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*

The gene and the amino acid sequence of the structural and regulatory region of the Pseudomonas aeruginosa with different resistance patterns were analyzed. Six strains with different resistance patterns were selected and the AmpC beta-lactamase was identified. The objective gene fragment was amplified by colonies PCR. The sequences of the PCR-products were analyzed. The DNA sequence of the structural gene ampC and the regulatory genes ampR, ampD and ampE was detected. The 6 strains and the wild-type Pseudomonas aeruginosa are highly homogeneous in structural and regulatory region. Some new mutant points were found.
Bacterial Proteins/*genetics ; DNA, Bacterial/genetics ; Point Mutation/*genetics ; Pseudomonas aeruginosa/enzymology ; Pseudomonas aeruginosa/*genetics ; Sequence Analysis, DNA ; beta-Lactam Resistance/*genetics ; beta-Lactamases/*genetics

Members of the ferric uptake regulator (Fur) protein family are bacterial transcriptional repressors that control iron uptake and storage in response to iron availability, thereby playing a crucial role in the maintenance of iron homeostasis. The fur null mutants of Pseudomonas aeruginosa could not be obtained because fur is an essential gene. In this study, We constructed a Fur inducibly expression strain Δfur/attB::PBAD-fur in order to study the effect of fur on the growth, biofilm formation, motilities and oxidative stress response of P. aeruginosa. The results showed that a low level of fur expression retarded the growth of P. aeruginosa at an iron-depleted condition, or under high concentration of iron, or in the presence of H2O2. Fur affected the biofilm formation and the motilities (swimming, twitching, and swarming) of strain PAO1. The production of pyoverdine is regulated by Fur. Interestingly, proteins from Magnetospirillum gryphiswaldense MSR-1, which shares homology with Fur, can partially recover the pyoverdine production of strain Δfur/attB::PBAD-fur. This study provides new clues for the prevention and treatment of P. aeruginosa infections.
Bacterial Proteins/genetics* ; Hydrogen Peroxide ; Magnetospirillum ; Pseudomonas aeruginosa/genetics* ; Repressor Proteins/genetics*

Manipulation of genes, including knock-out or knock-in, replacement of gene elements (such as promoters), fusion with a fluorescent protein gene, and construction of in situ gene reporter, is required in most of the biotechnological laboratories. The widely used gene manipulating methods based on two-step allelic exchange are cumbersome in terms of constructing plasmids, transforming and screening. In addition, the efficiency of using this method for long fragment knockout is low. To simplify the process of gene manipulation, we constructed a minimized integrative vector pln2. When a gene needs to be inactivated, an internal fragment of the target gene (non-frameshift) is cloned into the pln2 plasmid. Once the single-crossover recombination between genome and the constructed plasmid occurs, the endogenous gene is segmented by the plasmid backbone and thus inactivated. We developed a toolbox based on pln2 that can be used for different genomic operation mentioned above. With the help of this toolbox, we successfully knocked out large fragments of 20-270 kb.
Genetic Vectors/genetics* ; Pseudomonas aeruginosa/genetics* ; Plasmids/genetics* ; Promoter Regions, Genetic ; Genome

OBJECTIVETo investigate the resistance genes and antibiotic resistance patterns against beta-lactams in Pseudomonas aeruginosa prevalent in burn ward.

METHODSK-B method was performed to test bacterial resistance patterns against 9 species of beta-lactams in Pseudomonas aeruginosa isolated from wounds and dressings of the patient in burn wards. Seven species of resistance genes against beta-lactams were detected with PCR. Tazobactam-inhibited piperacillin resistance test was performed to study whether the above strains produce extended spectrum beta-lactams.

RESULTSAll 12 strains of bacteria with resistance genes detected were resistant to penicillin and cephalosporins (100%), among them 11 were resistant to all antibiotics. Tazobactam-inhibited piperacillin resistance test demonstrated that all strains with resistance genes were ESBLs.

CONCLUSIONHigh incidence of beta-lactams resistance genes is found in Pseudomonas aeruginosa isolated from burn ward, and they have close relationship with the occurrence of multiple drug-resistance.

