- Volume 131, Issue 10, 1985
Volume 131, Issue 10, 1985
- Biochemistry
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Langmuir and Scatchard Parameters Do Not Describe the Binding of Actinomyces viscosus to Saliva-treated Hydroxyapatite
More LessSummary: The binding of Actinomyces viscosus T14V to saliva-treated spheroidal hydroxyapatite (SHA) beads was studied. The association constant (K) and the total number of binding sites (N) obtained from the Langmuir plots were in good agreement with those reported by other workers (approx. 3 × 10−8 and 3 × 108, respectively). The values for N obtained from Scatchard plots differed from those obtained from Langmuir plots by factors of 106 or more. These results suggest that either these equations are inappropriate to describe binding or certain assumptions regarding this system are not being met. The use of these models requires, among other constraints, that the process be reversible and that measurements be taken at equilibrium. A method was developed which allowed a close examination of the equilibrium dynamics without perturbation of the system. The results suggest that the adsorption process is only poorly reversible. Adsorption to SHA was not at equilibrium after 1.5 h. Even when bacteria were allowed to adsorb for longer periods, and the system appeared to approach equilibrium, the increased time of adherence did not significantly alter the derived K or N values. Our results suggest that the use of Scatchard and Langmuir plots is inappropriate to describe binding of A. viscosus to SHA.
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Occurrence of Corynomycolic Acids in Strains of Nocardia otitidis-caviarum
More LessSummary: Studies of the hydroxylated fatty acids in strains of Nocardia otitidis-caviarum showed variations in mycolic acid patterns, some strains containing only nocardomycolic acids and other strains having both nocardomycolic and corynomycolic acids. Mass spectroscopy showed that most of the nocardomycolic acids contained 52 to 56 carbon atoms and two double bonds in the main chain; the corynomycolic acids had saturated or monounsaturated chains and 30 to 34 carbon atoms. In the strains having both types of mycolic acid, only nocardomycolic acids were found as cell wall components.
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- Development And Structure
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Factors Regulating the Encystment Enhancing Activity (EEA) of Acanthamoeba castellanii
More LessSummary: An extracellular encystment enhancing activity (EEA) greatly stimulates cyst formation by Acanthamoeba castellanii under several different conditions. The activity appears when conditions are suboptimal for growth. EEA does not induce encystment by itself, but enhances differentiation initiated by other factors. EEA titres and their relationship to differentiation are described for encystment induced by: (1) berenil: (2) glucose starvation; and (3) total nutrient starvation. Extracellular EEA was required for maximum encystment by low density cultures in (1) and (2), but not in (3). It was required during the period of visible cyst wall formation rather than for earlier events in encystment. The effectiveness of EEA was reduced under some conditions by spontaneous changes in cellular sensitivity, and by extracellular inhibitors.
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- Ecology
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Xylan-digesting Bacteria from the Rumen of Sheep Fed Maize Straw Diets
More LessSummary: Representatives of a large number of bacterial isolates that produced clearings in xylan agar medium in an earlier study were characterized in greater detail. Butyrivibrios and ruminococci predominated. Three different biotypes of Butyrivibrio fibrisolvens, all non-cellulolytic, were identified on the basis of net acetate production or uptake, or the production of propionate. The only variable characteristic observed within the group of Ruminococcus albus isolates was the ability to solubilize cellulose. In contrast, all typical Ruminococcus flavefaciens isolates were cellulolytic. Other bacteria that occurred in smaller numbers belonged to the genera Eubacterium, Coprococcus, Fusobacterium, Bacteroides, Treponema and Clostridium.
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Regulation of Toxin Biosynthesis by Plasmids in Vibrio cholerae
More LessSummary: Vibrio cholerae strain 569B Inaba harbouring P plasmid produced less toxin than the parent strain. To examine the effect of plasmid loss on toxin production, temperature-sensitive (ts) mutants of P, unable to replicate at 42 °C, were isolated. One ts plasmid was unstable at 42 °C and its loss yielded a cured strain that resumed a normal level of toxin biosynthesis characteristic of the plasmid-free parent strain. Toxin production was again suppressed in the cured strain after reacquisition of P plasmid. This suggested a role for plasmid-borne genes in the regulation of toxin biosynthesis. A mutant of strain 569B Inaba that produced mutant toxin was isolated by transfer of P and V plasmids. The mutant toxin was similar to choleragenoid because it did not give rise to symptoms of cholera but induced antitoxin immunity in rabbits.
