- Volume 128, Issue 2, 1982
Volume 128, Issue 2, 1982
- Biochemistry
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An Asymmetric Archaebacterial Diether Lipid from Alkaliphilic Halophiles
More LessArchaebacterial halophiles from alkaline soda lakes were shown to possess substantial amounts of a core diether lipid differing from the C20,C20 diether lipid characteristic of Halococcus and Halobacterium spp. This novel diether lipid was shown to be an asymmetric C20,C25 diether (2-O-sesterterpanyl-3-O-phytanyl-sn-glycerol). The implications of this unusual lipid for membrane structure are discussed.
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Repression of the Alkaline Phosphatase of Vibrio cholerae
More LessThe synthesis of alkaline phosphatase by two strains of Vibrio cholerae belonging to the Inaba and Ogawa serotypes has been examined in relation to the phosphate concentration of the culture medium. The synthesis of the enzyme in both strains was repressed in cells grown in the presence of a high concentration of inorganic phosphate. Lowering the phosphate content of the growth medium led to a derepression of enzyme activity. The presence of glucose in low phosphate medium stimulated the degree of derepression. The synthesis of the enzyme by strain Inaba 569B was more sensitive to inorganic phosphate than that of strain Ogawa 154. The enzyme was presumably located in the periplasmic space since it was released when the organisms were converted to spheroplasts.
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- Development And Structure
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Isolation and Characterization of the Sheath from the Cyanobacterium Chlorogloeopsis PCC 6912
More LessA sheath fraction was isolated from cells of the cyanobacterium Chlorogloeopsis PCC 6912. It had a fibrillar fine structure, a high density and contained 38% (w/w) carbohydrates and 22% (w/w) protein. Glucose, mannose, galactose, arabinose, xylose, an unknown sugar and glucuronic acid were the major carbohydrates. Lipids were almost entirely absent. The protein was not solubilized by treatment with hot phenol/water (68 °C; 20 min) or Triton X-100 (2%, w/v; 20 °C; 3 h). The carbohydrates of the sheath belong to two different polysaccharides. The major one was not separated from the protein moiety of the sheath by hot phenol/water. It contained glucose as the main constituent, arabinose and xylose but little galactose, in addition to the other carbohydrates. In contrast, the minor polysaccharide was soluble in the water phase of hot phenol/water extracts and contained galactose as a major constituent in addition to glucose and mannose, but very little arabinose and xylose. The polysaccharide enriched in galactose could also be obtained from the water phase by precipitation with Cetavlon when sheath-containing whole cells were extracted by phenol/water.
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Polysaccharide Containing 6-O-Methyl-d-mannose in Chlorogloeopsis PCC 6912
More LessA high molecular weight polysaccharide sedimenting at 105 000 g was extracted into the water phase of phenol/water extracts of Chlorogloeopsis PCC 6912 cells. The main sugars of the heteropolysaccharide were mannose, 6-O-methyl-d-mannose, glucose, rhamnose, and glucuronic and galacturonic acids. 6-O-Methyl-d-mannose was present partly in terminal linkage and partly 1,3-chain-linked, as revealed by methylation analysis. 3-O-Methyl-mannose, however, was found in only trace amounts and exclusively occupied terminal positions. The heteropolysaccharide presumably represented a polysaccharide of the cell wall. There was no indication of a lipid moiety. Application of procedures commonly used for lipopolysaccharide extraction from whole cells resulted in very low yields or even indicated complete lack of a typical lipopolysaccharide.
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Nucleoids of Caulobacter crescentus CB15
More LessNucleoids isolated from heterogeneous populations of Caulobacter crescentus were invariably associated with the cell envelope. The cell types from which nucleoids were derived were easily identifiable because of the distinctive dimorphic cell cycle of this organism. Sucrose density gradient centrifugation of an exponentially growing culture, gently lysed at 10 °C, gave two classes of envelope-associated nucleoids. One, a broad slow sedimenting band with a sedimentation coefficient of 5600S, was comprised primarily of nucleoids from stalked cells. The other, a tight fast sedimenting band with a sedimentation coefficient of 7100S, was comprised of nucleoids from flagellate and pre-divisional cells. The DNA packing and nucleoid morphology of each class of nucleoids was examined by electron microscopy.
