spontaneous mutants resistant to the glucose analog, 2-deoxyglucose. although some of these mutations relied on the glk gene, the full effect of the mutagenic treatment remains unknown. therefore, to isolate the “glk” effect, the glk gene was deleted in the s. coelicolor m wild-type generating the sco∆glk strain. in addition, the z. mobilis glk gen was cloned into the plasmid pij generating the plasmid punamzm. the plasmids pij and punamzm were independently transformed into the sco∆glk mutant generating the sco and scozm strains. a qualitative in vivo visualization of glk activity using bpg agar medium displayed a clear glk activity in strains scom and scozm while sco was unable to grow in this medium. the capacity of z. mobilis glk to reestablish growth of the Δglk mutant was quantitatively confirmed measuring the specific growth rate, μ =   h− for scom and μ =   h− for scozm. next, the complemented mutant scozm was used in the transcriptomic assays. global transcriptomic analysis as introduced above, even when ccr has been largely studied in s. coelicolor, neither the glk protein role nor its precise regulatory mechanism has been elucidated. therefore, to evaluate the effect of scglk in ccr, the gene expression of scom and the scozm, were compared under repressed conditions at the exponential growth phase,. additionally, to evaluate the effect of glucose on gene expression, a global transcriptomic analysis was performed using a classical glucose-repressive condition over agar utilization, in exponential growing phase cultures using a classical glucose repressive condition over agar utilization. thus, the transcriptomic profile of scom was compared between repressive. additionally, to evaluate the scglk participation in ccr, the gene expression of scom and the scozm, were compared under repressive conditions at the exponential growth phase. gta was applied to four independent biological replicates using the 104 k microarray from oxford gene technology ltd.fig. scheme showing the number of differentially expressed genes in comparisons between strains and carbon sources. orientation of the longer arrows shows the comparison between both conditions. differential transcription values were obtained by rank product analysis at a pfp cutoff ≤  value. the small arrows up or down oriented indicate the number of genes up or down-expressed in each condition, respectively. a scom strain grown under repressive conditions. b scom strain grown under non-repressive conditions. c scozm mutant grown under repressive conditions average expression data were compared, using the rank products analysis method. for all expression data, the rpa method calculated a pfp value. those genes with a pfp value ≈  have the highest probability of biological relevance. differentially expressed genes were identified as having a pfp value less than or equal to, equal to a false discovery rate of approximately 15 %, as previously reported. this statistical analysis involving four biological replicates resulted in the identification of up-regulated and down-regulated genes in the glc/agar comparison and, up-regulated and down-regulated genes in the scom145/scozm comparison. in total, genes were differentially expressed between both comparisons.fig. a classification based on go biological processes for glk/agar and m145/scozm comparisons. in all cases the most representatives go are those corresponding to metabolic processes, transport and regulation of biological processes. b classification of genes coding for enzymes included in the go metabolic processes the list of differentially expressed genes was submitted to the protein center to group broadly them on the basis of its go-biological function term. half of the annotated genes were related to the go metabolic processes in both comparisons. the go term “metabolic process”, includes anabolic and catabolic chemical reactions and pathways. looking closer inside the go metabolic process, genes encoding enzymes were also classified. a considerable number of oxidoreductases, hydrolases and transferases were repressed by glucose in glc/agar comparison. comparison between scom and the scozm mutant revealed that the presence of a glk protein, but not its enzyme activity, stimulated or repressed mostly the expression of oxidoreductases and transferases. considering their possible role in signal transduction, we have also looked for potential sensor kinases in this group. for the glc/agar comparison, regarding to agar, the sensor kinase gene sco was down-regulated by glucose. in the scom14/scozm comparison, the putative sensor kinase gene sco was up-regulated. metabolic processes and catalysis as previously mentioned glucose is mostly transported inside the cell by the glcp transporter, most probably bound to scglk. in this hypothesis, the glucose is transported and phosphorylated in a single step. in the glc/agar comparison, the genes glcp, and glcp showed the highest stimulation