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16S Ribosomal RNA Analysis of Filibacter limicola Indicates a Close Relationship to the Genus Bacillus
More LessSummary: The phylogenetic relationship of the Gram-negative, filamentous gliding bacterium Filibacter limicola was analysed by 16S rRNA oligonucleotide cataloguing. In contrast to the proposed membership of this asporogenous species in the Flexibacteriaceae, Filibacter limicola clusters phylogenetically with the Gram-positive eubacteria Bacillus pasteurii, Sporosarcina ureae and the asporogenous species Planococcus citreus. The genetic relationship is supported by several common phenotypic properties.
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- Genetics And Molecular Biology
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Conjugation Systems of IncT Plasmids
More LessSummary: Four IncT plasmids were compared for various chatacters, in particular pilus synthesis and function at different temperatures. The prototype Rtsl differed in some respects from the others (R402, R394, pIN25). At 37 °C, the supposedly temperature-sensitive conjugation systems of the plasmids could still function efficiently on a surface, but not in a liquid. Long conjugative pili were synthesized at 30 °C, but only short ones (approx. 200 nm) were produced at 37 °C. The long pili converted two surface-obligatory conjugation systems to surface + liquid ones at 30 °C.
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Colicin E2 Production and Release by Escherichia coli K12 and Other Enterobacteriaceae
More LessSummary: Previous work has shown that Escherichia coli K 12 ColE2+ cells undergo a form of partial lysisand exhibit increases in lysophosphatidylethanolamine (lysoPE) and free fatty acid content due to activation of phospholipase A when induced to produce and release colicin E2. The increase in lysoPE content was assumed to be essential for efficientcolicin release. These same characteristics are also presented by some natural ColE2+ isolates, and by other representatives of the Enterobacteriaceae after transformation with derivatives of a ColE2 plasmid. However, Salmonella typhimurium strains carrying ColE2 plasmids released colicin without partial lysis and without increasing their lysoPEcontent. A previously undetected minor phospholipid, which appeared in these and other strains only when they were induced to produce colicin, may be an important factor in colicin release. In ColE2+ E. coli K 12, production of this new lipid was dependent on phospholipase A activation following expression of the ColE2 lysis gene. Some other ColE2+ strains did not respond to induction of colicin production in the same way as ColE2+ E. coli K 12. These strains were less sensitive to inducer (mitomycin C) or unable to produce increased amounts of colicin in response to induction, or unable to degrade colicin once it was released. In general, the results suggest that colicin release occurs by the same or similar processes in the various strains tested, and support the continued use of E.coli K 12 as the model strain for studying the mechanisms of colicin release.
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Regulation of NAD Biosynthesis in Salmonella typhimurium: Expression of nad–lac Gene Fusions and Identification of a nad Regulatory Locus
More LessSummary: Regulation of NAD biosynthesis was examined through the construction of nad–lac fusions in Salmonella typhimurium. The nad A (17 unit map position) and nadB (55 units) genetic loci involved with quinolinic acid biosynthesis were both found to be regulated by the product of a nadR locus (99 units) in a repression/derepression manner while nadC (3 units) expression appeared constitutive at the transcriptional level. Increases in nadAB transcription directly correlated with decreases in intracellular NAD(P) levels, and kinetic studies indicated that the NAD analogue 6-aminoNAD was ineffective in repressing either nadA or nadB. The presence of cAMP + cAMP receptor protein was essential for the complete derepression of nadA while no effect was evident upon nadB. Transfer of cultures from aerobic to anaerobic conditions, however, resulted in the partial derepression of both nadA and nadB. Thus, there appears to be a very complex set of controls regulating NAD biosynthesis.
