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Microbiology Society logo - Volume 142, Issue 4, 1996

Volume 142, Issue 4

Volume 142, Issue 4, 1996

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  • Antigens And Immunity
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  • Biochemistry

      • Polysaccharide lyases from gellan-producing spp.

        https://doi.org/10.1099/00221287-142-4-867

        A number of strains capable of synthesizing the bacterial exopolysaccharide gellan and related polymers were shown to possess constitutive gellanase activity. In each case, the degradation of deacylated gellan was due to extracellular, eliminase-type enzymes (lyases) which cleave the sequence …ß-D-glucosyl 1,4-ß-D-glucuronosyl… in the tetrasaccharide repeat unit of the substrate polysaccharides. Deacetylated rhamsan was an alternative substrate but there was little or no action against most other polysaccharides with similar structures. Slight differences were found between the specificities of the lyases from different strains. Activities of gellan lyase preparations were generally low. As well as the extracellular ‘gellanase’ activity, all the bacteria possessed varying amounts of ß-D-glucosidase and ß-D-glucuronidase activities apparently located in the periplasm. The products from deacylated gellan and the chemically deacylated form of polysaccharide S194 (rhamsan gum), which is effectively a gentiobiosylated form of gellan, closely resembled those recently obtained by the authors from other, gellandegrading, non-gellan-producing bacteria. The enzymes had negligible activity against the natural, acylated gellan and rhamsan polysaccharides from bacteria now designated as strains of .

    • Bacteriophage receptors on cells are the -acetylglucosamine and rhamnose substituents of teichoic acids or the peptidoglycan itself

      https://doi.org/10.1099/00221287-142-4-985

      Different approaches were used to examine the function of teichoic acids (TA) as phage receptors among selected strains, and to identify and characterize specific receptor structures of host cells belonging to different serovars. This included successive removal of cell wall constituents, preparation and purification of TA, and GLC analysis of TA components. Adsorption of bacteriophages could be inhibited by polyvalent antisera, specific lectins and addition of purified TA. The results confirmed the necessity of TA in general and of rhamnose and glucosamine in particular for adsorption of phage A118, which is a temperate Siphovirus (morphotype B1), attacking predominantly serovars 1/2. Host binding of siphoviral phage A500 (predominantly lysing serovars 4b), was also dependent on cell wall TA. A phage-resistant strain was shown to lack glucosamine in its TA. These results support the view that TA substituents may play an important role not only in antigenicity of cells, but also in specificity of host recognition by two temperate phages. In contrast, the broad-host-range virulent phage A511 (Myovirus, morphotype A1) uses the listerial peptidoglycan as primary receptor. This corresponds well with the observation that A511 is capable of lysing the majority of strains.

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  • Bioenergetics And Transport

      • The cytochrome bd quinol oxidase in has an extremely high oxygen affinity and two oxygen-binding haems: implications for regulation of activity by oxygen inhibition

        https://doi.org/10.1099/00221287-142-4-755

        Cytochrome is a respiratory oxidase in and many other bacteria. It contains cytochromes and as redox centres, and is thus unrelated to the haem-copper super-family of terminal oxidases. The apparent affinities ) for oxygen uptake by respiring cells and membranes from a mutant lacking the alternative oxidase cytochrome were determined by deoxygenation of oxyleghaemoglobin as a sensitive reporter of dissolved oxygen concentration. Respiration rates were maximal at oxygen concentrations of 25-50 nM, but the kinetics were complex and indicative of substrate (i.e. oxygen) inhibition. values were in the range 3-8 nM (the lowest recorded for a respiratory oxidase), and values between 0.5 and 1.8 μM were obtained. Low temperature photodissociation of anoxic, CO-ligated membranes confirmed the absence of cytochrome and revealed a high-spin -type cytochrome identified as cytochrome of the cytochrome complex. Photodissociation in the presence of oxygen revealed binding of a ligand (presumably oxygen) to cytochrome at a rate much greater than that of CO binding, and formation of the oxygenated form of cytochrome . The results confirm that both high-spin haems in the cytochrome complex bind CO and demonstrate that oxygen can also react with both haems. Substrate inhibition of oxidase activity, in addition to transcriptional regulation of oxidase synthesis, may play a crucial role in the regulation of partitioning of electron flux between the cytochrome and -terminated respiratory pathways.

