Excess SeqA leads to replication arrest and a cell division defect in Vibrio cholerae

Although most bacteria contain a single circular chromosome, some have complex genomes, and all Vibrio species studied so far contain both a large and a small chromosome. In recent years, the divided genome of Vibrio cholerae has proven to be an interesting model system with both parallels to and novel features compared with the genome of Escherichia coli. While factors influencing the replication and segregation of both chromosomes have begun to be elucidated, much remains to be learned about the maintenance of this genome and of complex bacterial genomes generally. An important aspect of replicating any genome is the correct timing of initiation, without which organisms risk aneuploidy. During DNA replication in E. coli, newly replicated origins cannot immediately reinitiate because they undergo sequestration by the SeqA protein, which binds hemimethylated origin DNA. This DNA is already methylated by Dam on the template strand and later becomes fully methylated; aberrant amounts of Dam or the deletion of seqA leads to asynchronous replication. In our study, hemimethylated DNA was detected at both origins of V. cholerae, suggesting that these origins are also subject to sequestration. The overproduction of SeqA led to a loss of viability, the condensation of DNA, and a filamentous morphology. Cells with abnormal DNA content arose in the population, and replication was inhibited as determined by a reduced ratio of origin to terminus DNA in SeqA-overexpressing cells. Thus, excessive SeqA negatively affects replication in V. cholerae and prevents correct progression to downstream cell cycle events such as segregation and cell division.     

Effect of permeabilizers on antibiotic sensitivity of Pseudomonas aeruginosa

Agents which had previously been shown to act as permeabilizers against Pseudomonas aeruginosa or other Gram-negative bacteria were tested to determine whether susceptibility to various antibiotics could be increased. In the absence of a permeabilizer, Ps. aeruginosa was resistant to several hydrophobic antibiotics and vancomycin, but not to gentamicin. Tartaric and gluconic acids had weak potentiating activity, whereas ethylenediamine tetraacetic acid and citric acid were more effective permeabilizers. However, sodium polyphosphate enhanced the activity of erythromycin, fucidin, novobiocin, rifampicin and methicillin; vancomycin was unaffected and the activity of gentamicin was reduced considerably.     

High temperature induced antibiotic sensitivity changes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, which was resistant to a wide variety of antibiotics, became sensitive to several of these antibiotics when grown and tested at 46 degrees C. Cell wall antibiotics such as penicillin G and ampicillin were only effective when added to cells growing at 46 degrees C prior to a temperature shift to 37 degrees C. Antibiotics which penetrate the cytoplasmic membrane to express their inhibiting action present a pattern different from those which are active against the outer cell wall. In order that these compounds be effective, the permeability of the cytoplasmic membrane must be further altered with agents such as EDTA which allow the penetration of actinomycin D. Inhibitors of protein synthesis, such as streptomycin and chloramphenicol, have increased access to their sites of action in cells grown at 46 degrees C. Cells grown at 46 degrees C have 40% less lipopolysaccharide (LPS) than cells grown at 37 degrees C and the LPS aggregates were of large molecular size in cells grown at 46 degrees C. Growth at 46 degrees C affects the permeability properties of the outer cell wall more than the permeability properties of the cytoplasmic membrane and this was due, in part, to the selective release of LPS of LPS-protein complexes at elevated growth temperatures.     

htpR-like gene controls cell division and proteolysis in Pseudomonas aeruginosa

As shown in hybridization experiments, the genome of Pseudomonas aeruginosa cells contains a htpR-like gene which controls the expression of heat shock genes in cells of Escherichia coli. By means of specially constructed plasmids, the synthesis of htpR antisense RNA has been found to disturb cell division and proteolytic processes in P. aeruginosa, suggesting the functional relationship of htpR genes in E. coli and Pseudomonas bacteria.     

Flagellation of Pseudomonas aeruginosa during the cell division cycle

Flagellation of Pseudomonas aeruginosa during the cell division cycle was examined by scanning electron microscopy. A new flagellum grows on an old polar end located at the opposite position of the parental flagellum in the late stage of the cell cycle.     

Organic solvent-tolerant mutants of Pseudomonas aeruginosa display multiple antibiotic resistance.

Organic solvent-tolerant mutants of Pseudomonas aeruginosa selected in the presence of hexane exhibited increased resistance to a variety of structurally unrelated antimicrobial agents, including beta-lactams, fluoroquinolones, chloramphenicol, tetracycline, and novobiocin, a phenotype typical of nalB multidrug-resistant mutants. Western immunoblotting with antibodies specific to components of the three known multidrug efflux systems in P. aeruginosa demonstrated that the solvent-tolerant mutants displayed increased expression of the MexAB-OprM system and decreased expression of the MexEF-OprN system. Sequence analysis of mexR, the repressor gene of mexAB-oprM efflux operon, identified a nonsense mutation and a point mutation in the mexR genes of two solvent-tolerant mutants. These results emphasize the importance of the MexAB-OprM efflux system in organic solvent tolerance and the ability of environmental pollutants to select bacteria with a medically relevant antibiotic-resistant phenotype.     