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Isolation, Characterization and Complementation Analysis of nir B Mutants of Escherichia coli Deficient Only in NADH-dependent Nitrite Reductase Activity
More LessSummary: Mutants have been isolated which lack NADH-dependent nitrite reductase activity but retain NADPH-dependent sulphite reductase and formate hydrogenlyase activities. These NirB−strains synthesize cytochrome c 552 and grow normally on anaerobic glyeerol–fumarate plates. The defects map in a gene, nir B, which is extremely close to cysG, the gene order being crp, nirB, cysG, aroB. Complementation studies established that nirB + and cysG + canbe expressed independently. The data strongly suggest that nirB is the structural gene for the 88 kDal NADH-dependent nitrite oxidoreductase apoprotein (EC 1.6.6.4).
The nirB gene is apparently defective in the previously described nirD mutant, LCB82. The nirH mutant, LCB197, was unable to use formate as electron donor for nitrite reduction, but NADH-dependent nitrite reductase was extremely active in this strain and a normal content of cytochrome c 552 was detected. Strains carrying a nirE, nirF or nirG mutation gavenormal rates of nitrite reduction by glucose, formate or NADH.
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Derepression of Sporulation and Synthesis of Mycobacillin and Dipicolinic Acid by Guanosine 3': 5'-Cyclic Monophosphate under Conditions of Glucose Repression in Bacillus subtilis
More LessSummary: Dibutyryl cyclic GMP, but not dibutyryl cyclic AMP, derepresses sporulation and synthesis of mycobacillin and dipicolinic acid under conditions of glucose repression in Bacillus subtilis strain 834. Neither of these compounds appears to affect sporulation and synthesis of mycobacillin and dipicolinic acid in this strain under normal physiological conditions. Mutants insensitive to glucose repression were indifferent to the addition of either of the nucleotides both in the presence and in the absence of glucose. A role for dibutyryl cyclic GMP in annulling the repressing effect of glucoseon sporulation and on synthesis of mycobacillin and dipicolinic acid is thus indicated.
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Isolation of Thermophilic Mutants of Bacillus subtilis and Bacillus pumilus and Transformation of the Thermophilic Trait to Mesophilic Strains
More LessSummary: Thermophilic mutants were isolated from mesophilic Bacillus suhtilis and Bacillus pumilus by platinglarge numbers of cells and incubating them for several days at a temperature about 10 °C above the upper growth temperature limit for the parent mesophiles. Under these conditions we found thermophilic mutant strains that were able to grow at temperatures between 50 °C and 70 °C at a frequency of less than 10−10. The persistence of auxotrophic and antibiotic resistance markers in the thermophilic mutants confirmed their mesophilic origin. Transformation of genetic markers between thermophilic mutants and mesophilic parents was demonstrated at frequencies of 10−3 to 10−2 for single markers and about 10−7 for two unlinked markers. With the same procedure we were able to transfer the thermophilic trait from the mutant strains of Bacillus to the mesophilic parental strains at a frequency of about 10−7, suggesting that the thermophilic trait is a phenotypic consequence of mutations in two unlinked genes.
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Potentiation of a Nucleolytic Activity in Bacillus subtilis
More LessSummary: In several strains of Bacillus subtilis extensive breakdown of chromosomal DNA may be potentiated by osmotic lysisof protoplasts. At its most severe, in strains originating from Farmer& Rothman’s thymine auxotroph, the rate of DNA breakdown was greater than 50% per hour at 40 °C. The rate of DNA breakdown in most other strains tested was approximately 5% per hour except for SP β-strains, in which the rate of DNA breakdown was only 0.3 %. DNA degradation was attributed to relaxation ofcontrol of a nuclease specified by the prophage of SP β or a related phage. The most potent nuclease in lysates was an ATP-activated protein of M r 280000. Derivatives of Farmer and Rothman’s strain containing integrated plasmids had the highest rate of DNA degradation. Although the chromosome was completely destroyed, covalently closed circular plasmids were generated from the integrated sequence. These showed massive deletions of the B. subtilis part of the integrated plasmid but the vector sequence remained intact. The nucleolytic activity therefore appears to recognize specific sequences in B. subtilis DNA. We suggest that activation of SP β genes during development of competence may be a cause of deletion ofcloned genes in the early stages of establishment of cloned sequences.
