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Microbiology Society logo - Volume 147, Issue 1, 2001

Volume 147, Issue 1

Volume 147, Issue 1, 2001

  • Pathogenicity And Medical Microbiology

      • GroEL (Hsp60) of is involved in cell adherence

        https://doi.org/10.1099/00221287-147-1-87

        Previous results have demonstrated that adherence of to tissue culture cells is augmented by various stresses; this study focussed on whether the GroEL heat shock protein is implicated in this process. The 1940 bp operon of was isolated by PCR. The 1623 bp gene is highly conserved between various isolates as determined by RFLP-PCR and DNA sequencing, and the operon is present in one copy on the bacterial chromosome. The 58 kDa GroEL protein was expressed in in fusion with glutathione -transferase and the fusion protein was purified from IPTG-induced bacterial lysates by affinity chromatography on glutathione–Sepharose. A polyclonal, monospecific antiserum was obtained for GroEL which established by immunoelectron microscopy, indirect immunofluorescence and immunoblot analysis that GroEL is released extracellularly after heat shock and can be surface associated. Cell fractionation experiments suggest that GroEL is predominantly cytoplasmic and membrane bound. GroEL-specific antibodies as well as the purified protein partially inhibited cell attachment and expression of the protein was induced by cell contact, suggesting a role for GroEL in cell adherence.

    • resistance of complex isolates to reactive oxygen species in relation to catalase and superoxide dismutase production

      https://doi.org/10.1099/00221287-147-1-97

      The complex comprises groups of genomovars (genotypically distinct strains with very similar phenotypes) that have emerged as important opportunistic pathogens in cystic fibrosis (CF) patients. The inflammatory response against bacteria in the airways of CF individuals is dominated by polymorphonuclear cells and involves the generation of oxidative stress, which leads to further inflammation and tissue damage. Bacterial catalase, catalase-peroxidase and superoxide dismutase activities may contribute to the survival of following exposure to reactive oxygen metabolites generated by host cells in response to infection. In the present study the authors investigated the production of catalase, peroxidase and SOD by isolates belonging to various genomovars of the complex. Production of both catalase and SOD was maximal during late stationary phase in almost all isolates examined. Native PAGE identified 13 catalase electrophoretotypes and two SOD electrophoretotypes (corresponding to an Fe-SOD class) in strains belonging to the six genomovars of the complex. Seven out of 11 strains displaying high-level survival after HO treatment had a bifunctional catalase/peroxidase, and included all the genomovar III strains examined. These isolates represent most of the epidemic isolates that are often associated with the cepacia syndrome. The majority of the isolates from all the genomovars were resistant to extracellular \(O_{2}^{{-}}\) , while resistance to intracellularly generated \(O_{2}^{{-}}\) was highly variable and could not be correlated with the detected levels of SOD activity. Altogether the results suggest that resistance to toxic oxygen metabolites from extracellular sources may be a factor involved in the persistence of in the airways of CF individuals.

    • Identification of the acid phosphatase () gene homologues in pathogenic and non-pathogenic spp. facilitates Tn mutagenesis

      https://doi.org/10.1099/00221287-147-1-111

      and are pathogens responsible for disease in both humans and animals. , while phylogenetically similar, is considered avirulent in comparison. These three species exhibit phosphatase activity when grown on media containing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (XP). Tn-OT182 mutagenesis has been utilized to isolate mutants of and unable to hydrolyse XP. Sequence analysis of these mutants revealed an ORF of 1734 nucleotides demonstrating a high degree of homology to the gene product of . PCR primers were designed based on the gene sequence and were used to amplify an homologue from . The predicted amino acid sequence of AcpA differed from those of the predicted AcpA and AcpA by 15 and 3 amino acids, respectively. Allelic exchange was used to construct Δ mutants in each of these spp. These mutants were shown to be devoid of phosphatase activity and have subsequently allowed for the implementation of fusion transposon mutagenesis systems. Two such systems have been successfully utilized in spp. for the identification of several genes encoding exported proteins.

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  • Physiology And Growth

      • Dimensional regulation of cell-cycle events in during steady-state growth

        https://doi.org/10.1099/00221287-147-1-171

        Two opposing models have been put forward in the literature to describe the changes in the shape of individual cells in steady-state growth that take place during the cell cycle: the model, which maintains that the regulating dimension is cell length, and the model, which asserts it to be cell volume. In addition, the former model envisages cell diameter as decreasing with length up to constriction whereas the latter sees it as being constrained by the rigid cell wall. These two models differ in the correlations they predict between the various cellular dimensions (diameter, length, volume) not only across the entire population of bacteria but also, and especially, within subpopulations that define specific cell-cycle events (division, for example, or onset of constriction); the coefficients of variation at these specific events are also expected to be very different. Observations from cells prepared for electron microscopy (air-dried) and for phase-contrast microscopy (hydrated) appeared qualitatively largely in accordance with the predictions of the model. To obtain a more quantitative comparison, simulations were carried out of populations defined by each of the models; again, the results favoured the model. Finally, in age-selected cells using membrane elution, the diameter–length and diameter–volume correlations were in complete agreement with the model, as were the coefficients of variation. It is concluded that, at least with respect to cell-cycle events such as onset of constriction and cell division, length rather than volume is the controlling dimension.

    • Morphological characterization of : growth, septation and fragmentation

      https://doi.org/10.1099/00221287-147-1-239

      The influence of the gene on the growth of has been investigated by characterizing and comparing the parental strain A28 () with the null mutant (Δ). The gene is known to affect the septation process in , therefore the Δ strain does not produce any septa during the first hours of growth. During batch cultivations Δ shows an abrupt decrease in specific growth rate and more pronounced fragmentation (in response to elevated stirrer speed) than the parental strain. Higher specific fragmentation rates ( ) were obtained for the Δ strain. The physiological reasons for the differences have been investigated by employing fluorescent stains. Computerized image analysis revealed that the more pronounced fragmentation in the mutant was due to the lower number and irregular spacing of septa (visualized by calcofluor white staining), which resulted in a weaker hyphal structure that is more vulnerable to shear stress and fragmentation than the parental strain. This led to a loss of active biomass (determined by Mag fura staining) from the hyphae of the mutant, which had failed to compartmentalize by formation of septa, in turn resulting in decreased specific growth rates for the culture.

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