Prodigiosin, a red linear tripyrrole pigment normally secreted by Serratia marcescens, has received attention for its reported immunosuppressive, antimicrobial and anticancer properties. Although several genes have been shown to be important for prodigiosin synthesis, information on the regulatory mechanisms behind this cellular process remains limited. In this work, we identified that the transcriptional regulator RcsB encoding gene BVG90_13250 (rcsB) negatively controlled prodigiosin biosynthesis in S. marcescens. Disruption of rcsB conferred a remarkably increased production of prodigiosin. This phenotype corresponded to RcsB negative control of transcription of the prodigiosin-associated pig operon probably by binding to the promoter region of the prodigiosin synthesis positive regulator FlhDC. Moreover, using transcriptomics and further experiments, we revealed that RcsB also controlled some other important cellular processes, including swimming and swarming motilities, capsular polysaccharide production, biofilm formation, and acid resistance in S. marcescens. Collectively, this work proposes that RcsB is a prodigiosin synthesis repressor in S. marcescens and provides insight into the regulatory mechanism of RcsB on cell motility, capsular polysaccharide production, and acid resistance in S. marcescens. Importance RcsB is a two-component response regulator in the Rcs phosphorelay system, and plays versatile regulatory functions in Enterobacteriaceae. However, information on the function of the RcsB protein in bacteria, especially in S. marcescens remains limited. In this work, we illustrated experimentally that the RcsB protein was involved in diverse cellular processes in S. marcescens, including prodigiosin synthesis, cell motility, capsular polysaccharide production, biofilm formation, and acid resistance. Additionally, the regulatory mechanism of the RcsB protein on these cellular processes was investigated. In conclusion, this work indicated that RcsB could be a regulator for prodigiosin synthesis and provides insight into the function of RcsB protein in S. marcescens.

Serratia marcescens is a Gram-negative bacterium of the Enterobacteriaceae family that can produce numbers of biologically active secondary metabolites. However, our understanding of the regulatory mechanisms behind secondary metabolites biosynthesis in S. marcescens remains limited. In this study, we identified an uncharacterized LysR family transcriptional regulator, encoding gene BVG90_12635, here we named psrA, that positively controlled prodigiosin synthesis in S. marcescens. This phenotype corresponded to PsrA positive control of transcriptional of the prodigiosin-associated pig operon by directly binding to a regulatory binding site (RBS) and an activating binding site (ABS) in the promoter region of the pig operon. We demonstrated that L-proline is an effector for the PsrA, which enhances the binding affinity of PsrA to its target promoters. Using transcriptomics and further experiments, we show that PsrA indirectly regulates pleiotropic phenotypes, including serrawettin W1 biosynthesis, extracellular polysaccharide production, biofilm formation, swarming motility and T6SS-mediated antibacterial activity in S. marcescens. Collectively, this study proposes that PsrA is a novel regulator that contributes to antibiotic synthesis, bacterial virulence, cell motility and extracellular polysaccharides production in S. marcescens and provides important clues for future studies exploring the function of the PsrA and PsrA-like proteins which are widely present in many other bacteria.

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In Serratia marcescens JNB5-1, prodigiosin was highly produced at 30°C, but it was noticeably repressed at ≥37°C. Our initial results demonstrated that both the production and stability of the O-methyl transferase (PigF) and oxidoreductase (PigN) involved in the prodigiosin pathway in S. marcescens JNB5-1 sharply decreased at ≥37°C. Therefore, in this study, we improved mRNA stability and protein production using de novo polynucleotide fragments (PNFs) and the introduction of disulfide bonds, respectively, and observed their effects on prodigiosin production. Our results demonstrate that adding PNFs at the 3′-UTR of pigF and pigN significantly improved the mRNA half-life of these genes, leading to an increase in the transcript and expression levels. Subsequently, the introduction of disulfide bonds in pigF improved the thermal stability, pH stability, and copper ion resistance of PigF. Finally, shake flask fermentation showed that the prodigiosin titer with the engineered S. marcescens was increased by 61.38% from 5.36 g/L to 8.65 g/L when compared to the JNB5-1 strain at 30°C, and significantly, the prodigiosin yield increased 2.05-fold from 0.38 to 0.78 g/L at 37°C. In this study, we revealed that the introduction of PNFs and disulfide bonds greatly improved the expression and stability of pigF and pigN , hence efficiently enhancing prodigiosin production with S. marcescens at 30°C and 37°C. Importance This study highlights a promising strategy to improve mRNA/enzyme stability, and to increase production using de novo PNF libraries and the introduction of disulfide bonds into the protein. PNFs could increase the half-life of target gene mRNA and effectively prevent its degradation. Moreover, PNFs could increase the relative intensity of target genes without affecting the expression of other genes; as a result, it could alleviate the cellular burden compared to other regulatory elements such as promoters. Additionally, we obtained a PigF variant with improved activity and stability by the introduction of disulfide bonds into PigF. Collectively, this work demonstrates a novel approach for improving mRNA/enzyme stability using PNFs, which results in enhanced prodigiosin production in S. marcescens at 30°C.