exerted by glucose. the transcriptional regulation of glcp was dependent on the presence of glucose but scglk independent since in the scom145/scozm comparison no changes were detected for glc transporters. previous reports have demonstrated a constitutive expression of scglk. in agreement with this result, we did not observe differential expression of scglk in the glc/agar comparison. as expected, this difference was detected when comparing m145/scozm due to scglk deletion. no other glucokinases were found being regulated by glucose or scglk. once glucose-6-phosphate is formed, it can enter to central carbon catabolic pathways.fig. profile of glucose dependent genes involved in both, glycolysis and pentose phosphate pathways. a schematic representation of the central carbon metabolism pathways in s. coelicolor, in red are genes stimulated and in blue are genes repressed. b the comparison between conditions is indicated as ratios of glc/agar. results are given as log ratios, thus a positive number indicates up-regulation and a negative number down-regulation, respectively fig. selected genes encoding transporters and their expression profiles depending upon glucose and glk presence. a glc/agar comparison reflects the glucose effect and the m145/scozm comparison shows glk dependence. the comparison between conditions is indicated as ratio of glc/agar and scom145/scozm. results are given as log ratio, thus a positive number indicates up-regulation and a negative down-regulation, respectively. b putative products of regulated genes are shown. genes that showed the same pattern of repression/stimulation across glc/agar and m145/scozm comparisons, are indicated in bold. genes that exhibit an opposite pattern of stimulation or repression between comparisons are underlined glycolysis and gluconeogenesis glycolysis or the embden-meyerhof-parnas pathway usually consists of ten reactions in which glucose is degraded to pyruvate. this pathway is present in all domains of life and hence is considered one of the most ancient pathways for sugar degradation. the expression of eight glycolytic steps involved in glucose degradation was up-regulated by glucose. this differential expression was not observed in the scom145/scozm comparison, suggesting that glycolysis proceeded more actively when glucose was present, but it was not affected by the absence of endogenous scglk. in detail, two of the three isoforms of the glyceraldehyde-3-phosphate dehydrogenase genes showed induction by glucose while sco was glucose repressed. expression of the sco gene encoding the gluconeogenic phosphoenolpyruvate carboxykinasewas glucose repressed. the genes with greater changes were those encoding a pyruvate kinase isoform and phosphoenolpyruvate carboxykinase, which were strongly induced and repressed by this carbohydrate, respectively. the large changes in the expression of these genes reflected their importance as regulatory points for the glucose/gluconeogenesis pathways. it is generally known that enzymes of metabolic pathways catalyzing irreversible steps are potential sites for regulatory controls. pyk catalyzes the transfer of phosphate from phosphoenolpyruvate to adp, producing pyruvate and atp. this reaction has a large negative free energy change and represents the third regulated and irreversible enzyme of glycolysis. the strong repression of glucose over the pepck gene whose product is involved in the conversion of oxalacetate to phosphoenolpyruvate, may reflect the strict control exerted by this carbohydrate, as this enzyme catalyzes the rate-controlling step of gluconeogenesis, and likely is the first committed step in all organisms. in b. subtilis, pepck is also reported to operate in reverse direction and might function in the anaplerotic node linking tca cycle and glycolysis/gluconeogenesis. the tricarboxylic acid cycle the tricarboxylic acid cycle is the final common pathway for molecules from which energy is generated. molecules enter tca as acetyl-coa and leave as co in the glc/agar comparison, just the putative succinate dehydrogenase complex encoded by sco0922-sco was induced, while in the tested conditions, the expression of other genes coding or enzymes of the tca, remained unchanged, suggesting that the tca cycle may be regulated mostly by the metabolic intermediates rather than by transcriptional regulation. proteomic studies have previously suggested that the tca enzymes are not regulated by protein expression. pentose phosphate pathway this pathway, also known as the phosphogluconate pathway or hexose monophosphate shunt, is another way to metabolize carbon sources like glucose, generating reducing power in the form of nadph and c carbohydrates. the ribulose-5-phosphate is an important precursor of atp, coa, nad+, fad+, dna and rna production. in s. coelicolor, there are two major clusters encoding enzymes involved in the pentose phosphate pathway. in the first cluster, sco1935-sco neither the presence