Pyocin S2 and S4 in Pseudomonas aeruginosa use the same uptake channels as the pyoverdine does in bacteria, indicating a possible connection between them. In this study, we characterized the single bacterial gene expression distribution of three S-type pyocins (Pys2, PA3866, and PyoS5) and examined the impact of pyocin S2 on bacterial uptake of pyoverdine. The findings demonstrated that the expression of the S-type pyocin genes was highly differentiated in bacterial population under DNAdamage stress. Moreover, exogenous addition of pyocin S2 reduces the bacterial uptake of pyoverdine so that the presence of pyocin S2 prevents the uptake of environmental pyoverdine by non-pyoverdine synthesizing 'cheaters', thereby reducing their resistance to oxidative stress. Furthermore, we discovered that overexpression of the SOS response regulator PrtN in bacteria significantly decreased the expression of genes involved in the synthesis of pyoverdine, significantly decreasing the overall synthesis and exocytosis of pyoverdine. These findings imply a connection between the function of the iron absorption system and the SOS stress response mechanism in bacteria.
Pyocins/metabolism* ; Pseudomonas aeruginosa/metabolism*

Lysis is a common functional module in synthetic biology and is widely used in genetic circuit design. Lysis could be achieved by inducing expression of lysis cassettes originated from phages. However, detailed characterization of lysis cassettes hasn't been reported yet. Here, we first adopted arabinose- and rhamnose-inducible systems to develop inducible expression of five lysis cassettes (S₁₀₅, A52G, C51S S76C, LKD, LUZ) in Escherichia coli Top10. By measuring OD₆₀₀, we characterized the lysis behavior of strains harboring different lysis cassettes. These strains were harvested at different growth stages, induced with different concentrations of chemical inducers, or contained plasmids with different copy numbers. We found that although all five lysis cassettes could induce bacterial lysis in Top10, lysis behaviors differed a lot at various conditions. We further found that due to the difference in background expression levels between strain Top10 and Pseudomonas aeruginosa PAO1, it was hard to construct inducible lysis systems in strain PAO1. The lysis cassette controlled by rhamnose-inducible system was finally inserted into the chromosome of strain PAO1 to construct lysis strains after careful screen. The results indicated that LUZ and LKD were more effective in strain PAO1 than S₁₀₅, A52G and C51S S76C. At last, we constructed an engineered bacteria Q16 using an optogenetic module BphS and the lysis cassette LUZ. The engineered strain was capable of adhering to target surface and achieving light-induced lysis by tuning the strength of ribosome binding sites (RBSs), showing great potential in surface modification.
Rhamnose/pharmacology* ; Plasmids/genetics* ; Pseudomonas aeruginosa ; Escherichia coli/metabolism*

OBJECTIVETo investigate the relationship among oxygen concentration, quorum sensing system, type secretion system, and biofilm production of Pseudomonas aeruginosa.

METHODSA total of 23 clinical strains of Pseudomonas aeruginosa were cultured at different levels of environmental oxygen for three days. Then biofilm mass and alginate were quantified. The expression levels of LasI and RhlI were detected by real time polymerase chain reaction (PCR). The secretion of exoenzyme S was examined by Western blot.

RESULTSBoth the biofilm mass (R=0.455, P=0.000) and alginate (R=0.367, P=0.000) were positively correlated with oxygen concentration. Real time PCR showed that the expression levels of LasI and RhlI were not significantly correlated with oxygen concentration (R=0.025, P=0.794; R=-0.044, P=0.653), the production of biofilm (R=0.001, P=0.990; R=0.011, P=0.909), or alginate(R=0.029, P=0.770; R=0.193, P=0.064). Western blot showed that the optimal oxygen concentration range for exoenzyme S secretion of Pseudomonas aeruginosa ranged 10% to 30%.

CONCLUSIONSHyperoxia can promote the production of biofilm and alginate by Pseudomonas aeruginosa. Las/Rhl system may not participate in biofilm production at the early stage due to the low bacteria amount. The increased production of biofilm may inhibit the expression of Type Secretion system and thus inhibit bacterial virulence.

Alginates ; metabolism ; Biofilms ; drug effects ; Oxygen ; metabolism ; Pseudomonas aeruginosa ; metabolism ; physiology ; Quorum Sensing ; drug effects ; physiology

随着铜绿假单胞菌（铜绿）的耐药性逐年增强，铜绿感染已经成为公共医疗卫生的重点关注问题。线粒体自噬及其介导的线粒体功能障碍在多种细菌感染中已被报道，但线粒体功能障碍在宿主调控铜绿感染中的作用尚不明确。因此，本研究建立铜绿刺激小鼠巨噬细胞感染模型和小鼠急性铜绿感染模型，探讨铜绿是否通过诱导线粒体自噬改变线粒体功能，进而影响宿主免疫炎症反应和细胞毒性，并通过监测生存率和肺组织病理学变化进一步确定线粒体自噬在小鼠铜绿体内感染模型中的作用。结果表明，铜绿引起小鼠腹腔巨噬细胞线粒体功能障碍，并通过线粒体自噬途径清除铜绿刺激引起的活性氧（ROS）累积，从而抑制铜绿引起的促炎性细胞因子分泌并增强细胞毒性。体内实验进一步确认线粒体自噬在铜绿体内感染中的作用。
Mice ; Animals ; Reactive Oxygen Species/metabolism* ; Pseudomonas aeruginosa ; Macrophages/metabolism* ; Mitochondria ; Cytokines/metabolism*

