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Volume 138,
Issue 5,
1992
Volume 138, Issue 5, 1992
- Biochemistry
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Bacterial rhodopsins of newly isolated halobacteria
More LessSUMMARY: Several new halobacterial strains were isolated from crude solar salts commercially produced in Mexico and Australia. The presence of bacteriorhodopsin (BR)- and halorhodopsin (HR)-like pigment in their total membrane fraction was measured by flash spectroscopy and light-induced ion pumping activity. Two of these strains contained both BR- and HR-like pigments; the others contained only BR-like pigment. DNA hybridization analysis with probes from bacterioopsin and haloopsin genes revealed that the genes encoding the BR- and HR-like pigments were not homologous to those found in Halobacterium halobium R1. In addition, the presence of sensory rhodopsin (SR)- and phoborhodopsin (PR)-like pigment in these membranes was detected by flash spectroscopy. The kinetics of the cyclic photoreaction of the SR-like pigment was more than 15 times slower than that of H. halobium. A PR-like pigment existed in two strains, and the kinetics of the cyclic photoreaction of the PR-like pigment was similar to that found in H. halobium.
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- Development And Structure
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Bacterial ice nucleation activity after T4 bacteriophage infection
More LessSUMMARY: The changes in ice nucleation activity of transformed Ina+ Escherichia coli K12 after infection with T4D bacteriophage have been examined. Within 2 min after infection class A nucleation activity (measured at — 4 °C) fell about 1000–100-fold whilst class B (measured at — 5.5 °C) and class C (measured at — 9 °C) nucleation activities increased 50–100-fold and then rapidly decreased. These changes also occurred after interaction with T4D ghost particles or T4D 11-/12-particles. Since ghost particles lack DNA and 11-/12-particles lack short tail fibres, the T4D particles appear to be exerting their effect by the attachment of the phage long tail fibres to the cell. The changes were not influenced by the addition of chloramphenicol.
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- Ecology
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Detection of micro-organisms in soil after in situ hybridization with rRNA-targeted, fluorescently labelled oligonucleotides
More LessSUMMARY: rRNA sequences were used as targets for synthetic oligonucleotides labelled with the fluorescent dye tetramethylrhodamine isothiocyanate (Tritc) for in situ hybridizations to detect micro-organisms directly in soils that have different contents of soil minerals and organic material. Introduced Pseudomonas aeruginosa cells were directly fixed in soils and applied to slides after separation of large soil minerals only. Remaining soil minerals (clay minerals) and organic material (up to 8%) did not significantly interfere with signal expression after hybridization. Background signals were mainly caused by autofluorescence of organic material. Non-specific binding of labelled oligonucleotides to soil particles was not observed. In situ detection of introduced cells of Pseudomonas cepacia in a sandy loam spiked with a mixture of selected soil micro-organisms was possible after hybridization with a specific probe. Analysis of natural bacterial populations in soil, however, was not possible by in situ hybridization without activation of these micro-organisms by adding nutrients. Growing cells, e.g. Streptomyces scabies hyphae growing in amended soil, were easily detected.
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- Genetics And Molecular Biology
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The 23S/5S ribosomal RNA genes (rrl/rrf) are separate from the 16S ribosomal RNA gene (rrs) in Borrelia burgdorferi, the aetiological agent of Lyme disease
More LessSUMMARY: DNA fragments containing the rRNA genes for Borrelia burgdorferi strain B31 were cloned in bacteriophage λ EMBL3. A restriction map of the fragments was constructed and the organization of the rRNA genes was determined by Southern hybridization. One genomic DNA fragment contained a single copy of the rrs sequence and another cloned fragment contained both rrl and rrf sequences. The results revealed that the rrs gene is located separately from the set of rrl/rrf genes, suggesting that these rRNA genes are expressed independently in B. burgdorferi
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Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization
More LessSUMMARY: Transposon Tn5, on a mobilizable ColE1 plasmid, on a Ti plasmid derepressed for bacterial transfer, and on the bacteriophage fd genome, was used to construct pathogenicity mutants of the fire blight pathogen Erwinia amylovora. Eleven nonpathogenic mutants were isolated from 1600 independent mutants screened. These mutants were divided into three types: auxotrophs, exopolysaccharide (EPS)-deficient mutants and a mutant of the dsp phenotype. According to their insertion sites the Tn5 mutants were mapped into several classes. Some of the mutants could be complemented with cosmid clones from a genomic library of the parent strain for EPS production on minimal agar. EPS-deficient mutants and the dsp mutant could complement each other to produce virulence symptoms on pear slices.