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In vitro Transcription and Translation Directed by Caulobacter crescentus CB15 Nucleoids
More LessEnvelope-associated nucleoids isolated from Caulobacter crescentus contained DNA-dependent RNA polymerase and nascent RNA. In the presence of nucleotide substrates, and under suitable conditions, RNA chain elongation took place at similar rates on both fast sedimenting (FS) and slow sedimenting (SS) nucleoids. Reinitiation of transcription did not occur in vitro except when exogenous holoenzyme was added to the assay system. Three major classes of RNA were synthesized in vitro. The two larger species were heterodisperse and had electrophoretic mobilities equivalent to 16-20S and 23-35S. The third class contained RNA in the region of 3-4S. The high molecular weight components were absent from RNA synthesized on FS nucleoid templates whereas the 3-4S fraction was much increased.
Caulobacter crescentus nucleoids were also examined for the presence of mRNA in a protein-synthesizing system derived from Escherichia coli cells. The activity of heterologous mRNA was detectable only after the conversion of E. coli cell lysates into an mRNA-dependent translation system by treatment with micrococcal nuclease. Incorporation of [35S]methionine into trichloroacetic acid-insoluble material reflected synthesis of bona fide template-specific polypeptides by several criteria showing that SS and FS envelope-associated nucleoids contained biologically active mRNAs. Since the nucleoids were transcriptionally inactive during protein synthesis, the assay reflected the presence of mRNAs produced at different stages in the Caulobacter cell cycle. The translational capacity of the nucleoids increased markedly after transcription indicating that some RNA synthesized in vitro was mRNA. The specific activity of transcribed nucleoids as templates for protein synthesis supported the contention that the predominant RNAs synthesized in vitro were rRNAs and their precursors.
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A Mutant of Dictyostelium discoideum with Alternative Pathways of Differentiation Depending on Culture Conditions
More LessA mutant (dev-1515), which forms either spores or stalk cells depending on the cultural conditions, was isolated from dev-1510, a mutant of Dictyostelium discoideum NC-4. When grown with Escherichia coli on a nutrient-rich medium, dev-1515 formed a hemispherical cell mass in which most cells differentiated into spores. Culture with E. coli on a nutrient-intermediate medium inhibited amoebae from aggregating normally and they differentiated into stalk cells only. Development of washed vegetative cells of the mutant proceeded further without bacteria on either medium. Cells cultured in a roller tube were profoundly affected by the ionic strength of the medium. Almost all the cells in an agglutinate differentiated into stalk cells in a medium of low ionic strength, while most cells differentiated into spores in a medium of high ionic strength.
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- Ecology
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Stimulation of the Growth of Cutaneous Strains of Peptococcus saccharolyticus by Iron, Haematin and Blood
More LessThe growth of nine strains of Peptococcus saccharolyticus was assessed quantitatively by culture on Trypticase Soy/yeast extract/Tween 80 agar (TSY-TW) with and without supplementation with iron or haematin and on blood agar, in aerobic, reduced O2 (3% O2 with 8% CO2, 8% H2 and 81% N2) and anaerobic atmospheres. All strains grew better anaerobically and under reduced O2 conditions than aerobically on supplemented or unsupplemented TSY-TW. Supplementation of TSY-TW with iron or haematin resulted in an average 44-fold increase in bacterial count in a reduced O2 atmosphere and an average 42-fold increase under anaerobic conditions. Under aerobic conditions the increase in count ranged from 0 to >5000-fold, as some strains failed to grow on unsupplemented TSY-TW but responded well to the supplements of iron or haematin. The highest bacterial counts were obtained on Columbia blood agar incubated anaerobically. However, P. saccharolyticus failed to grow aerobically on plain or heated Columbia blood agar with or without supplements. TSY-TW blood agar supported the growth of the one strain tested under all three atmospheric conditions. The type strain (ATCC 14953) differed from all others in its failure to grow aerobically or in a reduced O2 atmosphere on supplemented or unsupplemented media. Colony size varied greatly on different media, in different atmospheres and from strain to strain, being greatest in a reduced O2 atmosphere on Columbia blood agar. There was no correlation between the viable bacterial count and colony size.