With the increasing use of animal and plant proteins, pollution due to nitrogen sources is attracting increasing attention. In particular, the amount of nitrogen-containing sewage discharged into the environment has increased significantly, causing eutrophication of water bodies and environmental degradation of water quality. Traditionally, nitrifying bacteria perform ammonia nitrification under aerobic conditions, while denitrifying bacteria perform nitrate/nitrite denitrification under anaerobic conditions. However, heterotrophic nitrifying and aerobic denitrifying microorganisms (HNADs) perform ammonia nitrification and nitrate/nitrite denitrification under the same aerobic conditions using an organic carbon source, which is a much simpler and more efficient process. In this review, the distribution and evolutionary relationships of novel HNADs strains are presented, and the influencing factors, metabolic pathways, key enzymes, and practical applications of HNADs are reviewed.

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Wuxistatin, a novel statin and more potent than lovastatin, was converted from lovastatin by Amycolatopsis sp. (CGMCC 1149). Product I, an intermediate product, was found in the fermentation broth, and the structure analysis showed that product I had an additional hydroxyl group at the methyl group attached to C3 compared to lovastatin, which indicates that product I is one isomer of wuxistatin. Isotope tracing experiment proved that hydroxyl group of wuxistatin was provided by product I and the reaction from product I to wuxistatin was an intramolecular transfer. Hydroxylation reaction established in a cell-free system could be inhibited by CO and enhanced by ATP, Fe(2+), and ascorbic acid, which were consistent with the presumption that the hydroxylase was an induced cytochrome P450. Study on proteomics of Amycolatopsis sp. CGMCC 1149 suggested that three identified proteins, including integral membrane protein, Fe-S oxidoreductase, and GTP-binding protein YchF, were induced by lovastatin and required during hydroxylation reaction. In conclusion, bioconversion mechanism of wuxistatin by Amycolatopsis sp. CGMCC 1149 was proposed: lovastatin is firstly hydroxylated to product I by a hydroxylase, namely cytochrome P450, and then product I is rearranged to wuxistatin by isomerases.

Nitrogen pollution in water bodies is becoming increasingly serious, and how to remove nitrogen from water bodies economically and effectively has become a research hotspot. Especially in recent years, with the gradual expansion of aquaculture in China, the content of nitrite and other nitrogen-containing substances in water bodies has been increasing, which inhibits the growth of farm animals and is one of the causes of eutrophication in water bodies. In this study, a strain of bacteria was isolated from the sludge of an aquaculture fishpond and identified as Pseudomonas stutzeri, which can efficiently degrade nitrite. After continuous domestication in nitrite mixed solution, the nitrite nitrogen reduction capacity of P. stutzeri was significantly improved. Univariate experiments aiming to optimize the degradation conditions indicate that the optimal culture conditions for strain F2 are: medium with a carbon source of sodium succinate; C/N of 18; pH of 8; culture temperature of 28 °C; and shaking speed of 210 rpm in the shaker. Under the optimal culture conditions, the NO2−-N concentration of the culture solution was 300 mg/L, and the nitrite removal rate reached 98.67%. Meanwhile, the results of the nitrogen balance test showed that the strain converted 6.1% of the initial nitrogen into cellular organic nitrogen and 62.3% into gaseous nitrogen.