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Plasmid Transfer and Behaviour in Acinetobacter calcoaceticus EBF65/65
More LessSummary: At least one plasmid from each of the incompatibility groups B, C, FIV, H2/S, Iα, Iδ, P, W and X was shown to be capable of transfer from Escherichia coli K12 to Acinetobacter calcoaceticus EBF65/65. Transfer was influenced by the presence of pAV2 (thought to encode a restriction-modification system) in the recipient strain; however, not all plasmids belonging to a particular incompatibility group behaved identically. All plasmids were unstable to varying degrees in A. calcoaceticus EBF65/65, but under suitable conditions were capable of transfer to further strains of EBF65/65 and re-transfer to E. coli K12. Of 40 recently isolated trimethoprim R plasmids 31 transferred successfully from E. coli K12 to A. calcoaceticus EBF65/65, but 17 of these 31 required the introduction of a second mobilizing plasmid for re-transfer to occur.
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Protoplast Fusion as a Tool for Grenetic Analysis in Cephalosporium acremonium
More Lesssummary: Protoplasts of nutritionally complementary strains of Cephalosporium acremonium were fused and plated onto media which supressed the growth of both parents. The regenerating colonies were used for genetic analysis and were found to be of two types, stable haploid recombinants and unstable heterozygotes (aneuploids and/or diploids). Analysis of these colonies provided evidence for eight linkage groups and a relatively high rate of mitotic crossing-over. The gene order for three of the markers on one linkage group was also determined.
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Molecular Cloning and Expression of a Xylanase Gene of Alkalophilic Aeromonas sp. no. 212 in Escherichia coli
More LessSummary: A gene coding for a xylanase activity of alkalophilic Aeromonas sp. no. 212 (ATCC 31085) was cloned in Escherichia coli HB101 with pBR322. Plasmid pAX1 was isolated from transformants producing xylanase, and the xylanase gene was located in a 6.0 kb Hind III fragment. The pAX1-encoded xylanase activity in E. coli HB101 was about 80 times higher than that of xylanase L in alkalophilic Aeromonas sp. no. 212. About 40% of the enzyme activity was observed in the periplasmic space of E. coli HB101. The pAX1-encoded xylanase had the same enzymic properties as those of xylanase L produced by alkalophilic Aeromonas sp. no. 212, but its molecular weight was lower (135000 vs 145000, as estimated by SDS polyacrylamide gel electrophoresis).
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Molecular Cloning of a Bacillus subtilis Gene Involved in Spore Outgrowth
More LessSummary: A λ 4A derivative carrying the outB gene of Bacillus subtilis has been identified by transformation of a B. subtilis mutant temperature-sensitive in spore outgrowth. The cloned region is a single EcoRI fragment 14 kb in legnth. In addition to outB, the cloned DNA includes at least part of the amyE and aroI loci.
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Lysis of Escherichia coli by β-Lactam Antibiotics: Deletion Analysis of the Role of Penicillin-binding Proteins 1A and 1B
More LessSummary: Deletions of the ponA and ponB genes of Escherichia coli have been constructed in vitro and recombined into the chromosome to produce strains that completely lack penicillin-binding protein 1A or penicillin-binding protein 1B. In each case a DNA fragment internal to the gene was replaced by a fragment encoding an antibiotic resistance. The ponA and ponB deletions can therefore be readily introduced into other E. coli strains by P1 transduction of the antibiotic resistance. Although the complete absence of penicillin-binding protein 1A or penicillin-binding protein 1B was tolerated, the absence of both of these proteins was shown to result in bacterial lysis.
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- Immunology
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Interaction between Human Polymorphonuclear Leucocytes and Chlamydia trachomatis Elementary Bodies: Electron Microscopy and Chemiluminescent Response
More LessSummary: Incubation of human polymorphonuclear leucocytes (HPMN) with highly purified Chlamydia trachomatis serotype L2/434/Bu elementary bodies (EB), in the presence and absence of specific antibody, resulted in a 103-fold reduction of viable count after 24 h incubation. Electron microscopy observations indicated activation of the HPMN by the EB. Attachment of the EB to the HPMN cell membrane, formation of a cytoplasmic cup and EB-containing vacuoles were observed. In addition, two types of phagocytic vacuoles were observed after 30 min incubation; in one type, a single EB was tightly surrounded by the vacuolar membrane, while the other type was enlarged and held one or more intact EB or degenerated EB or both. A fuzzy coat was observed on EB located in the HPMN vacuoles only in the presence of specific antibody. Empty vacuoles containing degenerated EB were observed in the HPMN after 24 h incubation. HPMN exposed to EB of C. trachomatis produced a marked chemiluminescent response with a peak 14 times greater than the peak value of the control. A second stimulation with phorbol 12-myristate 13-acetate and zymosan was achieved. The chemiluminescent peak value in the presence of heat-treated EB (56 °C. 20 min) was 50% of that obtained in the presence of untreated EB. The significance of the chemiluminescent response in the killing mechanism of C. trachomatis EB by HPMN is discussed.