of glucose nor the scglk have an effect on its expression level. the second cluster, sco6658-sco was entirely stimulated by glucose, and of these, the most noticeable change observed was in the sco gene, encoding a transaldolase with a log2 = . it has been proposed that this cluster may be involved in providing nadph for secondary metabolite production. in concordance with the reported capacity of glk from z. mobilis to reestablish the glycolytic flux, no changes were observed in the transcriptomic data of glycolysis, ppp, tca cycle or gluconeogenesis in the scom145/scozm comparison. therefore, regarding central carbon metabolism the glycolysis and ppp were highly activated only by glucose. enzymes in the tca cycle practically did not change, while gluconeogenesis was inhibited by glucose, supporting a preferential channeling of glucose to form essential building blocks. in this regard, a good correlation between our transcriptomic data and previously reported proteomics data was observed. additionally, a putative gluconokinase coding gene was also stimulated by glucose in the glc/agar comparison, while no changes were detected in the scom145/scozm comparison. the finding of up-regulation in the glnk encoding gene sco, correlated well with a glucose positive regulation of sco, encoding a gluconate transporter. in streptomyces lividans, the consumption of gluconate as a sole carbon source has been reported by. however, to our knowledge, no studies on the regulation of the gluconate catabolic genes in s. coelicolor have been previously performed. in several genera of bacteria such as pseudomonas, gluconobacter, acetobacter, and various fungi, gluconate is produced from glucose through a simple dehydrogenation reaction catalyzed by a glucose oxidase. in general, bacterial growth on gluconate requires two specific enzymes: a gluconate permease for extracellular gluconate incorporation and a gluconate kinase, which phosphorylates gluconate to 6-phosphogluconate. in streptomyces, gluconate can eventually be degraded by ppp to generate reducing power, needed for secondary metabolite biosynthesis. in agreement with this observation, glucose activation of this ppp branch was in synchrony with the over-expression of both sco and sco genes. ccr at the operon-specific mechanisms in many bacteria ccr may operate at operon-specific mechanisms by both a) inducer exclusion, i.e., prevention of the internalization of non-preferred carbon sources to thwart induction of their catabolic operons, and b) induction prevention, i.e., control of the activity of operon-specific transcription factors. a) in the case of induction exclusion, approximately 10 % of the down or up-regulated genes from each transcriptome were implicated in transport functions. glucose repression was detected over the maltose transporter malg and malf genes and the putative operon sco3482-sco were repressed. it is noteworthy mentioning that only a limited number of sugar transporter genes were detected in this study, probably due to the lack of specific inducers in the system. interestingly, the xylose transporter, showed 10-fold stimulation, just after the glcp transporter. this oddly up-regulation of the xylose transporter was also accompanied by changes in its transcriptional regulator rok7b. recently, it has been shown that the protein rok7b is also involved in s. coelicolor ccr, but its function in this phenomenon remains unclear. the genes involved in glutamate incorporation, encoding glud, gluc, glub and glua, respectively), were strongly stimulated by glucose. it was not surprising to find that glucose did not exert a repressive effect over the glutamate transporter since this amino acid is a preferred carbon source, even over glucose. besides, in the scom145/scozm comparison, this transporter was also up-regulated in scozm when compared to scom however, it is unclear why and how glucose stimulates the glutamate permease and how is the scglk effect mediated. likely, this is the result of transcriptional changes in the arginine repressor, which will be discussed later. for induction prevention, it is well known that glucose represses glycerol kinase production by negatively influencing transcription of the glycerol-inducible glpfkdx operon. this operon comprises genes for a putative glycerol transporter, a glycerol kinase, a glycerol-3-phosphate dehydrogenase, and a gene of unknown function. in the glc/agar comparison, the glycerol kinase encoded by sco was repressed while the glycerol operon repressor protein was up-regulated by glucose. in addition, a transcriptional repression of glpk was observed, confirming the findings of hindle and smith and supporting that the gylr effect is due to an operon specific mechanism rather than a pleiotropic one. interestingly, no transcriptional changes were detected in this operon when comparing the scom with scozm, suggesting a scglk-independent repression of glpk or induction of gylr. since