Molecular mechanism underlying the function of AfsQ1/Q2 on secondary metabolism in S. coelicolor
In the present work, we described the dissection of the molecular basis of AfsQ1/Q2 on antibiotic biosynthesis in S. coelicolor. It was revealed that AfsQ1/Q2 activates ACT, RED and CDA biosynthesis directly through actII-ORF4, cdaR and redZ respectively. The precise AfsQ1 binding sequences in the promoter regions of these three genes were determined. A characteristic AfsQ1 binding motif (with a length of 16 nt) comprising two 5-nt direct repeats separated by six variable nucleotides was identified in the respective upstream regions of cdaR and redZ. However, the AfsQ1 binding sequence in actII-ORF4 promoter region lacks similar binding motif, indicating that there exists other binding mode for AfsQ1 regulation, which yet to be discovered in the future. The diversity of the binding motifs for AfsQ1 regulation was also described for other TCS response regulator. Sola-Landa et al. (2008) identified three types of PhoP binding motifs which are composed of two to six 11-nt direct repeat units (DRus). The most complex binding motif (Class III) contains six DRus with poor conservation (Sola-Landa et al., 2008).
Besides AfsQ1, several other activators as well as repressors have also been identified involved in regulation of ACT, RED and CDA production through the pathway-specific activator genes in S. coelicolor (Uguru et al., 2005; McKenzie and Nodwell, 2007; Rigali et al., 2008). For instance, AbsA2, the response regulator paired with histidine kinase AbsA1, represses the biosynthesis of ACT, RED and CDA by directly interfering with the expression of actII-ORF4, redZ and cdaR, respectively, in a phosphorylation-dependent manner (McKenzie and Nodwell, 2007). However, the precise binding site for AbsA2 in these promoter regions is still unclear. DasR, a GntR-family member, is another key regulator of antibiotic biosynthesis, which can bind to the dre site (DasR binding box: 5′-TGGTCTAGACCA-3′) presented in the promoters of actII-ORF4 and redZ to repress their transcription (Colson et al., 2007; Rigali et al., 2008). AtrA, a TetR-family transcriptional regulator, functions as an activator of ACT production through a direct interaction with the regions flanking the actII-ORF4 promoter and two binding sites of AtrA were identified within the coding region of actII-ORF3 and actII-ORF4 respectively (Uguru et al., 2005). Interestingly, the identified AfsQ1 binding site (5′-GAAAC-N6-GTATC-3′) upstream of redZ gene overlaps with the DasR binding dre site. However, the AfsQ1 binding sequence in the actII-ORF4 promoter region is located separately from all of the binding sites identified so far. It will be interesting to decipher how AfsQ1/Q2 and other global regulators interact with each other in regulation of the biosynthesis of these antibiotics in S. coelicolor.
AfsQ1/Q2 might also play a positive role in yCPK biosynthesis, a yellow-pigmented secondary metabolite encoded by cpk gene cluster (SCO6273-SCO6288). Intriguingly, the function of AfsQ1/Q2 on yCPK production may be achieved through directly regulating the expression of two structural genes, including cpkA encoding a putative type I polyketide synthase subunit and cpkD encoding a putative secreted protein but not through pathway-specific activator gene kasO (data not shown). It was previously reported that there are other regulators involved in the control of cpk genes in S. coelicolor. For instance, we showed that DraR-K repressed the transcription of cpk genes via interaction with the promoter region of the pathway-specific activator gene kasO (Yu et al., 2012). Similarly, two γ-butyrolactone receptors ScbR and ScbR2 were identified to function as repressors directly via regulating the transcriptional level of kasO (Takano et al., 2005; Xu et al., 2010). In addition, intriguingly PhoP/R has recently been found to enhance cpk transcription possibly via binding to three specific regions internal to two polyketide synthase genes (cpkB and cpkC) in cpk gene cluster (Allenby et al., 2012). Moreover, it was also reported that DasR and ArgR may influence the transcription of cpk genes, although their exact mechanism is still to be determined (Rigali et al., 2008; Perez-Redondo et al., 2012). The data described above clearly reveal the complicated regulatory network of yCPK biosynthesis in S. coelicolor, which allows the bacteria to integrate different environmental stress signals in the regulation of yCPK biosynthesis.
One of the AfsQ1 target genes, abrC3, encodes a response regulator of an atypical TCS SCO4596/4597/4598 (AbrC1/C2/C3) consisting of two histidine kinases and a response regulator that was reported to have a positive role in both antibiotic production and morphological differentiation (Yepes et al., 2011). AfsQ1 positively regulates its expression, suggesting that the function of AfsQ1/Q2 might be partly mediated by AbrC1/C2/C3. The role of AbrC1/C2/C3 in AfsQ1/Q2-mediated signal transduction system is yet to be determined.