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- Pathogenicity And Medical Microbiology
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IncP-l R Plasmids Decrease the Serum Resistance and the Virulence of Pseudomonas aeruginosa
More LessSummary: Pseudomonas aeruginosa strain PAOl was compared to PAOl strains containing an IncP-l R plasmid (RPl. R68, or R68.45) in an experimental mouse burn infection model. All R plasmids tested caused a 10-to 400-fold increase in mean lethal dose (LD50). The decrease in virulence produced by plasmids R68 and R68.45 was significantly greater than the decrease caused by the closely related plasmid RPl. All plasmids also led to an increased sensitivity of strain PAOl to human serum bactericidal activity. Virulence and serum resistance of strain PAOl were restored by curing of the entire plasmid R68.45 but not by deletions in the plasmid’s transfer gene regions.
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The Transfer of Genes Encoding Production of Mannose-resistant Haemagglutinating Fimbriae from Uropathogenic Enterobacteria
More LessSummary: Two hundred and thirty-seven bacterial strains, isolated from patients with urinary tract infections, were examined for the presence of plasmid-determined fimbrial adhesins. Ninety-nine strains were capable of producing mannose-resistant haemagglutination. Seventeen of these strains possessed transferable resistance plasmids and 11 of these were also able to transfer the mannose-resistant haemagglutination gene, suggesting that it was carried on the R plasmid or had been mobilized by it.
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- Physiology And Growth
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Environmental Regulation of Carbohydrate Metabolism by Streptococcus sanguis NCTC 7865 Grown in a Chemostat
More LessSummary: Carbohydrate metabolism by the oral bacterium Streptococcus sanguis NCTC 7865 was studied using cells grown in a chemostat at pH 7.0 under glucose or amino acid limitation (glucose excess) over a range of growth rates (D = 0.05 h−1−0.4 h−1). A mixed pattern of fermentation products was always produced although higher concentrations of lactate were formed under amino acid limitation. Analysis of culture filtrates showed that arginine was depleted from the medium under all conditions of growth; a further supplement of 10 mm-arginine was also consumed but did not affect cell yields, suggesting that it was not limiting growth. Except at the slowest growth rate (D = 0.05 h−1) under glucose limitation, the activity of the glucose phosphotransferase (PTS) system was insufficient to account for the glucose consumed during growth, emphasizing the importance of an alternative method of hexose transport in the metabolism of oral streptococci. The PTS for a number of sugars was constitutive in S. sanguis NCTC 7865 and, even though the cells were grown in the presence of glucose, the activity of the sucrose-PTS was highest. The glycolytic activity of cells harvested from the chemostat was affected by the substrate, the pH of the environment, and their original conditions of growth. Glucose-limited cells produced more acid than those grown under conditions of glucose excess; at slow growth rates, in particular, greater activities were obtained with sucrose compared with glucose or fructose. Maximum rates of glycolytic activity were obtained at pH 8.0 (except for cells grown at D = 0.4 h−1 where values were highest at pH 7.0), while slow-growing, amino acid-limited cells could not metabolize at pH 5.0. These results are discussed in terms of their possible significance in the ecology of dental plaque and the possible involvement of these bacteria in the initiation but not the clinical progression of a carious lesion.