In order to investigate the role of the MexA-MexB-OprM efflux pump system in the pathogenesis of Pseudomonas aeruginosa (PA)-induced pulmonary infection, pulmonary infection models were established by intratracheal injection of K767 (wild type), nalB (MexA-MexB-OprM up-regulated mutant), and ΔmexB (knockout) strains, separately. All mice were treated with Meropenem (intraper Δ itoneal injection, 100 mg/kg body weight, twice every day), and strain-related pathology, bacteria count, cytokine level, myeloperoxidase (MPO, indicator of neutrophil recruitment) activity, and macrophage inflammatory protein-2 (MIP-2) expression were evaluated at early (3rd day post-infection) and late (7th and 14th day post-infection) stages of infection. E-test showed that ΔmexB was more significantly Δ sensitive to panipenan (ETP), meropenem (MP) and imipenem (IP) than K767 and nalB strains. There was no significant difference in sensitivity to cefepime (TM) among the three stains. In contrast to the K767 and nalB groups, the ΔmexB group showed decreased bacteria burden over time and less exte Δ nsive pathological change. Additionally, MPO activity and levels of inflammatory cytokines (IL-1b, IL-12, and TNF-α) were increased at the early stage (day 3) and decreased at the later stage (day 14). Serum MIP-2 expression level was steadily increased in all three groups from early to late stages, but significantly higher in ΔmexB group than in K767 and nalB groups ( Δ P<0.05). In conclusion, the MexA-MexB-OprM efflux pump system might play an important role in PA-induced chronic pulmonary infection. High expression of the MexA-MexB-OprM efflux pump could increase antibacterial resistance and promote infection.
Animals ; Bacterial Outer Membrane Proteins ; metabolism ; Lung ; microbiology ; Membrane Transport Proteins ; metabolism ; Mice ; Mice, Inbred BALB C ; Pseudomonas Infections ; metabolism ; microbiology ; Pseudomonas aeruginosa ; metabolism

OBJECTIVETo investigate the characteristic and biochemical mechanism about the phenol biodegradation by bacterial strains ZD 4-1 and ZD 4-3.

METHODSBacterial strains ZD 4-1 and ZD 4-3 were isolated by using phenol as the sole source of carbon and energy, and identified by 16S rDNA sequence analysis. The concentrations of phenol and total organic carbon (TOC) were monitored to explore the degradation mechanism. The biodegradation intermediates were scanned at 375 nm by using a uv-vis spectrophotometer. The enzyme assays were performed to detect the activities of dioxygenases.

RESULTSBacterial strains ZD 4-1 and ZD 4-3 were identified as Comamonas testosteroni and Pseudomonas aeruginosa by 16S rDNA sequence analysis, respectively. The growth of the two strains was observed on a variety of aromatic hydrocarbons. The strains ZD 4-1 and ZD 4-3 metabolized phenol via ortho-pathways and meta-pathways, respectively. In addition, the results of enzyme assays showed that the biodegradation efficiency of phenol by meta-pathways was higher than that by ortho-pathways. Finally, the results of induction experiment indicated that the catechol dioxygenases, both catechol 1,2-dioxygenase (C120) and catechol 2,3-dioxygenase (C230), were all inducible.

CONCLUSIONThe strains ZD 4-1 and ZD 4-3 metabolize phenol through ortho-pathways and meta-pathway, respectively. Furthermore, the biodegradation efficiency of phenol by meta-pathways is higher than that by ortho-pathways.

Biodegradation, Environmental ; Comamonas testosteroni ; physiology ; Disinfectants ; metabolism ; Oxygenases ; pharmacology ; Phenol ; metabolism ; Pseudomonas aeruginosa ; physiology ; Water Pollutants ; metabolism

PURPOSE: Two Korean nationwide studies showed that metallo-beta-lactamases (MBLs)-producing-Pseudomonas spp. are not rare. The aim of this study was to assess the trends of MBL-producing isolates among imipenem-resistant isolates of Pseudomonas spp. MATERIALS AND METHODS: Imipenem-resistant clinical isolates were collected from 23 hospitals and one commercial laboratory participating in the KONSAR program in 2005. Polymerase chain reaction (PCR) was used to detect MBL genes. RESULTS: Alleles of MBL genes were detected in 10.8% of 415 Pseudomonas aeruginosa and 66.7% of 12 P. putida isolates from 18 of 24 hospitals/laboratory. Among the 14 IMP-1-like and 39 VIM-2-like MBLs, emergence of IMP-6 was detected for the first time. CONCLUSION: Prevalence of MBL-producing P. aeruginosa has not significantly increased, but IMP-6 emerged in P. aeruginosa.
Anti-Bacterial Agents/pharmacology ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Imipenem/pharmacology ; Korea ; Polymerase Chain Reaction ; Pseudomonas Infections/*microbiology ; Pseudomonas aeruginosa/drug effects/genetics/*metabolism ; beta-Lactamases/genetics/*metabolism

OBJECTIVETo observe the release of DNA from Pseudomonas aeruginosa (P. aeruginosa) induced by different concentrations of piperacillin/tazobactam (Piper) in vitro.

