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.

Members of the Sphingomonadales are renowned for their ability to degrade polycyclic aromatic hydrocarbons (PAHs). However, little is known about the regulatory mechanisms of the degradative pathway. Using cross-feeding bioassay, a functional LuxI/LuxR-type acyl-homoserine lactone (AHL)-mediated quorum sensing (QS) system was identified from Croceicoccus naphthovorans PQ-2, a member of the order Sphingomonadales. Inactivation of the QS system resulted in a significant decrease in PAHs degradation. The QS system positively controlled the expression of three PAH-degrading genes (ahdA1e, xylE and xylG) and a regulatory gene ardR, which are located on the large plasmid. Interestingly, the transcription levels of these three PAH-degrading genes were significantly down-regulated in the ardR mutant. In addition, bacterial cell surface hydrophobicity and cell morphology were altered in the QS-deficient mutant. Therefore, the QS system in strain PQ-2 positively regulates PAH degradation via two mechanisms: (i) by induction of PAH-degrading genes directly and/or indirectly; and (ii) by an increase of bacterial cell surface hydrophobicity. The findings of this study improve our understanding of how the QS system influences the degradation of PAHs, therefore facilitating the development of new strategies for the bioremediation of PAHs.

Nitrite has been used as a bacteriostatic agent for centuries in food preservation. It is widely accepted that this biologically inert molecule functions indirectly, serving as a stable reservoir of bioactive nitric oxide (NO) and other reactive nitrogen species to impact physiology. As a result, to date, we know surprisingly little about in vivo targets of nitrite. Here, we carry out comparative analyses of nitrite and NO physiology in Escherichia coli and in Shewanella oneidensis , a Gram-negative environmental bacterium renowned for respiratory versatility. These two bacteria differ from each other in many aspects of nitrite and NO physiology, including NO generation, NO degradation, and unexpectedly, their contrary susceptibility to nitrite and NO. In cell extracts of both bacteria, most of the NO targets are also susceptible to nitrite, and vice versa. However, with respect to growth inhibition caused by NO, the targets are impacted distinctly; NO targets are responsible for the inhibition of growth of E. coli but not of S. oneidensis . More surprisingly, all proteins identified to be implicated in NO tolerance in other bacteria appear to play a dispensable role in protecting S. oneidensis against NO. These data suggest that S. oneidensis is equipped with a robust but yet unknown NO protecting system. In the case of nitrite, it is clear that the target of physiological significance in both bacteria is cytochrome heme-copper oxidase. IMPORTANCE Nitrite is toxic to living organisms at high levels, but such antibacterial effects of nitrite are attributable to the formation of nitric oxide (NO), a highly reactive radical gas molecule. Here, we report that Shewanella oneidensis is highly resistant to NO but sensitive to nitrite compared to Escherichia coli by approximately 4-fold. In both bacteria, nitrite inhibits bacterial growth by targeting cytochrome heme-copper oxidase. In contrast, the targets of NO are diverse. Although these targets are similar in E. coli and S. oneidensis , they are responsible for growth inhibition caused by NO in the former but not in the latter. Overall, the presented data, along with the previous data, solidify a proposal that the in vivo targets of NO and nitrite in bacteria are largely different.

Production of chromosome-encoded β-lactamases confers resistance to β-lactams in many Gram-negative bacteria. Some inducible β-lactamases, especially the class C β-lactamase AmpC in Enterobacteriaceae, share a common regulatory mechanism, the ampR-ampC paradigm. Induction of ampC is intimately linked to peptidoglycan recycling, and the LysR-type transcriptional regulator AmpR plays a central role in the process. However, our previous studies have demonstrated that the expression of class D β-lactamase gene blaA in Shewanella oneidensis is distinct from the established paradigm since an AmpR homolog is absent and major peptidoglycan recycling enzymes play opposite roles in β-lactamase expression. Given that lytic transglycosylases (LTs), a class of peptidoglycan hydrolases cleaving the β-1,4 glycosidic linkage in glycan strands of peptidoglycan, can disturb peptidoglycan recycling, and thus may affect induction of blaA. In this study, we investigated impacts of such enzymes on susceptibility to β-lactams. Deletion of three LTs (SltY, MltB and MltB2) increased β-lactam resistance, while four other LTs (MltD, MltD2, MltF, and Slt2) seemed dispensable to β-lactam resistance. The double LT mutants ΔmltBΔmltB2 and ΔsltYΔmltB2 had β-lactam resistance stronger than any of the single mutants. Deletion of ampG (encoding permease AmpG) and mrcA (encoding penicillin binding protein 1a, PBP1a) from both double LT mutants further increased the resistance to β-lactams. Notably, all increased β-lactam resistance phenotypes were in accordance with enhanced blaA expression. Although significant, the increase in β-lactamase activity after inactivating LTs is much lower than that produced by PBP1a inactivation. Our data implicate that LTs play important roles in blaA expression in S. oneidensis.

The growing problem of antibiotic resistance in Gram-negative bacteria urgently needs new strategies so that researchers can develop novel antibiotics. Microbial whole-cell biosensors are capable of sensing various stimuli with a quantifiable output and show tremendous potential for the discovery of novel antibiotics.