- Volume 137, Issue 10, 1991
Volume 137, Issue 10, 1991
- Genetics And Molecular Biology
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Plasmid-mediated NAD independence in Haemophilus parainfluenzae
More LessThe location of the genes coding for NAD independence in four unusual clinical isolates of Haemophilus parainfluenzae was determined by transferring these genes to plasmid-free Haemophilus influenzae Rd by transformation and analysing transformants for the presence of plasmids by agarose gel electrophoresis. All NAD-independent transformants were found to carry a single plasmid species. The plasmids, originally harboured by the four H. parainfluenzae isolates recovered from unrelated sources, were of the same size (5·25 kb). Spontaneous reversion to NAD dependence occurred with a low frequency (0·1 to 0·2% of the progeny of a single clone) in both H. parainfluenzae and H. influenzae Rd. The revertants had lost this small plasmid. Mitomycin C exhibited a plasmid ‘curing’ effect with a frequency of‘curing’ of between 1 and 6% of the surviving clones. It was concluded that the genes conferring NAD independence were located on the small 5·25 kb plasmid.
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Plasmid-encoded determinants for bacteriocin production and immunity in a Lactococcus lactis strain and purification of the inhibitory peptide
More LessLactococcin, a bacteriocin produced by Lactococcus lactis subsp. lactis ADRIA 85LO30, was purified as a 2·3–2·4 kDa peptide. Six non-bacteriocin-producing (Bac–) and non-immune (Imm–) strains were isolated after curing experiments. These strains had in common the loss or modification of two plasmids: pOS4 (32 kb) and pOS5 (70 kb). By comparing pOS5 and several modified plasmids, a DNA region from pOS5 of about 10 kb, which was necessary for wild-type bacteriocin production and immunity, was identified.
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The thn mutation of Schizophyllum commune, which suppresses formation of aerial hyphae, affects expression of the Sc3 hydrophobin gene
More LessThe spontaneous and recessive mutation thn in the basidiomycete Schizophyllum commune suppresses the formation of aerial hyphae in the monokaryon and, if present as a double dose, the formation of both aerial hyphae and fruit-bodies in the dikaryon. In the monokaryon, the mutation prevents accumulation of mRNA of the Sc3 gene, and in the dikaryon it also prevents the accumulation of fruiting-specific mRNAs, including mRNAs of the Sc1 and Sc4 genes, which are homologous to the Sc3 gene. These three genes code for hydrophobins, a family of small hydrophobic cysteine-rich proteins. In the thn monokaryon, the only detectable change in synthesized proteins is the disappearance of an abundant protein of apparent M r = 28 K from the culture medium and from the cell walls. Protein sequencing shows that this is the product of the Sc3 gene. The Sc3 hydrophobin is present in the walls of aerial hyphae as a hot-SDS-insoluble complex. Submerged hyphae excrete large amounts of the hydrophobin into the medium.
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- Pathogenicity And Medical Microbiology
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Recovery of viable but non-culturable Campylobacter jejuni
More LessSuspensions of Campylobacter jejuni became non-culturable after storage in sterilized pond water at 4 °C for periods between 18 and 28 d, depending on the strain. Suspensions of four strains of C. jejuni that had been in water for 6 weeks, and shown to be non-culturable, were fed to suckling mice. Colonization of mice was established with two of the strains and failed with the other two strains. Examination of these suspensions under the electron microscope showed some cocci having the appearance of being viable, but most cocci and all remaining spiral forms showed extensive degeneration. The results indicate that non-culturable coccal forms of C. jejuni are capable of infecting mice but that this property may differ between strains.
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The type 4 pilin of Moraxella nonliquefaciens exhibits unique similarities with the pilins of Neisseria gonorrhoeae and Dichelobacter (Bacteroides) nodosus
More LessMoraxella nonliquefaciens is a bacterium which is part of the normal flora of the human upper respiratory tract and is an occasional cause of disease. Using a previously cloned type 4 pilin gene (tfpQ) from Moraxella bovis as a hybridization probe, we have cloned an 826 bp Sau3AI fragment which contains an M. nonliquefaciens type 4 pilin gene (tfpA) from strain NCTC 7784. The pilin gene is expressed in Escherichia coli. We have examined NCTC 7784 and nine other M. nonliquefaciens strains by genomic Southern hybridization using tfpA as a probe, and they all appeared to have more than one pilin gene. While the predicted amino acid sequence of the M. nonliquefaciens tfpA pilin has conserved regions as compared to pilins of M. bovis and M. lacunata, it also shows similarities to both the type 4 pilin of Neisseria gonorrhoeae and the type 4 pilin of Dichelobacter nodosus (formerly Bacteroides nodosus).