METHODSThe minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of Piper against 1244 strain (ATCC 27317) of P. aeruginosa were determined, respectively. This strain of P. aeruginosa was separately cultured with Piper in different concentrations at 37 degrees C for 4 h and 24 h. The samples of cultural supernatant were filtered and electrophoresis was conducted in 1.8% agarose with SYBR Gold stain. Then the images of the gel sheets were photographed.

RESULTSThis strain of P. aeruginosa was sensitive to Piper. The bacterial DNA was not detected in 4-h cultured P. aeruginosa either with or without Piper by this method. The bacterial DNA molecules could be detected in 24 h samples in cultures without Piper, and they were displayed in two zones of molecular weight over 2000 base pairs (bp) and lower than 100 bp. Similar results were observed when the MIC of piper (0.002, 0.004 g/L) were under the MIC measured at the 3rd time (0.008 g/L), but there was much more bacterial DNA with molecular weight lower than 100 bp. When Piper concentration was higher than its MIC, only smaller quantities of bacterial DNA in the area with molecular weight lower than 400 bp could be detected in 24-h culture samples.

CONCLUSIONA certain amount of bacterial DNA was released from P. aeruginosa under its natural growth circumstance. Different concentrations of Piper showed different effects on DNA release, in regard to its quantity and molecular weight, from P. aeruginosa cultures.

Anti-Bacterial Agents ; pharmacology ; DNA, Bacterial ; metabolism ; Penicillanic Acid ; analogs & derivatives ; pharmacology ; Piperacillin ; pharmacology ; Pseudomonas aeruginosa ; drug effects ; metabolism

To explore the biofilm inhibitory efficacy of perifosine against Pseudomonas aeruginosa (P. aeruginos) and its mechanisms. Twenty-fourwell plate was used to form biofilms at the bottom and crystal violet staining was used to determine the biofilm inhibitory effects of perifosine against P. aeruginosa, the wells without perifosine was set as control group. Glass tubes combined with crystal violet staining was used to detect the gas-liqud interface related bioiflm inhibitory effects of perifosine, the wells without perifosine was set as control group. Time-growth curved was used to detect the effects of perifosine on the bacteial planktonic cells growth of P. aeruginosa, the wells without perifosine was set as control group. The interaction model between perifosine and PqsE was assessed by molecular docking assay. The inhibitory effects of perifosine on the catalytic activity of PqsE was determined by detection the production of thiols, the wells without perifosine was set as control group. Binding affinity between perifosine and PqsE was detected by plasma surface resonance. The biofims at the bottom of the microplates and air-liquid interface were effectively inhibited by perifosine at the concentration of 4-8 μg/ml. There was no influence of perifosine on the cells growth of P. aeruginosa. The resuts of molecular docking assay indicates that perifosine could interacted with PqsE with the docking score of -10.67 kcal/mol. Perifosine could inhibit the catalytic activity of PqsE in a dose-dependent manner. The binding affinity between perifosine and PqsE was comfirmed by plasma surface resonance with KD of 6.65×10^-5mol/L. Perifosine could inhibited the biofilm formation of P. aeruginosa by interacting with PqsE.
Anti-Bacterial Agents/pharmacology* ; Bacterial Proteins/metabolism* ; Biofilms ; Molecular Docking Simulation ; Phosphorylcholine/analogs & derivatives* ; Pseudomonas aeruginosa/metabolism* ; Quorum Sensing

Pseudomonas aeruginosa is an important pathogenic bacterium of nosocomial infections. The microbe easily produce biofilm which brings us much difficulties in clinical treatment. The formation processes of biofilm, including the stages of early bacteria planting, mushroom-like structure forming and extracellular matrix producing, are regulated by a series of molecules and genes. And quorum sensing system of the microbe is responsible for regulation of the whole process of biofilm formation. According to the process of biofilm formation and the mimitat associated regulation mechanism, several anti-biofilm therapeutic strategies have been applied in clinical medicine, and some novel drugs and methods are developed.
Biofilms ; growth & development ; Gene Expression Regulation, Bacterial ; Polysaccharides, Bacterial ; metabolism ; Pseudomonas Infections ; drug therapy ; microbiology ; Pseudomonas aeruginosa ; genetics ; physiology ; Quorum Sensing ; genetics ; physiology