Burn Units ; Burns ; microbiology ; Genes, Bacterial ; Humans ; Pseudomonas aeruginosa ; drug effects ; genetics ; isolation & purification ; beta-Lactam Resistance ; genetics

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

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

Interleukin-8 (IL-8) is an important activator and chemoattratant of neutrophils and has been implicated in airway inflammatory diseases. To explore the new gene therapeutic strategies for airway inflammation, plasmid expressing dominant negative myeloid differentiation protein (MyD88 DN) was constructed and transfected into human airway epithelial cell lines A549 and SPC-A-I. The cells were challenged with M. tuberculosis, P. aeruginosa or K. pneumoniae and the release of IL-8 was measured using ELISA. The results showed that the supernatants of M. tuberculosis and R. aeruginosa enhanced IL-8 release from the epithelial cells; and transfection of MyD88 DN diminished this effect. MyD88 DN also reduced IL-8 release from cells induced by live bacteria of P. aeruginosa or K. pneumoniae. These data suggest that MyD88 could be used as a target gene in the gene therapy of airway inflammation.
Cells, Cultured ; Epithelial Cells ; microbiology ; secretion ; Humans ; Interleukin-8 ; secretion ; Mycobacterium tuberculosis ; Myeloid Differentiation Factor 88 ; genetics ; Pseudomonas aeruginosa ; Transfection

OBJECTIVETo investigate class I integrons and integrated gene cassettes in metalloenzyme-producing Pseudomonas aeruginosa.

METHODSA total of 68 isolated clinical strains of Pseudomonas aeruginosa were subjected to PCR analysis with primers specific for bla(IMP-1) and bla(VIM). The positive strains then underwent examination for class I integrons and integrated gene cassettes with PCR with primers specific to class I integrase ((IntI)1) and integrated gene cassettes, followed by sequence analysis for some of the positive strains.

RESULTSOnly 1 isolated strain showed positive results for both bla(IMP-1) and bla IntI1 detection. Fifty-five strains were positive for bla(VIM), including 26 positive for bla (IntI)1. Of the 26 bla (IntI)1-positive strains, only 18 contained integrated gene cassettes, which were classified into 5 types according to agarose gel electrophoresis.

CONCLUSIONIt is the first time to identify IMP-1-producing Pseudomonas aeruginosa carring bla(Int)1 in West China. The class I integrons were widespread in these Pseudomonas aeruginosa and 69.2% of them carry the gene cassettes. These findings provide useful insights into the clinical spread of these drug-resistant genes.

Bacterial Proteins ; genetics ; metabolism ; DNA, Bacterial ; analysis ; genetics ; Drug Resistance, Bacterial ; genetics ; Electrophoresis, Agar Gel ; Humans ; Integrons ; genetics ; Polymerase Chain Reaction ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; enzymology ; genetics ; isolation & purification ; Species Specificity ; beta-Lactamases ; genetics ; metabolism

OBJECTIVETo study the clinical significance of virulence genes exo U and exo S of type III secretion system (TTSS) of Pseudomonas aeruginosa (PA).

METHODSOne hundred and eighty-nine clinical isolates of PA were collected from five hospitals. The incidence of virulence genes exo U and exo S in PA were determined with PCR. Minimum inhibitory concentration of anti-bacterial drug for PA was determined with microdilution method. The clinical features and outcomes of 60 hospitalized patients colonized or infected with exo U+/exo S- positive or exo U-/exo S+ positive PA isolated from sputum were analyzed retrospectively. Data were processed with chi-square test.

RESULTSAmong the 189 PA isolates, 85.2% (161/189) harbored TTSS genes, including exo U-/exo S+ type (120 isolates), exo U+/exo S- type (31 isolates), exo U-/exo S- type (7 isolates), and exo U+/exo S+ type (3 isolates). 72.0% (72/100) isolates from sputum and 81.5% (44/54) isolates from blood belonged to exo U-/exo S+ genotype. Compared with those of TTSS-negative isolates, the antimicrobial resistance of TTSS-positive isolates to cefoperazone/sulbactam, ceftazidime, amikacin, and cefepime were lower (with χ² value respectively 10.1, 16.1, 9.3, 33.8, P values all below 0.01). The antimicrobial resistance to all examined drug between exo U-/exo S+ type and exo U+/exo S- type isolates was close (with χ² values from 0.08 to 2.04, P values all above 0.05). Patients detected with exo U+/exo S- positive PA isolated from sputum were significantly associated with PA infection, and they usually had history of tracheal intubation, ICU hospitalization, and combined use of drugs for anti-infection treatment. Patients detected with exo U-/exo S+ positive PA isolated from sputum were significantly associated with PA colonization, which had basic lung disease and better outcome than the former infection type.

CONCLUSIONSThe TTSS exists in most clinical isolates of PA. Detection of exo U or exo S of PA isolated from sputum is helpful for the analysis of clinical features and outcome of patients.

ADP Ribose Transferases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Bacterial Secretion Systems ; genetics ; Bacterial Toxins ; genetics ; metabolism ; Drug Resistance, Bacterial ; Genes, Bacterial ; Humans ; Microbial Sensitivity Tests ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; genetics ; isolation & purification ; pathogenicity ; Retrospective Studies ; Virulence