Role of Mutation in Pseudomonas aeruginosa Biofilm Development

The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor development, and may help to explain why structural and genetic heterogeneity are characteristic features of bacterial biofilm populations.     

Mapping and characterization of two mutations to antibiotic supersusceptibility in Pseudomonas aeruginosa

Two mutations associated with antibiotic supersusceptibility in Pseudomonas aeruginosa strain Z61 were transferred separately into strain PAO222, using R68.45-mediated conjugation and phage F116L transduction. One mutation (absA) was 40% contransducible with pro-82 at 26 min on the P. aeruginosa chromosome and was associated with increased susceptibility to beta-lactams, gentamicin and hydrophobic agents. Strains carrying the absA mutation also displayed enhanced uptake of a hydrophobic fluorescent probe, 1-N-phenylnaphthylamine, and were found, by SDS-PAGE, to be altered in the pattern of lipopolysaccharide O-antigen distribution. The other mutation (absB), associated with increased susceptibility to beta-lactams and gentamicin but not to hydrophobic agents, was cotransducible with met-28 and proC at 20 min on the chromosome. The absB mutation caused a structurally undefined alteration in the physical interaction of EDTA and gentamicin with the outer membrane.     

Fitness landscape of antibiotic tolerance in Pseudomonas aeruginosa biofilms

Bacteria in biofilms have higher antibiotic tolerance than their planktonic counterparts. A major outstanding question is the degree to which the biofilm-specific cellular state and its constituent genetic determinants contribute to this hyper-tolerant phenotype. Here, we used genome-wide functional profiling of a complex, heterogeneous mutant population of Pseudomonas aeruginosa MPAO1 in biofilm and planktonic growth conditions with and without tobramycin to systematically quantify the contribution of each locus to antibiotic tolerance under these two states. We identified large sets of mutations that contribute to antibiotic tolerance predominantly in the biofilm or planktonic setting only, offering global insights into the differences and similarities between biofilm and planktonic antibiotic tolerance. Our mixed population-based experimental design recapitulated the complexity of natural biofilms and, unlike previous studies, revealed clinically observed behaviors including the emergence of quorum sensing-deficient mutants. Our study revealed a substantial contribution of the cellular state to the antibiotic tolerance of biofilms, providing a rational foundation for the development of novel therapeutics against P. aeruginosa biofilm-associated infections.     

Mutation of retS, encoding a putative hybrid two-component regulatory protein in Pseudomonas aeruginosa, attenuates multiple virulence mechanisms

Two-component regulatory systems play an important role in bacterial virulence. We report that mutation of a Pseudomonas aeruginosa gene designated retS (previously designated fimK; accession number PA4856) encoding a putative hybrid two-component regulator, attenuates multiple virulence mechanisms. The retS mutant was selected from a Tn5 transposon library of the cytotoxic P. aeruginosa strain PA103 based upon expression of a small-colony phenotype suggestive of reduced surface-associated "twitching" motility, a property dependent upon type IV pili. Subsequent analysis revealed that the mutant expressed pilin, albeit at lower levels than wild-type PA103. In a murine model of corneal infection, retS mutation was associated with delayed disease development and altered pathology. In vitro, retS mutants demonstrated loss of acute cytotoxic activity towards corneal epithelia as determined by trypan blue exclusion and by LDH release assays (P<0.0001). This coincided with loss of ExsA-regulated type III secretion. Mutation of retS also impaired ExsA-independent pathogenic mechanisms. When compared to the exsA mutant of PA103, retS mutants exhibited reduced epithelial adherence and invasion and reduced intracellular survival within the cells after invasion. Time-lapse video microscopy revealed that retS mutants, compared to exsA mutants, had a reduced capacity to access, and move along, the basal cell surfaces of corneal epithelial cell monolayers. Taken together, these data suggest that the protein encoded by retS regulates various properties of P. aeruginosa including both ExsA-dependent and ExsA-independent virulence mechanisms.     

利用抗生素对大肠埃希菌和铜绿假单胞菌的影响进行菌株区分

目的为了探讨抗生素对中心碳代谢的影响,我们研究了大肠埃希菌和铜绿假单胞菌在11种不同抗生素的刺激下,三羧酸循环相关有机酸的代谢变化。方法利用毛细管电泳技术对2种菌在不同抗生素作用下细胞内的主要有机酸进行检测,然后通过多变量统计分析对数据进行处理。结果通过多变量统计分析发现,2种细菌可以通过抗生素对其胞内有机酸的影响不同而得到区分。结论胞内有机酸的变化具有菌株特异性,可以用于细菌的区分。