many operons, specific ccr mechanisms require the presence of inductor we choose a classical and widely model to evaluate the effect of glucose and scglk over the utilization of an alternative carbon source looking for transcriptional changes in agar metabolism. utilization of an alternative carbon source agar metabolism agar utilization is one of the most widely used tests to assess for ccr. agar is constituted by a family of complex polysaccharides found in the cell wall of some red algae cells. primarily, this polymer consists of agarobiose and agaropectin. agarobiose is formed of linear chains of alternating residues of 3-o-linked β-d-galactopyranose and 4-o-linked 6-anhydro-α-l-galactose while agaropectin seems to be formed by sulfate esters, pyruvate acetal and methyl esters, but its exact structure remains obscure. the genomic context of a cluster involved in agar degradation is presented in fig. 5a. complete agar hydrolysis produces monomeric sugars such as d-galactose, la, and l-galactose-6-sulfate. although used as a model to measure ccr, the s. coelicolor agarolytic pathway has not been widely studied. the first enzyme described in this pathway was an endo-type agarase that degrades agar into neoagarotetraose and neoagarohexaose, β-agarase a, daga. its transcription, controlled by four different promoters, is induced by agar but repressed by glucose. a second agarase, was recently identified. this enzyme is an exo and endo-type β-agarase that degrades agarose, neoagarotetraose and neoagarohexaose into neoagarobiose. in the proposed agarolytic pathway daga and dagb can hydrolyze agarose into neoagarobiose in a cooperative way. likely, neoagarobiose is transported into the cell by an unknown transporter and once inside the cell, a cytosol glycoside hydrolase hydrolyze neoagarobiose into gal and la. the reconstructed agarolytic pathway proposed by chi et al. is summarized in fig. 5b.fig. a genomic context of a cluster possible involved in agar degradation. b the agar metabolism. c average expression changes observed among experimental conditions. glc/agar comparison reflects the glucose effect and m145/scozm comparison shows the glk dependence. the comparison between these conditions is indicated as ratios of glc/agar and scom145/scozm. results are given as the log ratio, thus a positive number indicates up-regulation and a negative number down-regulation, respectively as expected, the genes encoding for agarases, were repressed by glucose in glc/agar comparison. this effect was observed in a large genomic region, except for the sco that encodes for a laci-transcriptional regulator. this region contains nine possible transcription units, although the function of only three of them has been described. previous attempts to find the transcriptional regulator of daga have failed. however, in our tested conditions, no changes were detected in the only gene encoding a putative transcriptional regulator present in this agar degradation cluster. the functions of this putative regulator are unknown, but apparently it is not transcriptionally regulated by glucose. in the scom145/scozm comparison, genes from the agar degradation cluster showed higher repression in the scom strain compared to scozm, particularly that of sco which codes for dagb implying, as previously reported, a repressive effect dependent on scglk per se. it is hard to establish the mechanism by which the scglk protein itself could exert a regulatory function. as mentioned before, at the central carbon metabolism, and hence the metabolites produced, operates in a similar way when endogenous scglk is present and when is substituted by a non-related glk. so the repression, at least that of agar metabolism, most be not dependent on the glycolytic flux, as previously proposed, but dependent on the expression of dna binding proteins and its interactions with scglk. it is becoming evident that besides the specific operon regulators, the presence of other pleiotropic or general regulators such as the recently described rok7b may be important in understanding the ccr in streptomyces and also in establishing the scglk effect. in the next section, the transcriptional changes in genes coding for putative transcriptional regulators responsive to glucose and scglk are described. interestingly, in this study, a possible transporter associated with neoagarobiose incorporation, was found. since this chromosomal region is implicated in agar metabolism, genes sco3482- showed the same expression pattern observed for agarases and genes in this region seemed to respond to a common regulation. the gene products of sco and sco have transmembrane domains and are therefore predicted to be membrane proteins. on the other hand, sco does not have any tm and, therefore, it is predicted to be the sugar binding protein. likely sco is the transporter associated with neoagarabiose internalization. regarding to agar utilization, this is the first report describing