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Microbial Ecology of Pectin Decomposition in Anoxic Lake Sediments
More LessAnaerobic digestion of pectin by bacteria was examined in two freshwater lakes in Wisconsin and in defined laboratory cultures of species prevalent in the lake sediment. The turnover times for pectin biodegradation to methane in sediments incubated at in situ temperature were much longer (100 h in Lake Mendota and 185 h in Knaack Lake) than either that observed for glucose (12 h in Lake Mendota) or previously reported for acetate (0·22 h in Lake Mendota). The numbers of pectinolytic anaerobes varied seasonally in both sediments (102–105 and 103–105 ml−1 in Knaack Lake and Lake Mendota, respectively), and were highest during the fall after sedimentation of algal blooms and/or leaf detritus. Clostridium butyricum was identified as a prevalent pectinolytic anaerobe in both lakes. In mono-culture pectin fermentations, C. butyricum produced methanol, H2/CO2, acetate, ethanol and butyrate; growth stopped in the presence of excess energy source when the pH fell to 43. In co-culture pectin fermentations of C. butyricum/Methanosarcina barkeri, H2/CO2, methanol and acetate were detected as intermediary metabolites, and pectin was completely degraded to CH4 and CO2, the pH remaining neutral. 14C-radiotracer analysis substantiated the simultaneous conversion of H2/CO2, methanol and acetate to CH4 by M. barkeri as these metabolites were generated from pectin hydrolysis by C. butyricum.
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Limits to Microbial Growth in Soil
More LessMore organic carbon was released into solution from soils that had been air-dried and rewetted than from the natural soils. Irradiation and autoclaving of a range of air-dried or field soils greatly increased the amount of carbon that was released into solution. The carbon released appeared to provide substrates for fungal spore germination and bacterial growth, thereby relieving microbiostasis. The percentage germination of Penicillium citrinum spores was at least doubled in sterilized soil and halved in untreated soil compared with spores germinating in the absence of soil. In general, a streptomycin-resistant strain of Agrobacterium radiobacter made significant growth only in sterilized soils.
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- Genetics And Molecular Biology
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Molecular Homology and Incompatibility in the IncFI Plasmid Group
More LessThe usual grounds for the inclusion of a plasmid in a particular incompatibility group are its mutual incompatibility with a type plasmid of that group, and, in some cases, the demonstration of shared regions of specific homology, presumed to be related to DNA replication. We have found that some plasmids classified as IncFI on genetical grounds share no homology with the previously described incompatibility regions of F on the basis of hybridization of specific radioactive probes to restriction enzyme digests of DNA from these plasmids. Others show homology with some or all of the regions of the F plasmid that can express incompatibility. The incompatibility behaviour of these plasmids has been examined to determine the relationship between the possession of regions of homology and the expression of incompatibility. Three plasmids, ColV3-K30, pHH507 and Entp307, show homology only with the secondary replicon of F and appear to use sequences homologous with the secondary F replicon in their replication. The results are consistent with the propositions that some contemporary IncFI plasmids arose by the integration of several replicons, and, in general, the replicon not being used for replication expresses its incompatibility, as does the replicon being used for replication. We conclude that incompatibility of two plasmids with F does not necessarily demonstrate relatedness of the plasmids to each other, and that inclusion within the IncFI group can result from the possession of any of several combinations of inc sequences.
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Genetic Instability of R Plasmids in Relation to the Shift of Drug Resistance Patterns in Salmonella johannesburg
More LessObservation of the resistance of Salmonella johannesburg to the six drugs ampicillin (A), streptomycin (S), tetracycline (T), chloramphenicol (C), kanamycin (K) and sulphadiazine (Su) was made over the 7 years from 1973 to 1979. Strains with ASTCKSu-and ASCKSu-resistance patterns predominated in the years 1973–1975 and 1976–1979, respectively. These resistances were found to be mediated by autotransferring plasmids belonging to the incompatibility group FI me. The ASTCKSu-resistance plasmids were unstable, giving rise to deletion variants at a much higher frequency than ASCKSu-resistance plasmids either of natural origin or derived in vitro from the ASTCKSu-resistance plasmids. Thus, the ASCKSu-resistance plasmid might be a deletion variant of the ASTCKSu-resistance plasmid. This is supported by the extensive similarity of their cleavage patterns produced by specific restriction endonucleases.
