ROK family regulator NagC promotes prodigiosin biosynthesis independent of N-acetylglucosamine in Serratia sp. ATCC 39006.

Serratia sp. ATCC 39006 is an important model strain for the study of prodigiosin production, whose prodigiosin biosynthesis genes (pigA-O) are arranged in an operon. Several transcription factors have been shown to control the transcription of the pig operon. However, since the regulation of prodigiosin biosynthesis is complex, the regulatory mechanism for this process has not been well established. In most γ-proteobacteria, the ROK family regulator NagC acts as a global transcription factor in response to N-acetylglucosamine (GlcNAc). In Serratia sp. ATCC 39006, NagC represses the transcription of two divergent operons, nagE and nagBAC, which encode proteins involved in the transport and metabolism of GlcNAc. Moreover, NagC directly binds to a 21-nt region that partially overlaps the -10 and -35 regions of the pig promoter and promotes the transcription of prodigiosin biosynthesis genes, thereby increasing prodigiosin production. Although NagC still acts as both repressor and activator in Serratia sp. ATCC 39006, its transcriptional regulatory activity is independent of GlcNAc. NagC was first found to regulate antibiotic biosynthesis in Gram-negative bacteria, and NagC-mediated regulation is not responsive to GlcNAc, which contributes to future studies on the regulation of secondary metabolism by NagC in other bacteria. IMPORTANCE: The ROK family transcription factor NagC is an important global regulator in the γ-proteobacteria. A large number of genes involved in the transport and metabolism of sugars, as well as those associated with biofilm formation and pathogenicity, are regulated by NagC. In all of these regulations, the transcriptional regulatory activity of NagC responds to the supply of GlcNAc in the environment. Here, we found for the first time that NagC can regulate antibiotic biosynthesis, whose transcriptional regulatory activity is independent of GlcNAc. This suggests that NagC may respond to more signals and regulate more physiological processes in Gram-negative bacteria.

cAMP and c-di-GMP synergistically support biofilm maintenance through the direct interaction of their effectors.

Nucleotide second messengers, such as cAMP and c-di-GMP, regulate many physiological processes in bacteria, including biofilm formation. There is evidence of cross-talk between pathways mediated by c-di-GMP and those mediated by the cAMP receptor protein (CRP), but the mechanisms are often unclear. Here, we show that cAMP-CRP modulates biofilm maintenance in Shewanella putrefaciens not only via its known effects on gene transcription, but also through direct interaction with a putative c-di-GMP effector on the inner membrane, BpfD. Binding of cAMP-CRP to BpfD enhances the known interaction of BpfD with protease BpfG, which prevents proteolytic processing and release of a cell surface-associated adhesin, BpfA, thus contributing to biofilm maintenance. Our results provide evidence of cross-talk between cAMP and c-di-GMP pathways through direct interaction of their effectors, and indicate that cAMP-CRP can play regulatory roles at the post-translational level.

Organic Hydroperoxide Induces Prodigiosin Biosynthesis in Serratia sp. ATCC 39006 in an OhrR-Dependent Manner.

The biosynthesis of prodigiosin in the model prodigiosin-producing strain, Serratia sp. ATCC 39006, is significantly influenced by environmental and cellular signals. However, a comprehensive regulatory mechanism for this process has not been well established. In the present study, we demonstrate that organic hydroperoxide activates prodigiosin biosynthesis in an OhrR-dependent manner. Specifically, the MarR-family transcriptional repressor OhrR (Ser39006_RS05455) binds to its operator located far upstream of the promoter region of the prodigiosin biosynthesis operon (319 to 286 nucleotides [nt] upstream of the transcription start site) and negatively regulates the expression of prodigiosin biosynthesis genes. Organic hydroperoxide disassociates the binding between OhrR and its operator, thereby promoting the prodigiosin production. Moreover, OhrR modulates the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide by regulating the transcription of its own gene and the downstream cotranscribed ohr gene. These results demonstrate that OhrR is a pleiotropic repressor that modulates the prodigiosin production and the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide stress. IMPORTANCE Bacteria naturally encounter various environmental and cellular stresses. Organic hydroperoxides generated from the oxidation of polyunsaturated fatty acids are widely distributed and usually cause lethal oxidative stress by damaging cellular components. OhrR is known as a regulator that modulates the resistance of bacteria to organic hydroperoxide stress. In the current study, organic hydroperoxide disassociates OhrR from the promoter of prodigiosin biosynthesis gene cluster, thus promoting transcription of pigA to -O genes. In this model, organic hydroperoxide acts as an inducer of prodigiosin synthesis in Serratia sp. ATCC 39006. These results improve our understanding of the regulatory network of prodigiosin synthesis and serve as an example for identifying the cross talk between the stress responses and the regulation of secondary metabolism.

Fnr Negatively Regulates Prodigiosin Synthesis in Serratia sp. ATCC 39006 During Aerobic Fermentation.

The well-known Crp/Fnr family regulator Fnr has long been recognized as an oxygen sensor to regulate multiple biological processes, including the switch between aerobic/anaerobic metabolism, nitrogen fixation, bioluminescence, infection, and virulence. In most cases, Fnr was found to be active under anaerobic conditions. However, its role in aerobic antibiotic metabolism has not yet been revealed. In this research, we report that in the model organism, Serratia sp. ATCC 39006, Fnr (Ser39006_013370) negatively regulates prodigiosin production by binding to the spacer between the -10 and -35 region in the promoter of prodigiosin biosynthetic gene cluster under aerobic conditions. Fnr was also shown to modulate the anti-bacterial activity and motility by regulating pathway-specific regulatory genes, indicating that Fnr acts as a global regulator in Serratia sp. ATCC 39006. For the first time, we describe that Fnr regulates antibiotic synthesis in the presence of oxygen, which expands the known physiological functions of Fnr and benefits the further investigation of this important transcriptional regulator.

The Cyclic AMP Receptor Protein, Crp, Is Required for the Decolorization of Acid Yellow 36 in Shewanella putrefaciens CN32.

Shewanella shows good application potentials in the decolorization and detoxification of azo dye wastewater. However, the molecular mechanism of decolorization is still lacking. In this study, it was found that Shewanella putrefaciens CN32 exhibited good decolorization ability to various azo dyes, and a global regulatory protein cAMP receptor protein (Crp) was identified to be required for the decolorization of acid yellow 36 (AY) by constructing a transposon mutant library. Then, the molecular mechanism of AY decolorization regulated by Crp was further investigated. RT-qPCR and electrophoretic mobility shift assay (EMSA) results showed that Crp was able to directly bind to the promoter region of the cymA gene and promote its expression. Riboflavin acting as an electron shuttle could accelerate the AY decolorization efficiency of S. putrefaciens CN32 wild-type (WT) but did not show a promoting effect to Δcrp mutant and ΔcymA mutant, further confirming that Crp promotes the decolorization through regulating electron transport chains. Moreover, the mutant with cymA overexpression could slightly enhance the AY decolorization efficiency compared with the WT strain. In addition, it was found that MtrA, MtrB, and MtrC partially contribute to the electron transfer from CymA to dye molecules, and other main electron transport chains need to be identified in future experiments. This study revealed the molecular mechanism of a global regulator Crp regulating the decolorization of azo dye, which is helpful in understanding the relationship between the decolorization and other metabolic processes in S. putrefaciens CN32.