Specialized and shared functions of diguanylate cyclases and phosphodiesterases in Streptomyces development.

The second messenger bis-3,5-cyclic di-guanosine monophosphate (c-di-GMP) determines when Streptomyces initiate sporulation. c-di-GMP signals are integrated into the genetic differentiation network by the regulator BldD and the sigma factor σWhiG . However, functions of the development-specific diguanylate cyclases (DGCs) CdgB and CdgC, and the c-di-GMP phosphodiesterases (PDEs) RmdA and RmdB, are poorly understood. Here, we provide biochemical evidence that the GGDEF-EAL domain protein RmdB from S. venezuelae is a monofunctional PDE that hydrolyzes c-di-GMP to 5'pGpG. Despite having an equivalent GGDEF-EAL domain arrangement, RmdA cleaves c-di-GMP to GMP and exhibits residual DGC activity. We show that an intact EAL motif is crucial for the in vivo function of both enzymes since strains expressing protein variants with an AAA motif instead of EAL are delayed in development, similar to null mutants. Transcriptome analysis of ∆cdgB, ∆cdgC, ∆rmdA, and ∆rmdB strains revealed that the c-di-GMP specified by these enzymes has a global regulatory role, with about 20% of all S. venezuelae genes being differentially expressed in the cdgC mutant. Our data suggest that the major c-di-GMP-controlled targets determining the timing and mode of sporulation are genes involved in cell division and the production of the hydrophobic sheath that covers Streptomyces aerial hyphae and spores.

The EAL domain protein YciR acts as a trigger enzyme in a c-di-GMP signalling cascade in E. coli biofilm control.

C-di-GMP-which is produced by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases (PDEs)-is a ubiquitous second messenger in bacterial biofilm formation. In Escherichia coli, several DGCs (YegE, YdaM) and PDEs (YhjH, YciR) and the MerR-like transcription factor MlrA regulate the transcription of csgD, which encodes a biofilm regulator essential for producing amyloid curli fibres of the biofilm matrix. Here, we demonstrate that this system operates as a signalling cascade, in which c-di-GMP controlled by the DGC/PDE pair YegE/YhjH (module I) regulates the activity of the YdaM/YciR pair (module II). Via multiple direct interactions, the two module II proteins form a signalling complex with MlrA. YciR acts as a connector between modules I and II and functions as a trigger enzyme: its direct inhibition of the DGC YdaM is relieved when it binds and degrades c-di-GMP generated by module I. As a consequence, YdaM then generates c-di-GMP and-by direct and specific interaction-activates MlrA to stimulate csgD transcription. Trigger enzymes may represent a general principle in local c-di-GMP signalling.

Molecular function and potential evolution of the biofilm-modulating blue light-signalling pathway of Escherichia coli.

Escherichia coli senses blue light via the BLUF-EAL protein BluF (YcgF). The degenerate EAL domain of BluF does not have cyclic-di-GMP phosphodiesterase activity, but BluF directly antagonizes the MerR-like repressor BluR (YcgE), which leads to expression of the ycgZ-ymgABC operon and activation of the Rcs system (Tschowri et al., 2009; Genes Dev 23: 522-534). While bluR, bluF and ycgZ have individual transcriptional start sites, comparative genome analysis indicates that the bluR-bluF-ycgZ-ymgAB region represents a functional unit in various enteric bacteria that is characterized by bluF alleles encoding degenerate EAL domains. Re-introducing conserved amino acids involved in phosphodiesterase activity of EAL domains did not restore enzymatic activity or c-di-GMP binding of BluF, but weakened its ability to antagonize BluR and improved a residual interaction with the BluR paralogue MlrA, which controls expression of the biofilm regulator CsgD and curli fibres. We identified the BluR binding site in the ycgZ promoter and observed that BluR also has residual affinity for the MlrA-dependent csgD promoter. Altogether, we propose that BluF evolved from a blue light-regulated PDE into a specific antagonist of a duplicate of MlrA that became BluR, which controls not only curli but various biofilm functions via the Ymg/Rcs pathway.

Expression Patterns, Genomic Conservation and Input Into Developmental Regulation of the GGDEF/EAL/HD-GYP Domain Proteins in Streptomyces.

To proliferate, antibiotic-producing Streptomyces undergo a complex developmental transition from vegetative growth to the production of aerial hyphae and spores. This morphological switch is controlled by the signaling molecule cyclic bis-(3',5') di-guanosine-mono-phosphate (c-di-GMP) that binds to the master developmental regulator, BldD, leading to repression of key sporulation genes during vegetative growth. However, a systematical analysis of all the GGDEF/EAL/HD-GYP proteins that control c-di-GMP levels in Streptomyces is still lacking. Here, we have FLAG-tagged all 10 c-di-GMP turnover proteins in Streptomyces venezuelae and characterized their expression patterns throughout the life cycle, revealing that the diguanylate cyclase (DGC) CdgB and the phosphodiesterase (PDE) RmdB are the most abundant GGDEF/EAL proteins. Moreover, we have deleted all the genes coding for c-di-GMP turnover enzymes individually and analyzed morphogenesis of the mutants in macrocolonies. We show that the composite GGDEF-EAL protein CdgC is an active DGC and that deletion of the DGCs cdgB and cdgC enhance sporulation whereas deletion of the PDEs rmdA and rmdB delay development in S. venezuelae. By comparing the pan genome of 93 fully sequenced Streptomyces species we show that the DGCs CdgA, CdgB, and CdgC, and the PDE RmdB represent the most conserved c-di-GMP-signaling proteins in the genus Streptomyces.

Probing the mechanism of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III mtFabH: factors influencing catalysis and substrate specificity.

Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These alpha-alkyl, beta-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C(24)-C(26) fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The beta-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein (ACP). The acyl-CoA chain length specificity of mtFabH was assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg(46) revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg(161) --> Ala substitution. Our structural studies suggested that His(258), previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys(122).