- Volume 123, Issue 1, 1981
Volume 123, Issue 1, 1981
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Synthesis and Order of Assembly of Spore Coat Proteins in Bacillus subtilis
More LessAnalysis of mature spores or their integuments by extraction with sodium dodecyl sulphate/dithioerythritol followed by electrophoresis shows that the coat contains four major proteins and about ten others. Nine of the 14 proteins begin to be synthesized in stage II or stage III and their synthesis must be controlled by stage II or stage III operons. Some of these proteins are incorporated into the spore structure from about t 4 onwards (i.e. 4 h after induction of sporulation). Their deposition in the coat between t 4 and t 7 is not stopped by chloramphenicol, with the exception of one protein (mol. wt 36000) which begins to be synthesized only at t 6. Spores were isolated at various stages from about t 5 onwards, and the surface proteins were labelled with 125I. The labelling patterns show that proteins which are exposed on the surface at t 5·3 are successively overlaid as the spores mature. It appears that the coat of the mature spore contains at least three layers. The outermost layer is mainly composed of an alkali-soluble protein (mol. wt 12 000), which is synthesized early (t 2), and the 36000 mol. wt protein, which is synthesized very late. Deposition of the former seems to require processing by proteolytic action, and both proteins are apparently necessary for the acquisition of resistance to lysozyme, though not for resistance to heat or organic solvents. The results are discussed in relation to the classification of sporulation events and the nomenclature of the genetic loci controlling sporulation.
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Complementation of Sporulation Mutations in Fused Protoplasts of Bacillus subtilis
More LessAsporogenous and oligosporogenous mutants of Bacillus subtilis blocked at stages III, IV and V and carrying mutations at 18 different genetic loci have been tested for complementation. This was done by preparing protoplasts 3 h after induction of sporulation (t 3) and fusing them with wild-type protoplasts prepared in parallel. After incubation to t 10 the suspension was assayed for heat-resistant (80 °C, 40 min) colony-forming units. Mutations in 9 of the 18 loci were complemented, i.e. the spore count was increased > 1000-fold. All of the complemented mutants, except for one stage V mutant, were blocked at stage IV. Spo− mutants that were complementable by wild-type were also complementable by other Spo− mutants provided that they did not carry the same spo mutation. Complementation analysis was applied to three alleles in the spoIVC locus. The complementation pattern suggests that there are at least two cistrons in the locus and this agrees with the mapping data.
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Dual Control of the gua Operon of Escherichia coli K12 by Adenine and Guanine Nucleotides
More LessThe gua operon of Escherichia coli K12 comprises structural genes for the two enzymes, IMP dehydrogenase and GMP synthetase, required for the biosynthesis of GMP from IMP. The specific activities of these enzymes were measured in various purine auxotrophs. GuaA-and guaB mutants (guanine-specific) were derepressed under conditions of growth limitation by guanine but were repressed by excess guanine. This suggests that formation of the enzymes is normally controlled by a guanine nucleotide. Derepression of the operon in purine-starved pur mutants depended on the type of mutant and on whether adenine or guanine was provided. A purA strain (adenine-specific) and strains with early blocks in purine biosynthesis (purF and purD) did not derepress. PurE or purC strains [5′-phosphoribosyl-5-aminoimidazole (AIR)-accumulating] derepressed only 4-fold. The operon was repressed in purH strains [5′-phosphoribosyl-5-amino-4-imidazolecarboxamide (AICAR)-accumulating] grown with limiting guanine or hypoxanthine, but derepressed by growth with limiting adenine. Two mutants (purA guaA and purA guaB) which can neither synthesize AMP and GMP de novo, nor interconvert them, were isolated. The specific activity of IMP dehydrogenase in one of these strains grown with different concentrations of guanine and adenine revealed that adenine induces the gua operon whereas guanine represses it. Intracellular purine nucleotide pools were measured in a purH mutant repressed (guanine-grown) and derepressed (adenine-grown) for IMP dehydrogenase. The guanylate pool was similar under the two growth conditions; however, the adenylate pool of the adenine-grown bacteria was two to three times greater than that of the guanine-grown cells. A dual mechanism for regulating expression of the gua operon, involving induction by AMP and repression by GMP, is proposed.