the genes and transcriptomic changes in a cluster involved in agar metabolism.fig. genes and its predicted products involved in cpk synthesis. on the right, transcriptomic expression of the cpk genes. glc/agar comparison reflects the glucose effect on cpk production and the m145/scozm comparison shows its glk dependence. comparisons between these conditions are indicated as ratios of glc/agar and scom145/scozm. results are given as log ratio, thus positive numbers indicate up-regulation and negative numbers down-regulation, respectively ccr and transcription regulators prokaryotic cells can adapt and respond to environmental conditions by modifying their genome expression pattern; therefore, as previously proposed, describing the transcriptional dynamics mediated by regulatory proteins could be an important clue to unraveling ccr mechanisms. in model organisms, transcriptional regulators operate both global and operon-specific mechanisms. in our study, around 15 % of all differentially expressed genes in glc/agar and scom145/scozm comparisons, comprised go regulation of metabolic processes and stimuli responses. transcriptional factors stimulated by glucose were found among the merr, laci, tetr, gntr, marr and asnc families, as well as sigma factors. on the other hand, transcriptional factors repressed by glucose included members of the gntr, marr, and deor families, but also sigma and anti-sigma factors. down-regulated transcriptional regulators with known functions included: the rna polymerase sigma factor sigu, the anti-sigma factor rsua, the sporulation transcription factor whih, as well as the rna polymerase sigma factor bldn. up-regulated transcriptional regulators were: the regulatory protein, the glycerol operon regulatory protein, and the regulatory protein rok7b previously discussed.table transcriptional regulators differentially expressed in the glc/agar comparison sco1541 sco0140 sco1699 sco2489 sco2209 sco2935 sco3134 sco3361 sco3933 sco3423 sco3986 sco3810 sco4122 sco3943 sco4412 sco4020 sco4677 sco4158 sco4920 sco4640 sco5785 sco4850 sco5811 sco5413 sco5819 sco5552 sco6162 sco5982 sco6992 sco7424 sco7727 sco1658 sco2954 sco2950 sco2953 sco4159 sco5819 sco4762 sco3323 sco6008 the transcriptional factors activated by glucose can be divided into three groups: a) pleiotropic regulators, b) regulators of specific operons and c) dna-associated proteins. regarding pleiotropic regulators, the glnr regulator functions as the main nitrogen regulator, but it is also involved in different metabolic processes such as carbon metabolism, synthesis of catabolic enzymes, and secondary metabolism. the recently described regulator rok7b was activated by glucose in the scom background, following the same activation pattern as the xylfgh operon. likely, rok7b may act as a positive regulator. thus, in addition to regulating the xylose operon, rok7b activates morphological development and actinorhodine production. on the contrary, this regulator blocked the calcium dependent antibiotic and prodigiosin formation. rok7b is also known to be involved in the ccr process. regarding regulators of specific operons, the only specific regulator gene observed with differential expression was the gylr gene. this regulator was stimulated by glucose and in agreement with this effect, the glycerol kinase gene was repressed. on dna associated proteins, hupa expression was stimulated by glucose. hupa is a protein found in vegetative mycelium growing in liquid culture and involved in gene expression, dna protection and nucleoid structuring. additionally, transcription of the chaperonine groel was also stimulated by glucose and was heat shock activated. an interesting regulator with an unknown function is sco a probable member of the luxr family, which showed more than 20-fold down-regulation as well as its cognate kinase. markedly, the divergent gene sco, exhibited the highest glucose repression. in the scom145/scozm comparison, the four genes encoding regulatory proteins that showed stimulation belonged to the gntr, luxr families and a transcriptional activator. the two regulators belonging to the gntr family, the sco and sco were highly overexpressed. remarkably, none of these up-regulated transcriptional factors have been previously described. the in silico prediction of the putative targets of sco has revealed a large set of sensitive genes involved in primary and secondary metabolism. actually, in a s. coelicolor ∆sco mutant constructed in our lab, a premature and high-level production of secondary metabolites was observed. on the other side, in the group of transcriptional down-expressed regulators, the arginine metabolism and its regulator argr, deserve special attention. in a recent transcriptomic and proteomic comparison between scom and its ∆argr mutant, the versatile functions of this regulator were demonstrated. beyond its regulatory role in the arginine and pyrimidine biosynthesis, argr was also able