    • Dissimilatory iron(III) reduction by

      https://doi.org/10.1099/00221287-142-4-765

      The photosynthetic proteobacterium was shown to be capable of dissimilatory Fe(III) reduction. Activity was expressed during anaerobic phototrophic and microaerobic growth with malate as the carbon source, but not during equivalent aerobic growth. A variety of Fe(III) complexes were demonstrated to act as substrates for intact cells and membrane fractions of strain N22DNAR using a ferrozine assay for Fe(II) formation. Rates of reduction appeared to be influenced by the reduction potentials of the Fe(III) complexes. However, Fe(III) complexed by citrate, which is readily reduced by , was a poor substrate for dissimilation by . The Fe(III)-reducing activity of was located solely in the membrane fraction. The reduction of Fe(III) complexes by intact cells was inhibited by 2-heptyl-4-hydroxyquinoline--oxide (HQNO), suggesting the involvement of ubiquinol: cytochrome oxidoreductases in the electron transport chain. Lack of sensitivity to myxothiazol plus data from mutant strains implies that the cytochrome complex and cytochrome are not obligatory for dissimilation of Fe(III)(maltol). Alternative pathways of electron transfer to Fe(III) must hence operate in . Using strain N22DNAR, the reduction rate of Fe(III) complexed by nitrilotriacetic acid (NTA) was elevated compared to that of Fe(III)(maltol), and moreover was sensitive to myxothiazol. However, these differences were not observed in the absence of the electron donor malate. The governing factor for the reduction rate of Fe(III)(maltol) thus appears to be the limited Fe(III)-reducing activity, whilst the reduction rate of Fe(III) complexed by NTA is controlled by the flux of electrons through the respiratory chain. The use of mutant strains confirmed that the role of the cytochrome complex in Fe(III) reduction becomes apparent only with the superior substrate. The energy-conserving nature of Fe(III) reduction by was demonstrated by electrochromic measurements, with the endogenous carotenoid pigments being employed as indicators of membrane potential generation in intact cells. Using Fe(III)EDTA as electron acceptor, periods of membrane potential generation were directly proportional to the quantity of complex added, and were extended in the presence of HQNO. Fe(III)-dependent carotenoid bandshifts were abolished by addition of the protonophoric uncoupler carbonyl cyanide -trifluoromethoxy-phenylhydrazone.

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  • Biotechnology

      • A new, broad-substrate-specificity aminopeptidase from the dairy organism SBT 2171

        https://doi.org/10.1099/00221287-142-4-799

        An aminopeptidase with a very broad substrate specificity was purified to homogeneity from SBT 2171 by FPLC. The enzyme was purified 144-fold from a cell-free extract with a yield of 16%. The purified enzyme appeared as a single band on an SDS-PAGE gel. It had a molecular mass of 95 kDa and an isoelectric point of 4.9. The enzyme hydrolysed a large range of naphthylamide- and nitroanilide-substituted amino acids, as well as several di-, tri- and oligopeptides. It also exhibited significant prolineiminopeptidase-like activity, since it hydrolysed several proline-containing peptides. Prolyl--nitroanilide was hydrolysed with a low affinity (Michaelis-Menten constant 0.6 mM) and a of 2.5 μmol min (mg protein) while lysyl--nitroanilide was hydrolysed with a high affinity 0.003 mM; 37.5 μmol min (mg protein)]. The aminopeptidase activity, which was optimal between pH 6.0 and 8.0 and at 50 °, was very stable at 30 ° for more than 7 d. The activity lost by treatment with the thiol-blocking reagents could be restored with ß-mercaptoethanol, while Co and Mn restored the activity of the EDTA-treated enzyme. Immunological experiments with antibodies raised against the aminopeptidases from and clearly showed that both aminopeptidases are at least immunologically different from each other.

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  • Development And Structure

      • A bacteriophage of

        https://doi.org/10.1099/00221287-142-4-979

        A bacteriophage, øBHG1, was isolated from a small eutrophic pond from which its host, , was originally obtained. It is a lytic bacteriophage specific for which also causes non-specific lysis of 8253. øBHG1 has an icosahedral head of 62 nm diameter and a short 39 nm tail. Caesium chloride density gradient centrifugation of infected cell lysates gave a single bacteriophage band at a density of 1.385 g cm, but also occasionally a second band was observed at a lower density. No differences were apparent between bacteriophage taken from either of the two bands. øBHG1 contained double-stranded DNA with a size of 48 kb and a G + C content of 50.6 mol%. The bacteriophage adsorbed to both photosynthetically and chemoheterotrophically grown at an identical rate of 1.39 × 10 ml min. One-step growth curves and kinetic studies of the bacteriophage under these physiological regimes showed no differences in the latent and rise periods and only slight changes in the burst size. Adsorption of this bacteriophage is cell-surface specific and attachment only occurs to the ‘older’ pole of the budding reproductive cell.