AfsQ1/Q2 has a pleiotropic role in primary metabolism
By genome screening using the known AfsQ1 binding sites upstream of sigQ, redZ and cdaR, in combination with EMSAs and transcriptional analysis, we identified a new set of genes within the AfsQ1/Q2 regulon. Functional analysis of these target genes pointed to the new roles of AfsQ1/Q2 in carbon, nitrogen and phosphate metabolism in S. coelicolor.
Three putative genes involved in carbon metabolism were identified as AfsQ1 direct targets, xysA (SCO0674), gap1 (SCO1947) and SCO2978. However, inactivation of afsQ1/Q2 resulted in the reduced expression of only SCO2978 that encodes a putative ABC transporter sugar binding protein, but did not affect the transcription of xysA and gap1. Nevertheless, binding of AfsQ1 to the presumptive promoter regions of these three carbon metabolism genes suggests a connection of AfsQ1/Q2 with carbon metabolism in S. coelicolor.
As reported previously, genes involved in nitrogen assimilation in S. coelicolor, including amtB, glnA, glnII, gdhA, ureA, nirB and nasA, are governed by the central nitrogen regulator GlnR under nitrogen-limited condition (Tiffert et al., 2008; Wang and Zhao, 2009). In addition, it was discovered that TCS PhoP/R exerts a negative control on the transcription of glnR, glnA, glnII and amtB upon phosphate limitation (Rodriguez-Garcia et al., 2009). Here, we revealed that AfsQ1 can bind specifically to the promoter regions of these seven nitrogen assimilatory genes and repress the transcription of amtB, glnA, glnII and ureA under the condition of MM supplemented with 75 mM Glu. This finding complements well the recent study of Nieselt et al. (2010), in which they showed that in a medium with an excess of glutamate, genes for nitrogen assimilation in S. coelicolor are repressed by other regulators or mechanisms other than that mediated via PhoR/P. Although nearly no effect on the expression of gdhA upon afsQ1/Q2 deletion, and no transcription of nirB and nasA in both the parental strain and the mutant was detected under the tested condition, we can not rule out the possibility that the effect of AfsQ1/Q2 on these nitrogen metabolism genes might be exerted under other conditions.
Competitive EMSAs revealed that the AfsQ1 binding site overlaps with that of GlnR in the promoter regions of glnA, indicating that AfsQ1 may function as a repressor of glnA transcription by blocking the GlnR activation. Further analysis of the AfsQ1 and GlnR binding sites upstream of glnA and nirB allowed us to distinguish the significant differences in the binding motif between AfsQ1 and GlnR. The sequence containing four 5-nt (a-b-a-b) required for GlnR recognition, only three (b-a-b) in the upstream region of nirB or two (the middle two 5-nt) in glnA promoter region are indispensable for AfsQ1 binding, further demonstrating the diversity of the AfsQ1 binding motif. In addition, we found that cross-regulation between AfsQ1 and GlnR is probably reciprocal, as GlnR could bind to the promoter regions of actII-ORF4, redZ and cdaR respectively (He et al., unpubl. data). Interestingly, we also found that the AfsQ1 binding site upstream of glnA also overlaps with that of PhoP; it is therefore still a possibility that the differential expression of glnA as well as other nitrogen metabolism genes in the ΔafsQ1/Q2 mutant might be partly ascribed to the function of PhoP.
It should be noted that regulation of nitrogen metabolism is quite complex; besides GlnR, AfsQ1/Q2 and PhoP/R, NnaR and AfsR are also involved (Rodriguez-Garcia et al., 2009; Amin et al., 2012; Santos-Beneit et al., 2012). NnaR, a new GlnR target, was identified recently as a new player in nitrogen metabolism. Four nitrate/nitrite assimilation genes, narK, nirB, nirA and nasA, were subject to direct regulation of NnaR, and a cooperative binding with GlnR to the nirB promoter was identified (Amin et al., 2012). In addition, AfsR, the global regulator of antibiotic biosynthesis in S. coelicolor (Lee et al., 2002), has recently been identified to bind to the promoter region of glnR and the AfsR binding site overlaps with that of PhoP (Santos-Beneit et al., 2012). In this study, we also showed that AfsQ1 positively controls the expression of pstS, which is a member of the PhoP regulon and also the direct target of AfsR (Santos-Beneit et al., 2009), indicating the possibility of cross-talk between these global regulators in phosphate metabolism. Overall, the cross-regulation in nitrogen and phosphate metabolism (also secondary metabolism) by global regulators, such as GlnR, PhoP, AfsR and AfsQ1, enables microbes to rapidly adapt their metabolism to the changing environments. The interaction between AfsQ1 and the other two regulators (PhoP and AfsR) will be the subject of our future research.