to regulate some different transcriptional factors from different families such as deor, arac and gntr. therefore, stimulation or repression of argr affected secondary metabolite production, morphology and synthesis of proteases and peptidases. the versatile functions of this regulator and its higher expression in the absence of scglk suggest a complex regulatory network. to make this panorama more complex, when scom145/scozm were compared, none of the differentially expressed genes of the putative transcriptional regulators seemed to be the transcriptional regulators found by pérez-redondo.table differentially expressed genes found in the scom145/scozm comparison sco0016 sco0138 sco0555 sco0556 sco1570 sco1576 sco1577 sco1578 sco1579 sco1580 sco1815 sco2126 sco2296 sco2513 sco2930 sco2986 sco3138 sco3411 sco3413 sco3985 sco4173 sco4174 sco4175 sco4317 sco4903 sco4950 sco5025 sco5026 sco5367 sco5536 sco5839 sco5976 sco6266 sco6268 sco6288 sco7036 sco7262 sco7530 sco7586 sco7587 sco7698 role of scglk in carbon catabolite repression as previously mentioned, the transcriptional response to glucose included differentially expressed genes while in the scglk response differentially expressed genes were identified. from the total of differentially expressed genes in scom145/scozm comparison, were included in the glc/agar comparison. this result conferred only a limited regulatory function to this enzyme in ccr, since specifically affected only out of a total of genes. this contrasted with previous results, which proposed a significant role of glk in ccr. as seen in additional file 1: table s several of this shared genes were involved in transport functions and agar metabolism. concerning transport functions, these included genes encoding abc transporters, cation transporters like sco3717-sco amino acid transporters as the glutamate permease and also the transporter putatively associated with neoagarabiose internalization. the genes encoding the abc transporter were stimulated by glucose in the wild-type strain, but repressed in the scom145/scozm comparison. as mentioned previously, the genes involved in agar metabolism were repressed by glucose and also were affected by scglk. aside from primary metabolic pathways and transport systems, significant transcriptional changes in the yellow cryptic polyketide cluster was also observed in both transcriptomes, and, therefore, deserved special mention. the repressive effects exerted by glucose on differentiation and secondary metabolite production are well known. however, in our experimental conditions, relative to agar, the cpk cluster was up-regulated by glucose. previously, pawlick et al. reported that the presumptive product of the cpk polyketide synthase is a yellow compound secreted into the medium. its production depended on the medium composition, inoculum density and required the absence of glucose. in parallel, gottelt et al. reported in s. coelicolor that cpk production is enhanced by glutamate supplementation. recently, in a s. coelicolor engineered strain, the product of this cluster has been named coelimycin p and suggested to be a glutamate adduct. in the glc/agar comparison, glucose exerted a stimulatory effect on the genes sco6272-sco and sco6282-sco. additionally, in the scom145/scozm comparison, sco and sco, also showed increased expression in the scom strain, relative to the scozm mutant and this effect was not observed in the glc/agar comparison. although pawlick et al. previously reported that glucose exerts a negative effect on the synthesis of ycpk, in our experimental conditions this carbohydrate exerted an up-regulation on the cpk genes. interestingly, in the absence of scglk a reduced expression of cpk genes was observed in scozm, relative to scom in the model proposed by gubbens et al., cpk synthesis is activated by glucose + mannitol and repressed by glucose + fructose. it would be interesting to test the scozm in glucose + fructose or any other carbon source combinations to evaluate their effect on the cpk cluster expression. as gubbens et al. anticipated for the cpk secondary metabolite production, the influence of glk likely involves a complex regulatory mechanism, also dependent on nutritional signals. from the proposed genes involved in the cpk synthesis, the only one that showed a down-regulation in scom relative to scozm, was sco which encodes a butenolide synthase. additionally, in the scom145/scozm comparison, sco and sco, also showed increased expression in the scom strain, relative to the scozm mutant and this effect was not observed in the glc/agar comparison. depending on the used carbon source, proteomic studies have also reported the differential expression of proteins of this cluster. regarding the genes that were exclusively found in the scom145/scozm comparison, as previously discussed, the gene encoding the versatile regulator argr was down-regulated in the scom strain, when compared to scozm. it is interesting