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Phage DNA synthesis and host DNA degradation in the life cycle of Lactococcus lactis bacteriophage c6A
More LessSUMMARY: Bacteriophage c6A is a lytic phage that infects strains of Lactococcus lactis. Infection of L. lactis strain C6 resulted in inhibition of culture growth within 10 min, mature intracellular phage particles appeared after 17.5 min, and cell lysis occurred after 25 min. A culture of strain C6 carrying 3H-labelled DNA was infected with c6A, and the fate of the radiolabel was monitored. The results showed that degradation of host cell DNA began within 6 min of infection and that the breakdown products were incorporated into progeny c6A DNA. Quantitative DNA hybridizations indicated that synthesis of phage DNA began within 6 min of infection and continued at an approximately constant rate throughout the latent period.
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Development of a host-vector system in a Rhodococcus strain and its use for expression of the cloned nitrile hydratase gene cluster
SUMMARY: Two different types of plasmid were isolated from strains of Rhodococcus rhodochrous. Two plasmids, of the same type but from different strains, were combined with Escherichia coli plasmids carrying antibiotic resistance markers to develop E. coli-Rhodocdccus shuttle vectors. The ampicillin and kanamycin resistance markers served for selection in Rhodococcus. Electroporation was used to introduce recombinant plasmid DNA into R. rhodochrous ATCC 12674 at a frequency of 5 × 107transformants per μg DNA. With these host-vector and transformation systems, the nitrile hydratase and amidase genes of a Rhodococcus strain were introduced into the host strain and were efficiently expressed.
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A dominant mutation in the gene for the Nag repressor of Escherichia coli that renders the nag regulon uninducible
More LessSUMMARY: The gene nagC encodes the repressor for the nag regulon. A point mutation within the gene, which confers a superrepressor phenotype and makes the repressor insensitive to the inducer, N-acetylglucosamine 6-phosphate, has been characterized. The mutation is semi-dominant since heterozygous diploids have reduced growth rates on glucosamine and N-acetylglucosamine compared to the wild-type strain.
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- Pathogenicity And Medical Microbiology
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Outer-membrane protein and lipopolysaccharide variation in Pasteurella haemolytica serotype A1 under different growth conditions
More LessSUMMARY: Growth characteristics, as well as outer-membrane protein (OMP) and lipopolysaccharide (LPS) profiles in SDS-polyacrylamide gels, of two serotype A1 isolates of Pasteurella haemolytica were examined under different in vitro growth conditions. The two isolates were chosen as representatives of disease (S/C 82/1) and non-disease (W/D 83/4) isolates, respectively. The growth rates and final cell densities of both isolates increased as the degree of aeration increased. In particular, the final cell densities varied significantly according to the degree of aeration. Under anaerobic conditions, however, both the growth rate and final cell density were significantly reduced. There was reduced expression of a 40.5 kDa protein under anaerobic conditions in both isolates, whereas in S/C 82/1 expression of the 71, 77 and 100 kDa iron-regulated proteins increased as aeration decreased. There were also differences in low-molecular-mass components of LPS between cells grown anaerobically and those grown aerobically. Growth in the presence of 5% CO2 did not significantly alter the growth rate and had little, if any, affect on OMPs or LPS. Differences in the expression of certain proteins occurred as growth progressed from the exponential to the stationary phase. Growth in the presence of the iron chelators 2,2′-dipyridyl, ethylenediaminedihydroxyphenylacetic acid (EDDA), desferrioxamine mesylate (desferal), ovotransferrin (conalbumin) and bovine transferrin was inhibited within a very narrow concentration range. In the presence of 2,2′-dipyridyl, EDDA or desferal, 71 and 100 kDa iron-regulated OMPs increased in both isolates whereas a 77 kDa protein increased in isolate S/C 82/1 only. In the presence