Growth curve analysis revealed that AfsQ1/Q2 has a negative role in S. coelicolor growth; the mutant with inactivation of afsQ1/Q2 accumulated a higher biomass in comparison with the original strain M145. Two possible reasons are: one is that the negative control of AfsQ1/Q2 on nitrogen assimilation relieved in ΔafsQ1/Q2 probably accelerated the primary metabolism; another is that reduced ACT and RED in the mutant would result in increased malonyl-CoA and possibly also acetyl-CoA supplies being available to primary metabolism, such as lipid biosynthesis and ATP production by TCA cycle, which would lead to better growth of the ΔafsQ1/Q2 mutant. However, such a hypothesis still needs further proof.
Plausible mechanism for the function of AfsQ1/Q2 on morphogenesis
Four genes involved in morphological differentiation were identified as the targets of AfsQ1, including bldM, whiD, amfC and SCO2529. whiD is required for the late stages of sporulation in S. coelicolor (Molle et al., 2000). Its transcriptional divergent gene bldM encodes a response regulator required for aerial mycelium formation (Bibb et al., 2000; Molle and Buttner, 2000). SCO2529 encodes for a possible metalloprotease with a peptidase family M4 (thermolysin family) conserved domain. Enzymes of the thermolysin family are secreted by both Gram-positive and Gram-negative bacteria to degrade extracellular proteins and peptides for bacterial nutrition, especially prior to sporulation. Experiments with proteinase inhibitor have revealed the importance of extracellular proteases in sporulation in Bacillus subtilis (Dancer and Mandelstam, 1975). amfC encodes an aerial mycelium-associated protein which is important in development (Kudo et al., 1995). Thus, we suggested that the remarkably increased expression of SCO2529 and whiD, and decreased bldM expression at 48 h (cells enter the stage of sporulation) in the ΔafsQ1/Q2 mutant might account for the rapid spore formation of the ΔafsQ1/Q2 mutant (Shu et al., 2009). Interestingly, bldM is also a member of the BldD regulon (den Hengst et al., 2010). BldD functions to repress its expression during vegetative growth through interaction with a predicted BldD binding site which is separated from the identified AfsQ1 binding site. It was predicted that they may control bldM expression by responding to different signals.
Proposed AfsQ1/Q2-mediated signal transduction system in S. coelicolor
A tentative regulatory model of AfsQ1/Q2 in S. coelicolor is proposed (Fig. 10). Under the MM condition supplemented with high concentration of glutamate as the sole nitrogen source, signals that might be an intermediate of nitrogen metabolism or the ratio of C/N/P ratio could be generated as we proposed previously (Yu et al., 2012). In response to the signals, the sensor histidine kinase AfsQ2 autophosphorylates and then activates its cognate response regulator AfsQ1. Activated AfsQ1 then issues its roles on antibiotic biosynthesis through binding to the promoter regions of its target genes, including actII-ORF4, redZ, cdaR and cpkA/cpkD directly, thereby stimulating the biosynthesis of ACT, RED, CDA and yCPK, respectively, directly controlling the expression of whiD/bldM and SCO2529 to affect aerial mycelium formation and sporulation. As for its role in primary metabolism, we speculated that AfsQ1/Q2 might be involved in the overall co-ordination of S. coelicolor metabolism. Under the MM condition supplemented with 75 mM Glu (with sufficient nitrogen supply), the cells are possibly required to repress the transcription of genes responsible for nitrogen assimilation (such as amtB, glnA and glnII), meanwhile channel more energy and resources (exerted by AfsQ1/Q2) to carbon and phosphate metabolism by activating the expression of genes involved in phosphate and carbon uptake (such as pstS and SCO2978), thus keeping the C/N/P ratio in an equilibrium state.
In conclusion, we report the comprehensive analysis of the AfsQ1/Q2 regulatory system involved in morphogenesis, primary and secondary metabolism and also the identification of AfsQ1/Q2 regulon in S. coelicolor. For the regulon, a conserved AfsQ1 binding motif (GTnAC-n6-GTnAC) comprising two 5-nt direct repeats separated by six variable nucleotides was defined. In addition, cross-regulation between AfsQ1/Q2 and GlnR was established, which further reveals the complex regulatory network of nitrogen metabolism in S. coelicolor. It is worth noting that for the target genes in the AfsQ1/Q2 regulon, not all in vitro AfsQ1 binding events could result in alterations in gene expression. This phenomenon has also been reported for other regulators in S. coelicolor, such as GlnR (Tiffert et al., 2008). There are two possible explanations for this. One is that other transcriptional regulators also influence the expression of these genes and the overall roles may lead to no obvious difference in transcription, and another is the specific experimental condition we employed here which might account for this result. It is likely that in other media, these genes might also show significant differential expression in the ΔafsQ1/Q2 mutant. Our future research will exploit high-throughput technologies, such as chromatin immunoprecipitation (ChIP) with microarray technology (ChIP on chip) or sequencing (ChIP sequencing) (Allenby et al., 2012) to fully identify the direct AfsQ1 targets, which would help to shed more light on the biological significance of the broader roles of AfsQ1/Q2 on morphological and physiological differentiation in S. coelicolor.