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- Physiology And Growth
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Thermal denaturation of whole cells and cell components of Escherichia coli examined by differential scanning calorimetry
More LessThermograms of whole cells of Escherichia coli obtained by differential scanning calorimetry contained ten main peaks (denoted f, l, m 1, m 2, m 3, n, p, q, r and s) occurring at temperatures of approximately 25, 54, 61, 71, 76, 81, 95, 105, 118 and 124 °C, respectively. After cooling to 5 °C and reheating, peaks denoted f r, m r and p r were observed at 23, 73 and 94 °C, respectively. By examining thermograms of different cell fractions we have identified the following thermal denaturation events. During primary heating there is a broad endotherm (f) beginning below 20 °C and extending to just above 40 °C that is caused by melting of membrane lipids. Superimposed on this is an exothermic process associated with a change of state of the peptidoglycan. The first irreversible denaturation event occurs just above 47 °C, associated with the onset of denaturation of the 30S ribosomal subunit and soluble cytoplasmic proteins. Ribosome melting is a complex process occurring between 47 and 85 °C and is characterized by peaks m 1, m 2 and n. Peak m 3 at 75–76 °C is of unknown identity but may possibly represent melting of tRNA. Peak p at 95 °C results from melting of a portion of the cellular DNA combined with denaturation of a cell wall component. Peak q at 105 °C is multicomponent and may be caused by melting of a different region of DNA together with denaturation of another cell wall component. The complex events denoted r and s at 118 and 125 °C, respectively, are associated with denaturation of a component of the cell envelope, and possibly also of DNA. Following cooling and reheating there is a broad endotherm with a maximum at 23 °C caused by remelting of membrane lipid and a very broad endotherm extending between 40 and 100 °C caused by the remelting of ribosomal RNA. Peak pr at 94 °C is caused by the melting of reannealed DNA. Additional features not appearing in whole cells were evident in some cell fractions. These observations should allow us to distinguish events that may lead to loss of viability from those that do not.
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Molar growth yields and bioenergetic parameters of extremely alkaliphilic Bacillus species in batch cultures, and growth in a chemostat at pH 10·5
More LessAlkaliphilic Bacillus species that grow at pH 10·5 must cope with a low protonmotive force (– 50 mV) due to a reversed transmembrane pH gradient at least 2 pH units more acid inside. Here we demonstrate that strictly alkaliphilic B. firmus RAB and two strains of B. alcalophilus (ATCC 27467 and DSM 485) grow exponentially in batch cultures with a doubling time of less than 1 h in 100 mm buffered medium, while the actual medium pH remains above 10·2. The ATCC strain continued to grow rapidly for at least 7 h, but the growth rate of the DSM strain declined dramatically after 3 h. However, both the B. alcalophilus strains, B. firmus RAB and facultatively alkaliphilic B. firmus OF4 were readily maintained for at least 24 h between pH 10·4 and 10·6 in a chemostat where nutrients were constantly replenished. A critical nutrient may be limiting in batch cultures of the DSM strain of B. alcalophilus. The facultative alkaliphile grew equally well in batch cultures at an initial pH of 7·5 or 10·5. Its molar growth yield (23 mg dry wt mmol−1) on malate (Y mal) was the same at the two pH values and was comparable to Y mal for B. subtilis grown at neutral pH. B. firmus RAB and B. alcalophilus ATCC 27467 grown at pH 10·5 also showed Y mal values at least as high as the neutralphile, indicating efficient use of the energy source even at low protonmotive force. Moreover, the phosphorylation potential of B. firmus OF4 grown at pH 7·5 (45·2 kJ mol−1)or pH 10·5 (46 kJ mol−1) was in a conventional range for bacteria.
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- Plant-Microbe Interactions
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Indole-3-acetic acid (IAA) synthesis in the biocontrol strain CHA0 of Pseudomonas fluorescens: role of tryptophan side chain oxidase
More LessPseudomonas fluorescens strain CHA0 is an effective biocontrol agent against soil-borne fungal plant pathogens. In this study, indole-3-acetic acid (IAA) biosynthesis in strain CHA0 was investigated. Two key enzyme activities were found to be involved: tryptophan side chain oxidase (TSO) and tryptophan transaminase. TSO was induced in the stationary growth phase. By fractionation of a cell extract of strain CHA0 on DEAE-Sepharose, two distinct peaks of constitutive tryptophan transaminase activity were detected. A pathway leading from tryptophan to IAA via indole-3-acetamide, which occurs in Pseudomonas syringae subsp. savastanoi, was not present in strain CHA0. IAA synthesis accounted for ⩽1·5% of exogenous tryptophan consumed by resting cells of strain CHA0, indicating that the bulk of tryptophan was catabolized via yet another pathway involving anthranilic acid as an intermediate. Strain CHA750, a mutant lacking TSO activity, was obtained after Tn5 mutagenesis of strain CHA0. In liquid cultures (pH 6·8) supplemented with 10 mm-l-tryptophan, growing cells of strains CHA0 and CHA750 synthesized the same amount of IAA, presumably using the tryptophan transaminase pathway. In contrast, resting cells of strain CHA750 produced five times less IAA in a buffer (pH 6·0) containing 1 mm-l-tryptophan than did resting cells of the wild-type, illustrating the major contribution of TSO to IAA synthesis under these conditions. In artificial soils at pH ~7 or pH ~6, both strains had similar abilities to suppress take-all disease of wheat or black root rot of tobacco. This suggests that TSO-dependent IAA synthesis is not essential for disease suppression.
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- Corrigendum
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