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Pattern of RNA Transcription during Bacillus subtilis Spore Outgrowth
More LessDuring early outgrowth of Bacillus subtilis spores, there is a period of RNA and protein synthesis in the absence of DNA replication. Two mutants of B. subtilis, PB2442 (gsp-4) and PB2452 (gsp-81), were used to study the pattern of RNA synthesis during this period. The two mutants are temperature-sensitive in the outgrowth phase, and show a limited amount of incorporation of [3H]uridine at 47 °C. The RNAs synthesized during a 2 min pulse with [3H]uridine were hybridized to Eco RI-digested DNA, after agarose gel electrophoresis and transfer to nitrocellulose paper (Southern technique). For both mutants the transcripts synthesized at 35 °C at different times were different. Differences were also observed in the transcripts made at 47 °C. For both mutants, in the presence of chloramphenicol, the same hybridization pattern was obtained for RNAs pulse-labelled at different periods during outgrowth.
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Envelope Protein Synthesis and Inhibition of Cell Division in Escherichia coli during Inactivation of the B Subunit of DNA Gyrase
More LessThe rates of synthesis of inner and outer membrane proteins of Escherichia coli K12 during inhibition of cell division have been studied. When cell division was inhibited, either by treatment of wild-type cells with the antibiotic clorobiocin (an inhibitor of the B subunit of DNA gyrase) or by a temperature shift of a gyrB-ts mutant, a 40% reduction in the rate of synthesis of total outer membrane protein relative to that of the inner membrane was observed. When a gyrB-ts mutant was shifted to high temperature under conditions which allowed continued cell division, this large reduction in the rate of synthesis of outer membrane protein relative to inner membrane protein was not observed. In contrast to the results obtained with clorobiocin, inhibition of cell division by the β-lactam antibiotic cefuroxime did not cause any detectable disturbance in the rate of synthesis of either inner or outer membrane protein. This demonstrates that inhibition of septum formation per se does not perturb synthesis of envelope protein.
The data obtained are consistent with a model in which the rate of synthesis and therefore expansion of outer membrane is one of many conditions which must be satisfied before septum formation can occur. The results are discussed in relation to such a model, and to previous findings which have shown that the rate of synthesis of outer membrane proteins displays a linear mode with an abrupt doubling in rate at a discrete point in the cell cycle.
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Secretion of Tyrosinase in Streptomyces glaucescens
More LessIn Streptomyces glaucescens, the intracellular and the extracellular enzyme forms of tyrosinase were found to be identical in molecular weight (29000), in copper content (0·21%), in the 19 amino acids at the amino-terminal end and in the ratio of cresolase to catecholase activity (0·005). The tyrosinase secretion process exhibited a constant rate of 0·15 units h−1 (mg protein)−1. Under highly induced conditions intracellular tyrosinase was accumulated. Mutations responsible for the non-melanogenic, tyrosinase-positive non-secretor mutant type are located chromosomally on the upper right arc of the S. glaucescens map near the ade-1 marker.
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Far Ultraviolet Light Sensitive Derivatives of Streptomyces clavuligerus
G. Saunders and G. HoltFive far ultraviolet light sensitive (Uvs) strains-CL77, CL89, CL90, CL104 and CL105-were isolated from an auxotrophic strain of Streptomyces clavuligerus (ATCC 27064). When compared to their progenitor, strain CL7, all exhibited a changed mutagenic response to far ultraviolet light (FUV). Strains CL77 and CL104 failed to yield detectable mutants, whereas induced mutants were obtained in strains CL90 and CL89, but only at low FUV doses (up to 20 J m−2). Strain CL89 was hypermutable at these low doses relative to strain CL7. Strain CL105 gave an increased response in terms of mutants per surviving cell. Caffeine increased the FUV-induced mutation frequency at a particular FUV dose in strain CL7 by up to 3000%. All the Uvs strains showed an altered response to caffeine compared with strain CL7.