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Osmoregulation in a Proline-producing Strain of Serratia marcescens
More LessSummary: A proline-producing strain of Serratia marcescens grew more rapidly than the wild-type strain in a medium of high osmolarity due to high concentrations of NaCl, KCl, Na2SO4, (NH4)2 HPO4, sodium glutamate, glucose or sucrose. Growth inhibition by NaCl was partially reversed by proline in the wild-type strain, and by glutamate and proline in the proline-producing strain. Intracellular proline and glutamate concentrations under conditions of high osmolarity were studied. The wild-type strain accumulated endogenously synthesized glutamate, and concentrated proline taken up from the external medium. In contrast, the proline-producing strain accumulated a large amount of endogenously synthesized proline. This increased proline content contributes to the osmotolerance of the proline-producing strain. The growth inhibition by NaCl was also reversed by glycinebetaine in S. marcescens wild-type and proline-producing strains.
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A Kinetic Study of the Colony Growth of Streptomyces coelicolor A3(2) and J802 on Solid Medium
More LessSummary: Colony growth kinetics of Streptomyces coelicolor A3(2), the wild-type strain, and J802, a mutant incapable of utilizing agar as the sole carbon and energy source, have been studied on solid medium. General features comparable to colony growth of filamentous fungi were observed in both streptomycete strains. Hyphal frequency at the margins of colonies was assessed as the number of hyphae crossing an are of defined length and increased in an exponential manner with distance from the margin, reaching a maximum at approximately 200 μm. Aerial hyphal initials were formed approximately 350 μm from the margin. Colony radial extension was linear, but in the wild-type strain grown at low medium depths, growth was sometimes multiphasic, with successive phases of linear growth each exhibiting a slower radial growth rate than that of the preceding phase. Hyphal frequency at the colony margin decreased as colony diameter increased, indicating significant changes in environmental conditions in the peripheral growth zone during colony growth. In the primary phase of linear growth, the colony radial growth rate of both strains was independent of medium depth and in the absence of a cellophane membrane was independent of glucose concentration. Colony radial growth rate of strain J802 growing on a cellophane underlay increased with glucose concentration. Hyphal frequency of strain A3(2) increased gradually with medium depth but was independent of glucose concentration. In strain J802, hyphal frequency exhibited a strong dependence on both medium depth and glucose concentration. A threshold glucose concentration for growth was not found and inhibition of strain A3(2) occurred above 1 gl−1. The results agree with many aspects of accepted theories for colony growth of both unicells and filamentous fungi, but suggest production of staling products and/or secondary metabolites to be of equal significance to nutrient limitation in controlling colony development.
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Protection of Saccharomyces cerevisiae against Cd2+ Toxicity by Ca2+
More LessSummary: Ca2+ protected yeast ceJls very effectively against the toxic effects of Cd2+; Mg2+had only a slight protecting effect as far as protection against Cd2+-induced release of K+ was concerned. Protection of the yeast cells against Cd2+ toxicity was due to a reduction in Cd2+ uptake in the presence of Ca2+. A single relationship existed between the relative rate of K+ release induced by Cd2+ and the cellular Cd2+concentration. Within the first few minutes of incubating cells with Cd2+. the molar ratio of K+ released and Cd2+ accumulated was 22 and was independent of the amount of CdC12 added. This ratio decreased during incubation of the cells with Cd2+. depending on the external Cd2+concentration.
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Cadmium Resistance in Pseudomonas putida: Growth and Uptake of Cadmium
More LessSummary: A strain of Pseudomonas putida resistant to low concentrations of cadmium (0.25 mm-Cd2+) adapted to growth in the presence of 3 mm-Cd2+ in a chemically defined medium. This increased resistance was rapidly lost if Cd2+ was omitted from the growth medium. P. putida concentrated109Cd2+ actively. Mechanisms appeared to exist for expelling cadmium from cells during the lag and exponential phases and for continued growth in the presence of high intracellular Cd2+ concentrations. Cd2+-resistant cells adapted so as to control both the extent and rate of Cd2+ uptake when compared to control cells.
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Accumulation of Aluminium by Anabaena cylindrica into Polyphosphate Granules and Cell Walls: an X-ray Energy-dispersive Microanalysis Study
More LessSummary: X-ray microanalysis of thin cryosections of the cyanobacterium Anabaena cylindrica showed that aluminium was rapidly taken up and accumulated into polyphosphate granules. In addition, aluminium was found in the cell walls but could not be detected in the cytoplasm. The concentration of phosphorus in the medium affected the accumulation pattern; more aluminium was bound in the polyphosphate granules and in the cell walls after growth in phosphorus-rich medium. The accumulation of aluminium in these structures may function as a detoxification mechanism. Treatment with aluminium for 24 h did not cause any significant changes in the elemental composition of polyphosphate granules or cell walls.
