The Serine Biosynthesis of Paenibacillus polymyxa WLY78 Is Regulated by the T-Box Riboswitch.

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.

Enhanced NADH Metabolism Involves Colistin-Induced Killing of Bacillus subtilis and Paenibacillus polymyxa.

The commonly believed mechanism of colistin against Gram-negative bacteria is to cause cell membrane lysis, whereas the mechanism of colistin against Gram-positive bacteria is extremely fragmented. In this study, we found that colistin treatment on Bacillus subtilis WB800, Paenibacillus polymyxa C12 and Paenibacillus polymyxa ATCC842 enhances not only the activities of α-ketoglutaric dehydrogenase and malate dehydrogenase in tricarboxylic acid (TCA) cycle, but also the relative expression levels of their encoding genes. Additionally, the oxaloacetate concentration also increases. Interestingly, the analysis of the relative expression of genes specific for respiratory chain showed that colistin treatment stimulates the respiratory chain in Gram-positive bacteria. Accordingly, the NAD⁺/NADH ratio increases and the oxidative level is then boosted up. As a result, the intensive oxidative damages are induced in Gram-positive bacteria and cells are killed. Notably, both rotenone and oligomycin, respectively, inhibiting NADH dehydrogenase and phosphorylation on respiratory chain can downgrade oxidative stress formation, thus alleviating the colistin-induced killing of Gram-positive cells. Besides, thiourea-based scavenging for reactive oxygen species also rescues the colistin-subjected cells. These data collectively demonstrate that colistin stimulates both TCA cycle and respiratory chain in Gram-positive bacteria, leading to the enhancement of NADH metabolism and resulting in the generation of oxidative damages in Gram-positive cells. Our studies provide a better understanding of antibacterial mechanism of colistin against Gram-positive bacteria, which is important for knowledge on bacterial resistance to colistin happening via the inhibition of respiratory chain and manipulation of its production.

Enhanced Production of Polymyxin E in Paenibacillus polymyxa by Replacement of Glucose by Starch.

Polymyxin E or colistin, produced by Paenibacillus polymyxa, is an important antibiotic against Gram-negative pathogens. The objective of this study is to evaluate the effect of starch in fermentation medium on colistin biosynthesis in P. polymyxa. The results indicated that replacement of glucose by starch stimulated colistin production and biosynthesis rate. Overall, the stimulation extent was starch concentration-dependent. As expected, addition of starch induced the expression of amyE encoding amylase and increased amylase activity in fermentation solution. Additionally, replacement of glucose by starch resulted in residue reducing sugar and pH of fermentation mixture low relative to glucose as the sole sugar source. At the molecular level, it was found that replacement of glucose by starch has enhanced the relative expression level of ccpA encoding catabolite control protein A. Therefore, the repression of starch utilization by glucose could be probably relieved. In addition, use of starch stimulated the expression of regulatory gene spo0A but repressed the expression of another regulatory gene abrB. As a result, the expression of genes directly involved in colistin biosynthesis and secretion increased, indicating that at the transcriptional level spo0A and abrB played opposite roles in regulating colistin biosynthesis in P. polymyxa. Taken together, our data demonstrated that starch instead of glucose can promote colistin production probably by affecting the expression of colistin biosynthesis-related genes, as well as reducing the repression of glucose to a secondary metabolic product.

Investigation of relationship between 2,3-butanediol toxicity and production during growth of Paenibacillus polymyxa.

Understanding the capacity of Paenibacillus polymyxa DSM 365 to tolerate increasing concentrations of 2,3-butanediol (2,3-BD) is critical to engineering a 2,3-BD-overproducing strain. Hence, we investigated the response of P. polymyxa to high 2,3-BD concentrations. In fed-batch cultures (6-L bioreactor) 2,3-BD was accumulated to a maximum concentration of 47g/L despite the presence of residual 13g/L glucose in the medium. Concomitantly, accumulation of acetoin, the precursor of 2,3-BD increased after maximum 2,3-BD concentration was reached, suggesting that 2,3-BD was reconverted to acetoin after the concentration tolerance threshold of 2,3-BD was exceeded. Cultures of P. polymyxa were then challenged with levo-2,3-BD (20, 40 and 60g/L) at 0h in a glucose medium, and a concentration dependent growth inhibition response to levo-2,3-BD was observed. The growth of P. polymyxa was completely inhibited by 60g/L levo-2,3-BD. Furthermore, P. polymyxa was challenged with incremental 2,3-BD concentrations (20, 40 and 60g/L at 12, 24 and 36h, respectively) to mimic 2,3-BD accumulation during fermentation. Interestingly, 2,3-BD was reconverted to acetoin when its concentration reached 60g/L, possibly to alleviate 2,3-BD toxicity. Collectively, our findings indicate that 2,3-BD-mediated toxicity is a major metabolic impediment to 2,3-BD overproduction, thus, making it an important metabolic engineering target towards rational design of a 2,3-BD-overproducing strain.