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- Immunology
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An Immunoelectrophoretic Approach to the Identification of Progressive and Fluctuating Isolates of the Hop Wilt Fungus Verticillium albo-atrum
More LessMycelial and culture filtrate antigens from 10 progressive and 10 fluctuating isolates of Verticillium albo-atrum were analysed by crossed immunoelectrophoresis and rocket-line immunoelectrophoresis using antisera raised against pooled mycelial and pooled culture filtrate antigens from 5 isolates of each type. In crossed immunoelectrophoresis, pooled antigens produced precipitin lines corresponding to about 30 mycelial and 25 culture filtrate antigens with homologous antisera. Cross-absorption of antisera with heterologous antigens indicated that the culture filtrate preparations from the two types of isolate were identical and that the mycelial extracts from the two groups differed in one antigen, antigen ‘21’. This antigen was present in mycelial preparations from 9 out of 10 progressive isolates but was either absent or present in low concentration in 7 out of 10 fluctuating isolates. Antigen ‘21’ was also present in culture filtrate preparations from 15 of the 20 isolates.
On the basis of the precipitin patterns of antigen ‘21’, three serological groups of V. albo-atrum may be described. It is suggested that the virulence of progressive isolates might be associated with the production of high concentrations of this antigen.
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- Pathogenicity And Medical Microbiology
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The Effect of Benzyl Penicillin on the Ultrastructure of Neisseria gonorrhoeae
J. J. Jamil and S. HafizElectron microscopy of Neisseria gonorrhoeae grown on solid medium with a subinhibitory concentration of penicillin suggested that the amount of penicillin reaching each pair of gonococci was different, as illustrated by the ultrastructural appearance of N. gonorrhoeae cells in the colony. This supports the view that the concentration of penicillin in different parts of the colony is not uniform, causing some cells to lyse while the others remain intact.
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Successful Treatment of Experimental Escherichia coli Infections in Mice Using Phage: its General Superiority over Antibiotics
More LessSUMMARY: Anti-K1 phages were more active in vitro and in vivo against an O18:K1:H7 ColV+ Escherichia coli strain, designated MW, than were other phages. A single intramuscular dose of one anti-K 1 phage was more effective than multiple intramuscular doses of tetracycline, ampicillin, chloramphenicol, or trimethoprim plus sulphafurazole in curing mice of a potentially lethal intramuscularly or intracerebrally induced infection of MW; it was at least as effective as multiple intramuscular doses of streptomycin. When MW and the phage were inoculated into different gastrocnemius muscles of the same mice, a rapid reduction in numbers of MW organisms occurred in the MW-inoculated muscle and in other tissues; the numbers of phage particles in the MW-inoculated muscle increased rapidly and greatly. MW failed to proliferate in the brains of intracerebrally infected mice that had been inoculated intramuscularly with the phage at the same time; many more phage particles were found in the brains of these mice than in other sites. The few phage-resistant mutants of MW found in the phage-treated mice were K1−; previous studies had shown such mutants to be of greatly reduced virulence.
The phage administered intramuscularly 3–5 d before challenge with a potentially lethal intramuscularly induced infection of MW was protective, the protective effect varying between phage propagated on different bacterial strains.
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The Fermentation of Lactulose by Colonic Bacteria
More LessSixty-four strains of intestinal bacteria were cultured under anaerobic conditions in lactulose-containing media to assess their ability to ferment lactulose. Some organisms were unable to metabolize the disaccharide, while others, e.g. clostridia and lactobacilli, metabolized lactulose extensively. Quantitative analyses of the fermentation products indicated that the major non-gaseous metabolites were acetic, lactic and butyric acids. Hydrogen and carbon dioxide were the only gases detected. Fermentation products were estimated for selected species throughout their growth cycles. The products of fermentation of lactulose by stool cultures varied with incubation conditions such as pH, but correlated well with those produced by pure cultures. These results are discussed in relation to the therapeutic uses of lactulose.
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