    • A mutational analysis of multicellular development

      https://doi.org/10.1099/00221287-142-4-993

      We have collected mutants that arrest in development after aggregation, but before first finger formation. A total of 118 mutant strains were isolated and are referred to as mound mutants. Nine complementation groups , containing 46 of the mutant strains, were defined by parasexual methods. A statistical analysis suggested that there are about 118 genes which, when mutated, confer the mound phenotype. Of these genes, about 60 are predicted to be mutated in our collection: the 9 assigned to complementation groups and another 51 unassigned mutants. and were assigned to linkage groups VII, IV, II and VI, respectively. Development of the mutant strains was characterized by terminal morphology, neutral red staining and expression of marker mRNAs for prespore and prestalk cells. Three broad classes were recognized. (1) Postaggregative mutants - those blocked early in multicellular development. They did not express any of the prestalk or prespore marker mRNAs and generally arrested as low mounds or ridges. (2) Pathway mutants - those blocked specifically in either prestalk or prespore differentiation. They expressed either prestalk or prespore marker mRNAs, but not both, and generally proceeded further morphologically than post-aggregative mutants. (3) Morphogenesis mutants - those apparently blocked in morphogenesis rather than cell differentiation. They expressed all the cell-type marker mRNAs tested. Most arrested as tight mounds lacking a tip and of defined upper size, but some mutants produced aberrant tips. The majority of mutants tested synergized with wild-type: 24/28 strains which cannot make spores when developed alone, were able do so when allowed to develop with an equal number of wild-type cells. We suggest that some of the morphogenesis mutants have a cytoskeletal defect which prevents first finger formation and that these mutants can be physically carried through development by the wildtype (synergy by ‘piggy-backing’).

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  • Environmental Microbiology

      • Stress resistance and recovery potential of culturable and viable but nonculturable cells of

        https://doi.org/10.1099/00221287-142-4-845

        The estuarine, human-pathogenic bacterium responds to low temperature by the formation of viable but nonculturable (VBNC) cells, while starvation at moderate temperatures allows for maintenance of culturability of this organism. Recovery of cold-incubated populations of was restricted to the culturable fraction in slide cultures and most probable number assays. These populations, however, gave between 1.1- and 8-fold higher c.f.u. counts on soft agar plates than on ordinary agar plates, indicating that a small and variable fraction of the cell population was injured rather than nonculturable. Thus, the population of cold-incubated cells is composed of culturable, injured and nonculturable cells, with the numbers of the culturable and injured cells rapidly decreasing during cold incubation. Recovery of nonculturable cells of the organism, however, could not be obtained by any combination of temperature and nutrient shifts in any of the assays. VBNC cells of the organism were assessed with regard to their persistence and stress resistance in comparison to growing and starved cells. The sonication resistance of VBNC cells was initially similar to that of growing cells, but increased during prolonged cold incubation. The final resistance of cold-incubated VBNC cells was equal to the markedly increased resistance of starving cells, which also displayed increased resistance against exposure to ethanol and mechanical stress. Our results indicate that in spite of the apparent absence of recovery under a wide range of laboratory conditions, VBNC cells of undergo changes at low temperature which potentially allow them to persist for extended periods.

    • Direct evidence for the involvement of extracellular proteins in the adhesion of

      https://doi.org/10.1099/00221287-142-4-855

      Adhesion of to glass and polystyrene was investigated by bringing the cells into contact with the support by sedimentation. Adhesion depended on time and temperature: lower adhesion densities were observed when the contact time was only 2 h or 6 h, as compared to 24 h, or when the test was performed at 4 C, as compared to 30 °. The influence of cell physiology was further demonstrated by the effect of tetracycline, which inhibited adhesion. Scanning electron microscopy showed that cells produced extracellular material when left in contact with a support for 24 h. The surface elemental composition of cells and of polystyrene supports after cell adhesion and subsequent detachment was determined by X-ray photoelectron spectroscopy; this provided information on the relative concentrations of proteins and polysaccharides at the surface. The protein concentration at the surface of a cell sediment increased as a function of time at 30 °, correlating with an increase of adhesion density. A similar correlation between protein concentration and adhesion density was found when comparing exponentialphase cells with stationary-phase cells. The surface composition of polystyrene supports examined after cell detachment was found to be rich in proteins, indicating that proteins are the major constituent at the support surface. Lowering the contact time, or performing adhesion under unfavourable metabolic conditions (4 °) or in the presence of tetracycline, resulted in a decrease in protein concentration at the support surface, which was correlated with a decrease in adhesion density. The correlation between protein concentration at the cell surface or at the support surface and adhesion density, under different experimental conditions, provides a direct demonstration of the involvement of extracellular proteins in the adhesion of to inert surfaces.

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  • Genetics And Molecular Biology
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