of ovotransferrin or bovine transferrin there was, in both isolates, increased expression of the 71 kDa protein, a slight increase in expression of the 100 kDa protein but no expression of the 77 kDa protein; there was also increased production of the 40.5 kDa protein, and synthesis of two additional proteins of 23 and 26 kDa. Other differences occurred after growth in foetal and newborn calf sera. In foetal calf serum there was enhanced expression of the 71 but not of the 100 kDa protein. In newborn calf serum there was no enhanced expression of the 71,77 or 100 kDa proteins, but expression of novel proteins of 97 and 98 kDa as well as a high-molecular-mass protein occurred. There were also slight quantitative differences in the LPS profiles of cells grown in foetal or newborn calf sera compared to those of cells grown in other media. Addition of FeCl3 to the various media caused repression of synthesis of the 71, 77 and 100 kDa proteins in the presence of EDDA and repression of the 71 kDa protein in the presence of ovotransferrin and in foetal calf serum, but caused no change to the expression of proteins in newborn calf serum. Thus, marked changes may occur in the OMP and LPS components of the outer membrane of P. haemolytica when growth conditions are altered; some of these changes may occur in vivo.
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Characterization of staphylococcal λ-lysin
More LessSUMMARY: λ-Lysin was purifed from Staphylococcus aureus strains Smith 5R and PG23 (a toxic shock syndrome isolate) by a combination of heparin-agarose and hydroxylapatite chromatography. Both strains produced two haemolytic components, designated λ1, and λ2. Though each component was weakly haemolytic they acted synergistically to potentiate haemolysis on rabbit, sheep and human blood. Rabbit and sheep erythrocytes were more sensitive to lysis by λ-lysin than human erythrocytes. The molecular mass of λ1 was 32 kDa and its pI value was 9.4. λ2 had a molecular mass of 36 kDa and a pI value of 9.3. While both trypsin and papain acted synergistically with λ2 to induce increased haemolysis, no such synergism was seen with λ1,. Also, protease inhibitors acted to inhibit synergism between λ1, and λ2. These findings suggest that λ1 could be a protease.
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- Physiology And Growth
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D-Alanyl-lipoteichoic acid in Lactobacillus casei: secretion of vesicles in response to benzylpenicillin
More LessSUMMARY: Vesicles containing lipoteichoic acid (LTA) have been isolated from Lactobacillus casei ATCC 7469 grown in the presence of either benzylpenicillin or D-cycloserine. These cell wall antibiotics enhanced the rate of LTA and lipid secretion 6.7 times, whereas chloramphenicol inhibited their release. The formation of these vesicles from peripheral and septal wall regions did not appear to be the result of bacteriolysis. The vesicle composition of LTA and lipid was similar to that of the cytoplasmic membrane whereas the protein composition was dissimilar. The size of these vesicles ranged from 20 to 40 nm and the length of LTA ranged from 5 to 50 glycerol phosphate residues. The isolation of these vesicles provides a potential in vitro acceptor system for studying the D-alanylation of lipoteichoic acid.
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Autolysis of Clostridium acetobutylicum ATCC 824
More LessSUMMARY: The optimum conditions for autolysis of Clostridium acetobutylicum ATCC 824 were determined. Autolysis was optimal at pH 6.3 and 55 °C in 0.1 M-sodium acetate/phosphate buffer. The ability of cells to autolyse decreased sharply at the end of the exponential phase of growth. Lysis was stimulated by monovalent cations and compounds that complex divalent cations, and inhibited by divalent cations. The autolysin of C. acetobutylicum, which was mainly cytoplasmic, was purified to homogeneity and characterized as a muramidase. The enzyme was identical to the extracellular muramidase in terms of Mr , isoelectric point and NH2-terminal amino acid sequence. The autolysin was inhibited by lipoteichoic acids and cardiolipin but not by phosphatidylethanolamine and phosphatidylglycerol. A mechanism of regulation and fixation involving lipoteichoic acid, cardiolipin and divalent cations is proposed.