to note that the absence of scglk somehow generates a higher transcription of the gene encoding the regulator argr in the scozm strain, relative to scom in agreement with the up-regulation of argr genes, the arginine biosynthetic pathway showed down-regulation on the mutant or, in other words, the arginine biosynthetic genes were up-regulated in the scozm when compared scom. rt-pcr validation of selected genes up- or down-regulated by glucose or scglk microarray data were validated by rt-qpcr utilizing glucose-stimulated and repressed genes in glc/agar comparison. to look for suitable reference genes, the sigma factor gene hrdb, the beta chain from dna polymerase iii, the dna gyrase subunit a gene, the recombinase a gene and the dna-directed rna polymerase alpha chain were utilized. to select the most stable reference genes, the bestkeeper program was utilized. this program calculates a bestkeeper index, which is the geometric mean of all housekeeping raw ct values. pearson correlations between individual genes and the bestkeeper index were calculated and reported as the bestkeeper correlation coefficient. genes with the highest correlation coefficient were considered the most stably expressed. in our conditions, reca, rpoa, and gyra were selected as reference genes due to their high bestkeeper index and low standard deviation. the geometrically averaged reference genes were used to normalize the following target genes: daga, glcp, rok7b sco and sco the relative transcript profile of the target genes corresponded well to the microarray analysis, confirming our results on glucose regulation. also, glcp and rok7b were largely induced by glucose, while expression of daga, sco and sco were largely repressed. their neighboring genes also were down-regulated by glucose. the sco and its cognate kinase sco constitute a two-component system. the response regulator belongs to the luxr family of transcriptional regulators and is conserved in many streptomyces species. its domain architecture is present in many activators. the two component systems are important to sense and respond to the environment. a great unexplored area in streptomyces ccr is the potential role of two-component systems. in other soil living or environmental strains such pseudomonas, sinorhizobium and bacillus, two-component systems are important in ccr control.  glucose can elicit significant metabolic and transcriptional changes in s. coelicolor. whereas glucose influx and its flow through glycolysis and pentose phosphate pathway were stimulated, gluconeogenesis was inhibited. glucose also triggered carbon catabolic repression by repressing a number of transporter systems and repressing transcription of enzymes needed for the utilization of secondary carbon sources. additionally, dna binding proteins, including transcriptional regulators and four two component systems were regulated by glucose and probably were involved in the signals and the effects elicited by this carbon source. the scglk enzyme per se was required for the glucose repression, at least in the agar model proposed by angel et al.. in this regard, our results confirm and update the agar model of ccr in streptomyces and its dependence on scglk per se. nevertheless, the expected regulatory function was not as global as thought before, but likely may be accompanied or coordinated by other transcriptional regulators. the remaining transcription factors, whose expression was affected by glucose or scglk with unknown functions, are an interesting area to describe in the s. coelicolor ccr. currently, we are constructing regulatory networks to find out the possible regulatory relations between genes regulated by glucose and by scglk. understanding the functions of the glucose-targeted transcriptional regulators and their transcriptional network may lead to unraveling one of the most fundamental regulatory mechanisms in the streptomyces metabolism. 
the transcriptomic study was extended to assess the scglk role in this model by comparing the transcriptomic profile of s. coelicolor m with that of a ∆glk mutant derived from the wild-type strain, complemented with a heterologous glk gene from zymomonas mobilis, insensitive to ccr but able to grow in glucose. this mechanism regulates the expression of genes involved in the uptake and utilization of alternative carbon sources. even when the outcome effect of ccr in different bacteria is the same, their essential mechanisms can be quite different. our results confirm and update the agar model of the ccr in streptomyces and its dependence on the scglk per se. while its entry and flow through glycolysis and pentose phosphate pathway were stimulated, the gluconeogenesis was inhibited. surprisingly, the expected regulatory function of scglk was not found to be as global as thought before, although may be accompanied or coordinated by other transcriptional regulators. this work identified the expression of s. coelicolor genes