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Formation of Two-spored Asci by Interrupted Sporulation in Saccharomyces cerevisiae
More LessSporulation in Saccharomyces cerevisiae was disturbed by transferring cells to water after 4 to 8 h incubation in potassium acetate medium, giving rise to almost exclusively two-spored asci at a high frequency. The (+ −) asci for a given auxotrophic marker (+ and − indicate the phenotypes due to the dominant and recessive alleles of the marker, respectively) predominated over the (+ +) and (− −) asci, and the (+ +) asci predominated over the (− −) asci. The frequencies of these three ascus types were related to the distance of the marker from the centromere. Most of the segregants showed a mating reaction with either a or α haploids, while a few were non- or omni-maters. These observations, along with statistical data on observed and expected frequencies of various ascus types, indicate that the two-spored asci are not generated from normal meiotic products by random abortion of two spores. An alternative mechanism is proposed in which the first meiotic division including crossing over at the four-chromatid stage occurs normally, but the second meiotic division is interrupted, possibly at centromere splitting and/or formation of the spindle apparatus. On this basis, equations describing theoretical frequencies of various ascus types were derived. Expected frequencies of the different ascus types for nine auxotrophic markers having various centromere distances and for mating types were calculated from the equations and compared with the data observed in 188 dyads from three different diploids. A uniformly good fit was found between expected and observed frequencies.
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A Phage for Generalized Transduction in Bacillus thuringiensis and Mapping of Four Genes for Antibiotic Resistance
More LessA phage (ϕ63) for generalized transduction in Bacillus thuringiensis was isolated from a soil sample. The diameter of the head was 95 nm and the tail length was 200 nm. The growth cycle required 75 min and the burst size was 330. Phage ϕ63 was stabilized by Ca2+ ions and phage stocks could be stored cold without loss of titre. The frequency of transduction of Leu+ into an auxotrophic strain was 2 × 10−6. The frequency of transduction of genes for resistance to nalidixic acid, rifampicin, streptomycin and spectinomycin was generally an order of magnitude lower. Phage ϕ3 could cotransduce these four genes for antibiotic resistance. Crosses were performed which established the gene order as nalA-rifA-strA-spcA. Heterologous cotransduction of rifA and strA at reasonable frequencies was obtained in three of six subspecies.
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Immunoelectrophoretic Separation of Spiroplasma Antigens
More LessAntigens from a number of spiroplasmas were solubilized in the neutral detergent Lubrol PX and compared by crossed immunoelectrophoresis. Samples were also run in tandem with the type species Spiroplasma citri, continuity of immunoprecipitin rockets indicating antigenic identity and serological relatedness of spiroplasmas. It was concluded that corn stunt spiroplasmas, the honeybee (BC3) spiroplasma and the tick (277F) spiroplasma were related serologically to S. citri but some flower isolates and the tick (SMCA) spiroplasma showed no obvious relationship. The serological relatedness of S. citri and the honeybee (BC3) spiroplasma was investigated in detail. These two spiroplasmas each contain a major membrane protein which differ slightly in molecular weight. The two proteins were purified under identical conditions and used to raise monospecific antisera. Using the sera obtained in this way with a modified immunoelectrophoretic system having sodium dodecyl sulphate-polyacrylamide gel electrophoresis as the first dimension separation, it was shown that the major membrane proteins of S. citri and the honeybee (BC3) spiroplasma are antigenically dissimilar. We suggest that spiralin, the major membrane protein of S. citri, and the major membrane protein of the honeybee (BC3) spiroplasma are dominant cell surface antigens, so that while these two spiroplasmas are known to be very similar, serological tests involving comparison of cell surface antigens indicate less relationship.
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Immunodiffusion Studies of Some Nocardia Strains
More LessForty-three strains of Nocardia, one of Actinomadura and two of Nocardiopsis were studied using the comparative immunodiffusion technique. Three reference precipitation systems were employed: one represented Nocardia asteroides N10, one N. asteroides ATCC 19247, and one N. otitidis-caviarum ATCC 14629. One tight cluster was formed by the N. otitidis-caviarum strains and another tight cluster was formed by some of the N. asteroides strains studied. However, other strains of N. asteroides were distinct from the latter cluster. Furthermore, N. asteroides ATCC 19247, which is the type strain, differed from most of the N. asteroides strains tested. Strains of the species N. asteroides, N. brasiliensis, N. farcinica and N. otitidis-caviarum were found to be closely related, while N. amarae strains differed slightly from this group. The strains referred to Actinomadura and Nocardiopsis were clearly distinct from the three Nocardia reference strains; nevertheless, three antigens common to these genera were revealed.
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A Rapid Procedure for the Detection of Archaebacterial Lipids in Halophilic Bacteria
More LessWhole-organism acid methanolysates of strains of halophilic bacteria were examined for glycerol diether moieties and other long-chain constituents by thin-layer chromatography. Glycerol diether moieties were detected only in acid methanolysates of archaebacterial halophiles. Eubacterial halophiles contained only fatty acid methyl esters. Spots on thin-layer plates attributed to diether moieties were further identified by infrared spectroscopy. Thin-layer chromatographic analysis of whole-organism methanolysates provides a simple and rapid method of distinguishing archaebacterial halophiles from eubacterial halophiles. The majority of archaebacterial halophiles produced only one chromatographically distinct glycerol diether moiety, but certain strains, including the recently described alkalophilic halophiles, produced two distinct moieties.