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Exogenous siderophore-mediated iron uptake in Pseudomonas aeruginosa: possible involvement of porin OprF in iron translocation
More LessSUMMARY: In addition to the two siderophores pyoverdine and pyochelin synthesized by Pseudomonas aeruginosa ATCC 15692 (strain PAO1), several siderophores produced by other bacteria or fungi, namely cepabactin, salicylic acid, desferriferrichrysin, desferriferricrocin, desferriferrioxamine B, desferriferrioxamine E and coprogen, were able to promote iron uptake with variable efficiencies into this bacterium. For most of these siderophores, these results were consistent with the growth stimulation produced by the same compounds in a plate bioassay. Desferriferrichrome A, enterobactin and desferriferrirubin, however, did not promote iron uptake, although enterobactin and desferriferrirubin stimulated bacterial growth. These paradoxical data are discussed in view of siderophore-inducible iron uptake systems, as demonstrated recently for enterobactin. Among the strains tested, including the wild-type PAO1, the pyoverdine-less mutant PAO6606 and the two porin-mutants P. aeruginosa H636 (oprF::Ω) and P. aeruginosa H673 (oprD::Tn501), only for the porin-OprF mutant were fewer siderophores able to promote iron uptake compared to the other strains. Such results suggest that beside specific routes for iron uptake P. aeruginosa is also able to take up siderophore-liganded iron through OprF.
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Osmotic adjustment in Brevibacterium ammoniagenes: pipecolic acid accumulation at elevated osmolalities
More LessSUMMARY: Brevibacterium ammoniagenes ATCC 6872 was grown aerobically in minimal defined glucose media of different osmolalities induced either by NaCl, other eletrolytes or non-electrolytes. Growth rate was slightly affected by elevation of medium osmolality up to 1 M-NaCl, but severely decreased at 1.5 M; however even at 2 M-NaCl, slow growth still occurred. Glycine betaine (or its precursor choline) did not stimulate growth in high osmotic media, although it accumulated intracellularly and was not metabolized. An organic solute which increased substantially in concentration during osmotic treatment with various osmolytes, was isolated and identified as pipecolic acid. No accumulation of this imino acid was observed when the medium concentration was raised by adding the permeant glycerol or when the culture medium was supplemented with glycine betaine (1 mM). While this non-proteic cyclic amino acid may play an important role in bacterial adaptation to environmental stress, it did not accumulate to a high level. Preliminary data suggest that the biosynthesis of pipecolic acid from lysine is strongly regulated by external osmolality.
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Energy conservation by succinate decarboxylation in Veillonella parvula
More LessSUMMARY: Veillonella parvula cannot grow with succinate as sole energy source. However, succinate decarboxylation simultaneous with malate or lactate fermentation increased growth yields by 2.4–3.5 g (mol succinate)-1. Malate was fermented stoichiometrically to acetate and propionate whereas lactate fermentation produced more acetate and considerable amounts of H2. Aspartate was utilized only in the presence of succinate as co-substrate. Methylmalonyl-CoA decarboxylase and ATP-dependent pyruvate carboxylase, but not methylmalonyl-CoA: pyruvate transcarboxylase, were detected in cell-free extracts of malate- or lactate-grown cells. The energetic aspects of these fermentation patterns are discussed.
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Growth and adaptation of the green alga Chlamydomonas reinhardtii on diesel exhaust particle extracts
More LessSUMMARY: We have examined the growth and photosynthesis of liquid cultures of the eukaryotic green alga Chlamydomonas reinhardtii incubated with iso-octane extracts of diesel particulate exhaust. At concentrations up to 0.125% (v/v), the extracts led to a dose-dependent growth retardation. Higher concentrations caused cell death. After a prolonged lag phase, air-grown illuminated algae, stressed with sublethal concentrations of diesel exhaust extracts, adapted to the pollutants. Further addition of toxic extract up to a concentration of 2.5 (v/v) affected neither growth nor photosynthesis. Media of adapted and non-adapted cells were analysed by HPLC and GC-MS and the concentrations of the remaining toxic compounds were determined. The results showed that the amounts of certain components decreased during incubation with adapted cells.