involved in primary metabolism that were influenced by glucose and/or scglk. the transcriptome of a classical model of ccr was studied in s. coelicolor to differentiate between the effects due to glucose or scglk in this regulatory mechanism. these findings offer new opportunities to study and understand the ccr in s. coelicolor by increasing the number of known glucose and scglk -regulated pathways and a new set of putative regulatory proteins possibly involved or controlling the ccr. in several streptomycetes glucose kinase represents the main glucose phosphorylating enzyme and has been regarded as a regulatory protein in ccr. interestingly, even when the scglk protein does not have dna binding domains and the glycolytic flux was restored by a heterologous glucokinase, the scglk replacement modified the expression of genes. aside from describing the metabolic pathways influenced by glucose and/or scglk, several unexplored transcriptional regulators involved in the ccr mechanism were disclosed. glucose also triggered the ccr by repressing transporter systems and the transcription of enzymes required for secondary carbon sources utilization. in the genus streptomyces, one of the most remarkable control mechanisms of physiological processes is carbon catabolite repression. ccr also affects the synthesis of secondary metabolites and morphological differentiation. to evaluate the paradigmatic model proposed for ccr in streptomyces, a high-density microarray approach was applied to streptomyces coelicolor m under repressed and non-repressed conditions. glucose elicited important metabolic and transcriptional changes in this microorganism.
 in the genus streptomyces, one of the most remarkable control mechanisms of physiological processes is carbon catabolite repression. this mechanism regulates the expression of genes involved in the uptake and utilization of alternative carbon sources. ccr also affects the synthesis of secondary metabolites and morphological differentiation. even when the outcome effect of ccr in different bacteria is the same, their essential mechanisms can be quite different. in several streptomycetes glucose kinase represents the main glucose phosphorylating enzyme and has been regarded as a regulatory protein in ccr. to evaluate the paradigmatic model proposed for ccr in streptomyces, a high-density microarray approach was applied to streptomyces coelicolor m under repressed and non-repressed conditions. the transcriptomic study was extended to assess the scglk role in this model by comparing the transcriptomic profile of s. coelicolor m with that of a ∆glk mutant derived from the wild-type strain, complemented with a heterologous glk gene from zymomonas mobilis, insensitive to ccr but able to grow in glucose.  microarray experiments revealed that glucose influenced the expression of genes. interestingly, even when the scglk protein does not have dna binding domains and the glycolytic flux was restored by a heterologous glucokinase, the scglk replacement modified the expression of genes. from these, were also affected by glucose while appeared to be under the control of scglk. this work identified the expression of s. coelicolor genes involved in primary metabolism that were influenced by glucose and/or scglk. aside from describing the metabolic pathways influenced by glucose and/or scglk, several unexplored transcriptional regulators involved in the ccr mechanism were disclosed.  the transcriptome of a classical model of ccr was studied in s. coelicolor to differentiate between the effects due to glucose or scglk in this regulatory mechanism. glucose elicited important metabolic and transcriptional changes in this microorganism. while its entry and flow through glycolysis and pentose phosphate pathway were stimulated, the gluconeogenesis was inhibited. glucose also triggered the ccr by repressing transporter systems and the transcription of enzymes required for secondary carbon sources utilization. our results confirm and update the agar model of the ccr in streptomyces and its dependence on the scglk per se. surprisingly, the expected regulatory function of scglk was not found to be as global as thought before, although may be accompanied or coordinated by other transcriptional regulators. aside from describing the metabolic pathways influenced by glucose and/or scglk, several unexplored transcriptional regulators involved in the ccr mechanism were disclosed. these findings offer new opportunities to study and understand the ccr in s. coelicolor by increasing the number of known glucose and scglk -regulated pathways and a new set of putative regulatory proteins possibly involved or controlling the ccr. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords transcriptometranscriptional factorscarbon catabolite regulationglucose kinasestreptomyces coelicolordgapa, papiit, unamin201413sánchez sergio conacytcb-219686sánchez sergio issue-copyright-statement© the author 2016 