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Filamentous Bacteria in Sediments of Lakes of Differing Degrees of Enrichment
More LessEstimates of sediment populations of filamentous bacteria were made by a variety of direct count procedures including the use of acridine orange and fluorescein diacetate. and by a viable (most probable number) technique. The counts with acridine orange showed an upward trend with increasing degree of enrichment of the lakes, particularly at the eutrophic end of the spectrum. The distribution pattern obtained with fluorescein diacetate was different, with an apparent upward trend in the intermediate lakes. The profundal and littoral zones of three lakes were examined both at the onset of thermal stratification and in late summer, when they were stratified and the hypolimnion of the eutrophic lake was anoxic. Consistently higher counts were obtained in the profundal zone, the difference being more marked in early summer. There were distinct differences between the lakes in the depth distribution of the filamentous bacteria in the sediments.
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The Distribution of Some Genera of Filamentous Bacteria in Littoral and Profundal Lake Sediments
More LessThe distribution of several genera of filamentous bacteria was determined in profundal and littoral sediments of three lakes of differing trophic status. The bacteria were tentatively identified on the basis of cytochemical tests and microscopic observations of organisms in wet mount preparations and on agar-coated and uncoated slides incubated in the sediment cores. The population density and the diversity of the filamentous bacteria increased with increasing nutrient content of the lakes. The general observations of higher numbers in the profundal than in the littoral zone and a decrease in population density with sediment depth were confirmed for most genera. Some of the genera may be microaerophiles. An affinity for surfaces was observed. Pseudanabaena usually occurred only in littoral samples and Beggiatoa only in the two richer lakes. Vitreoscilla was found mainly in the two richer lakes and Leptothrix was present in large numbers in all three lakes.
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Simulation of the Rat Intestinal Ecosystem using a Two-stage Continuous Culture System
More LessTo investigate the ecological mechanisms governing the community structure of the gut microbial ecosystem, we have attempted to simulate the rat gut ecosystem in vitro using a two-stage continuous culture. Extensive sampling of the rat hindgut has established a set of criteria with which the in vitro system may be compared. This paper discusses one of the criteria, the community composition and structure in vivo and in vitro. The experiments indicated that a gut microbial ecosystem could be satisfactorily mimicked in vitro. This was achieved using a two-stage continuous culture employing differential selection of species between the two stages on the basis of pH differences combined with cell recycling between stages.
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Dehalogenases in Soil Bacteria
More LessSixteen bacterial strains isolated from soil were able to grow on either 2-monochloropropionic acid or monochloroacetic acid as the sole carbon and energy source. The isolates were divided into five groups on the basis of differences in their dehalogenase activities towards four substrates - monochloroacetic acid, dichloroacetic acid, 2-monochloropropionic acid and 2,2-dichloropropionic acid. Disc gel electrophoresis of crude extracts identified four distinct dehalogenases with different electrophoretic mobilities: three isolates contained one, three or four dehalogenases, respectively, and the remaining 13 isolates contained different combinations of two dehalogenases. In some cases, dehalogenases with the same mobilities from different isolates appeared to be identical enzymes. In others, enzymes from different isolates with the same electrophoretic mobility had different substrate activity profiles. Pseudomonas putida PP3 was shown to contain one enzyme which was comparable with one of the dehalogenases detected in several of the newly isolated soil bacteria. The second enzyme was not found in soil bacteria and represented a fifth dehalogenase. The significance of these results in terms of the evolution of dehalogenase activity is discussed.
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The Maintenance of Plasmid-containing Organisms in Populations of Escherichia coli
More LessPopulations of Escherichia coli containing a small non-conjugative plasmid were grown in carbon-limited continuous culture. For all plasmids tested the presence of the plasmid lowered the growth rate of the host bacterium, and the proportion of plasmid-containing organisms in the total population declined initially. However, periodically, adaptive changes occurred in plasmid-containing organisms which increased their growth rate. This resulted in oscillations in the proportion of plasmid-containing organisms, and the delayed loss of the plasmid from the population.
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Involvement of Menaquinone in the Active Accumulation of Aminoglycosides by Bacillus subtilis
More LessAccumulation of aminoglycoside antibiotics by bacteria requires energy, and it appears that this must be derived from electron transport occurring within the cytoplasmic membrane. Dependence of aminoglycoside accumulation on cellular menaquinone content was examined using a menaquinone auxotroph of Bacillus subtilis. This dependence manifested itself only when the menaquinone concentration was decreased to less than 10% of normal. The restricted aminoglycoside accumulation observed under these conditions was closely correlated with susceptibility to growth inhibition by the antibiotics. Evidence of saturation of the accumulation system was observed at low menaquinone concentrations, an effect not seen when menaquinone deficiency was relieved by supplying adequate shikimic acid (a menaquinone precursor) to the auxotroph. Lipophilic quinones may play two roles in aminoglycoside accumulation by bacteria: (i) as a binding site or part of a carrier complex; and (ii) as a crucial component of the electron transport system in maintaining the proton electrochemical gradient.