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Kinetics and physiological implications of the growth behaviour of Eubacterium limosum on glucose/methanol mixtures
More LessSUMMARY: Batch-mode growth rates of the acetogenic anaerobe Eubacterium limosum on various substrates were dependent on the point of entry into the central metabolic network. During batch growth on glucose/methanol mixtures, simultaneous consumption of both substrates occurred at residual glucose concentrations below 6 mM following an initial period of growth on glucose alone. During this mixotrophic growth phase, significantly higher biomass yields and specific growth rates were obtained than on either substrate alone. This phenomena was confirmed in chemostat experiments and stoichiometric analysis based on specific rates indicated that carbon and energy flow was significantly altered during mixotrophic growth with little net flux through the pyruvate ferredoxin oxidoreductase. It is suggested that such an effect on both growth rate and biomass yield was due to the improved availability of both carbon and energy for anabolic reactions.
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Development of a synthetic medium for continuous anaerobic growth and ethanol production with a lactate dehydrogenase mutant of Bacillus stearothermophilus
More LessSUMMARY: A synthetic medium was developed by the pulse and medium-shift technique for the continuous cultivation of Bacillus stearothermophilus strain LLD-15 (NCIMB 12428) under anaerobic conditions. This mutant strain lacks L-lactate dehydrogenase activity, and is a promising candidate for the production of ethanol from pentoses and hexoses, using a high-temperature two-stage process. The final medium contained four amino acids and five vitamins, and growth characteristics in this medium compared well with those in complex medium containing yeast extract and tryptone. At 70 °C, the medium was capable of supporting good anaerobic and aerobic growth at 10 g input sucrose I-1. High ethanol production indicated that pyruvate metabolism probably occurred via the combined activity of the pyruvate-formate-lyase pathway and pyruvate dehydrogenase.
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Fatty acid desaturation-defective mutants of an arachidonic-acid-producing fungus, Mortierella alpina 1S-4
More LessSUMMARY: Three mutants, which were defective in the desaturation of fatty acids, were isolated from an arachidonic-acidproducing fungus, Mortierella alpina 1S-4, after treating wild-type spores with N-methyl-N′-nitro-N-nitrosoguanidine. They were designated Mut44, Mut48 and Mut49. Mut44 was a mutant with low Δ5-desaturase activity. It accumulated a high level of dihomo-λ-linolenic acid (DGLA) (28.6%, w/w) but a low level of arachidonic acid (Ara) (10.6%), compared with the wild type, which had levels of 6.3 and 47.0%, respectively. Mut48 was unable to desaturate oleic acid (18:1) to linoleic acid (18:2), i.e. Δ12-desaturation, and therefore a large amount of 18:1 (49.5%) accumulated and no fatty acid of the ω-6 family was detected. In addition, several fatty acids of the ω-9 family, such as 5,8,11-cis-eicosatrienoic acid, were found. In Mut49, 18:2 (46%) accumulated markedly, but only small amounts of DGLA and Ara were detected. Thus, Mut49 was considered to be defective in Δ6-desaturation. These mutants showed a somewhat longer lag phase than the wild type on cultivation at both 28 and 12 °C.
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Isolation and characterization of a ntrC mutant of Bradyrhizobium (Parasponia) sp. ANU289
More LessSUMMARY: A mutant of Bradyrhizobium (Parasponia) sp. ANU289 affected in the regulation of nitrogen metabolism was isolated. The mutant, designated ANU293, was unable to induce ammonium transport (Amt), nitrate reductase (NR) or glutamine synthetase II (GSII) activities under conditions that induce these activities in the wild-type. However, glutamine synthetase I (GSI), which is expressed constitutively in the wild-type, was present at normal levels in the mutant. The mutant also retained the ability to fix nitrogen in vitro and in planta, although nodule development on siratro (Macroptilium atropurpureum) was retarded. Southern blot analysis showed that ntrC, the product of which is involved in regulation of nitrogen metabolism, is the site of pSUP1021 insertion in ANU293. These results indicate that the transcriptional activator NtrC is required for the expression of Amt, NR and GSII, but not GSI or nitrogenase in Bradyrhizobium (Parasponia) sp. ANU289.
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