Exploring Regulation Genes Involved in the Expression of L-Amino Acid Oxidase in Pseudoalteromonas sp. Rf-1

Bacterial L-amino acid oxidase (LAAO) is believed to play important biological and ecological roles in marine niches, thus attracting increasing attention to understand the regulation mechanisms underlying its production. In this study, we investigated genes involved in LAAO production in marine bacterium Pseudoalteromonas sp. Rf-1 using transposon mutagenesis. Of more than 4,000 mutants screened, 15 mutants showed significant changes in LAAO activity. Desired transposon insertion was confirmed in 12 mutants, in which disrupted genes and corresponding functionswere identified. Analysis of LAAO activity and lao gene expression revealed that GntR family transcriptional regulator, methylase, non-ribosomal peptide synthetase, TonB-dependent heme-receptor family, Na⁺/H⁺ antiporter and related arsenite permease, N-acetyltransferase GCN5, Ketol-acid reductoisomerase and SAM-dependent methytransferase, and their coding genes may be involved in either upregulation or downregulation pathway at transcriptional, posttranscriptional, translational and/or posttranslational level. The nhaD and sdmT genes were separately complemented into the corresponding mutants with abolished LAAO-activity. The complementation of either gene can restore LAAO activity and lao gene expression, demonstrating their regulatory role in LAAO biosynthesis. This study provides, for the first time, insights into the molecular mechanisms regulating LAAO production in Pseudoalteromonas sp. Rf-1, which is important to better understand biological and ecological roles of LAAO

Precursor Amino Acids Inhibit Polymyxin E Biosynthesis in Paenibacillus polymyxa, Probably by Affecting the Expression of Polymyxin E Biosynthesis-Associated Genes

Polymyxin E belongs to cationic polypeptide antibiotic bearing four types of direct precursor amino acids including L-2,4-diaminobutyric acid (L-Dab), L-Leu, D-Leu, and L-Thr. The objective of this study is to evaluate the effect of addition of precursor amino acids during fermentation on polymyxin E biosynthesis in Paenibacillus polymyxa. The results showed that, after 35 h fermentation, addition of direct precursor amino acids to certain concentration significantly inhibited polymyxin E production and affected the expression of genes involved in its biosynthesis. L-Dab repressed the expression of polymyxin synthetase genes pmxA and pmxE, as well as 2,4-diaminobutyrate aminotransferase gene ectB; both L-Leu and D-Leu repressed the pmxA expression. In addition, L-Thr affected the expression of not only pmxA, but also regulatory genes spo0A and abrB. As L-Dab precursor, L-Asp repressed the expression of ectB, pmxA, and pmxE. Moreover, it affected the expression of spo0A and abrB. In contrast, L-Phe, a nonprecursor amino acid, had no obvious effect on polymyxin E biosynthesis and those biosynthesis-related genes expression. Taken together, our data demonstrated that addition of precursor amino acids during fermentation will inhibit polymyxin E production probably by affecting the expression of its biosynthesis-related genes

Enhanced NADH Metabolism Involves Colistin-Induced Killing of <em>Bacillus subtilis</em> and <em>Paenibacillus polymyxa</em>

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

Oxidative Stress Induced by Polymyxin E Is Involved in Rapid Killing of Paenibacillus polymyxa

Historically, the colistin has been thought to kill bacteria through membrane lysis. Here, we present an alternative mechanism that colistin induces rapid Paenibacillus polymyxa death through reactive oxygen species production. This significantly augments our understanding of the mechanism of colistin action, which is critical knowledge toward the yield development of colistin in the future

Antibacterial Mechanisms of Polymyxin and Bacterial Resistance

Multidrug resistance in pathogens is an increasingly significant threat for human health. Indeed, some strains are resistant to almost all currently available antibiotics, leaving very limited choices for antimicrobial clinical therapy. In many such cases, polymyxins are the last option available, although their use increases the risk of developing resistant strains. This review mainly aims to discuss advances in unraveling the mechanisms of antibacterial activity of polymyxins and bacterial tolerance together with the description of polymyxin structure, synthesis, and structural modification. These are expected to help researchers not only develop a series of new polymyxin derivatives necessary for future medical care, but also optimize the clinical use of polymyxins with minimal resistance development

Polymyxin E Induces Rapid Paenibacillus polymyxa Death by Damaging Cell Membrane while Ca2+ Can Protect Cells from Damage.

Polymyxin E, produced by Paenibacillus polymyxa, is an important antibiotic normally against Gram-negative pathogens. In this study, we found that polymyxin E can kill its producer P. polymyxa, a Gram-positive bacterium, by disrupting its cell membrane. Membrane damage was clearly revealed by detecting the leakage of intracellular molecules. The observation using scanning electron microscopy also supported that polymyxin E can destroy the cell membrane and cause an extensive cell surface alteration. On the other hand, divalent cations can give protection against polymyxin E. Compared with Mg2+, Ca2+ can more effectively alleviate polymyxin E-induced damage to the cell membrane, thus remarkably increasing the P. polymyxa survival. Our findings would shed light on a not yet described bactericidal mechanism of polymyxin E against Gram-positive bacteria and more importantly the nature of limited fermentation output of polymyxin E from P. polymyxa

Correlation between the detected H₂O₂ concentration and the diameters of Prussian blue holes.

<p>The distribution can perfectly be fitted with the exponential equation y = 0.673×^5.611, where x is the diameter of blue hole and y the H₂O₂ concentration. The inset showed that in the range of 0.5 mM≤H₂O₂≤20 mM, the change in diameter of blue hole was a function of logarithm of H₂O₂ concentration with linear fits under the equation y = 1.772x–1.890, where x is the diameter of blue hole and y the logarithm of H₂O₂ concentration.</p

Comparative Genomics of Methanopyrus sp. SNP6 and KOL6 Revealing Genomic Regions of Plasticity Implicated in Extremely Thermophilic Profiles

Methanopyrus spp. are usually isolated from harsh niches, such as high osmotic pressure and extreme temperature. However, the molecular mechanisms for their environmental adaption are poorly understood. Archaeal species is commonly considered as primitive organism. The evolutional placement of archaea is a fundamental and intriguing scientific question. We sequenced the genomes of Methanopyrus strains SNP6 and KOL6 isolated from the Atlantic and Iceland, respectively. Comparative genomic analysis revealed genetic diversity and instability implicated in niche adaption, including a number of transporter- and integrase/transposase-related genes. Pan-genome analysis also defined the gene pool of Methanopyrus spp., in addition of ~120-Kb genomic region of plasticity impacting cognate genomic architecture. We believe that Methanopyrus genomics could facilitate efficient investigation/recognition of archaeal phylogenetic diverse patterns, as well as improve understanding of biological roles and significance of these versatile microbes