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Surface Tension-like Forces Determine Bacterial Shapes: Streptococcus faecium
More LessThe same tendency that causes soap bubbles to achieve a minimum surface area for the volume enclosed seems to account for many of the features of growth and division of bacteria, including both bacilli and cocci. It is only necessary to assume that growth takes place in zones and that only in these zones does the tension caused by hydrostatic pressure create the strain that forces the cell to increase the wall area. The stress developed by osmotic pressure creates strains that significantly lower the free energy of bond splitting by hydrolysis or transfer. We believe this is sufficient to make growing wall have some of the properties ordinarily associated with surface tension. The feature common to all bacterial cell wall growth is that peptidoglycan is inserted under strain-free conditions. Only after the covalent links have been formed are the intervening stressed peptide bonds cleaved so that the new unit supports the stress due to hydrostatic pressure.
The present paper analyses the growth of Streptococcus faecium in these terms. This is a particularly simple case and detailed data concerning morphology are available. The best fit to the data is achieved by assuming that growth takes place in a narrow region near the splitting septum and that the septal material is already under tension as it is externalized and is twice as thick as the external wall throughout the development of the nascent poles. Constancy of the ratio of hydrostatic pressure to the effective surface tension, P/T, is also consistent with electron microscopic observations.
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Fatty Acids of Fusobacterium Species: Taxonomic Implications
More LessFatty acids of Fusobacterium species were examined by gas-liquid chromatography. Fusobacterium nucleatum, F. necrophorum, F. mortiferum, F. gonidiaformans and F. varium showed similar patterns, characterized by the presence of 3-hydroxytetradecanoate, n-tetradecanoate, hexadecenoate, n-hexadecanoate, octadecenoate, n-octadecanoate and a component having the properties of octadecadienoate. Fusobacterium nucleatum contained 3-hydroxyhexadecanoate as a distinctive character. Simpler fatty acid patterns characterized by the absence of 3-hydroxytetradecanoate and other hydroxy fatty acids were observed in F. plauti, the single strain of F. prausnitzii and in the majority of strains classified as F. russii and F. naviforme. Neither methyl-branched nor cyclopropane fatty acids could be detected in any of the strains examined. In addition to fatty acid methyl esters, the chromatographic profiles of all species except F. mortiferum, F. gonidiaformans and F. naviforme contained substantial amounts of fatty aldehyde dimethyl acetals of chain lengths C14 to C18.
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- Short Communication
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Oxygen Availability and Seasonal Migrations of Ciliated Protozoa in a Freshwater Lake
More LessMigrations of ciliated protozoa between the benthos and water column of a productive lake are reversible and linked to seasonal changes in oxygen availability. Maximum densities of planktonic populations are maintained for periods of several months at depths where the oxygen concentration is l mgl−1 or less. With the possible exception of one genus (Loxodes) there was no evidence for periodic vertical migration within a season. The supposed unconditional requirement for oxygen in large free-living ciliates is questioned.
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Human Serum Complement Requirements for Bacterial Killing and Protoplast Lysis of Escherichia coli ML308 225
R.J. Allen and G.K. ScottNormal human serum kills Escherichia coli ML308 225 and lyses protoplasts derived from this organism. Human serum which is depleted of complement component C9 or deficient in component C8 is not bactericidal, but C9-depleted serum will lyse protoplasts whereas C8-deficient serum will not. Bacterial lipopolysaccharide, which can protect bacteria from the serum bactericidal reaction, does not protect protoplasts from lysis by serum.
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Glycerol Utilization by Schizosaccharomyces pombe: Dehydrogenation as the Initial Step
More LessStrains NCYC 132 and 972h− of the fission yeast Schizosaccharomyces pombe both lacked glycerol kinase, but produced an NAD+-linked glycerol dehydrogenase. In both strains, production of the dehydrogenase was subject to catabolite repression, although in strain 972h− glycerol also appeared to exert some inductive effect.
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Molecular Cloning in Plasmid pBR322 giving Altered Expression of the Tetracycline Resistance Gene
More LessThe two HindIII fragments of polyoma virus DNA were cloned in the HindIII site of plasmid pBR322, a site located in the RNA polymerase promoter involved in the expression of tetracycline resistance. Although insertion of foreign DNA into this site did not always result in the complete loss of tetracycline resistance, Escherichia coli K12 strain χ1776 harbouring recombinant plasmids exhibited reduced growth properties in liquid culture with tetracycline and could easily be differentiated from bacteria transformed by non-recombinant plasmids. The formation of plasmid multimers increased the resistance to tetracycline at the level of the induction period, presumably as a result of a gene dosage effect.
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