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The Effects of Exogenous Amino Acids on Growth and Nitrogenase Activity in the Cyanobacterium Anabaena cylindrica PCC 7122
More LessSummary: Growth and nitrogenase activity were followed in batch cultures of Anabaena cylindrica PCC 7122 supplemented with a range of protein amino acids and urea at concentrations of 1 and 10 mm. Alanine, arginine, asparagine, aspartate, glutamate, glutamine and serine caused reductions in nitrogenase activity and showed evidence of acting as sources of nitrogen. Suppression of nitrogenase by other amino acids was accompanied by reduced growth. A number of amino acids appeared to be toxic at high concentrations. Cysteine and histidine were toxic at low concentrations.
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The Role of Haems and Related Compounds in the Nutrition and Zoosporogenesis of the Rumen Chytridiomycete Neocallimastx frontalis H8
More Lesssummary: The haem requirement for growth of the rumen chytridiomycete Neocallimastx Frontalis H8 was satisfied by haem, haemin, haematin, mesoporphyrin IX, coproporphyrins I and III, uroporphyrins I and III, but not deuteroporphyrin IX. Porphyrin degradation products and precursors of porphyrin synthesis did not support growth. Protoporphyrin IX and haemato-porphyrin IX supported growth, indicating that the organism contains ferrochetalase, but was unable to synthesize the porphyrin ring. Haem could be provided by catalase and peroxidase, but not by cytochrome C. Zoosporogenesis was induced in N. Frontalis H8 in rumen fluid principally by haem, haemin and haematin, and to a smaller extent by catalase, peroxidase and protoporphyrin IX. Haem precursors and degradation products were ineffective. Chlorophylls a and b and their ruminai degradation products, plant fraction 1 protein and chelated iron neither stimulated growth nor induced zoosporogenesis. Haem induced partial zoosporogenesis in haem-limited cultures and suspensions of sporangia grown In Vitro.
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Active Extrusion of Potassium in the Yeast Saccharomyces cerevisiae Induced by Low Concentrations of Trifluoperazine
Y. Eilam, H. Lavi and N. GrossowiczSummary: Trifluoperazine (TFP), the antipsychotic drug, induces substantial K+ efflux, membrane hyperpolarization and inhibition of H+-ATPase in the yeast Saccharomyces cerevisiae. Investigations on the mechanism of these effects revealed two different processes observed at different incubation conditions. At an acidic pH of 4.5 and an alkalinepH of 7.5, K+ efflux was accompanied by substantial proton influx which led to intracellular acidification and dissipation of ΔΨ formed by cation efflux. The results indicated nonspecific changes in membrane permeability. Similar results were also observed when cells were incubated at pH 5.5–6.0 with higher concentrations of TFP (above 75 μm). On the other hand, low concentrations of TFP (30–50 μm) at pH 5.5–6.0 caused marked membrane hyperpolarization and K+ efflux unaccompanied by the efflux of other cations and by H+ influx. Our experiments indicate that under these conditions K+ efflux was an active process. (1) K+ efflux proceeded only in the presence of a metabolic substrate and was inhibited by metabolic inhibitors. (2) When 0.3–0.9 mm-KCl was present in the medium at pH 6.0, the concentration of K+ within the cells (measured at the end of the incubation with TFP) was much lower than the theoretical concentration of if the distribution of K+ between medium and cell water was at equilibrium (at zero electrochemical gradient). (3) Valinomycin decreased the net K+ efflux and decreased the membrane hyperpolarization induced by TFP, probably by increasing the flux of K+ into the cells along its electrochemical gradient. (4) Conditions which led to active K+ efflux also led to a marked decrease in cellular ATP level. The results indicate that under a specific set of conditions TFP induces translocation of K+ against its electrochemical gradient.
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NADP+-dependent Glutamate Dehydrogenase Activity is Impaired in Mutants of Saccharomyces cerevisiae That Lack Aconitase
More LessSummary: A mutant of Saccharomyces cerevisiae lacking aconitase did not grow on minimal medium (MM) and had five-to tenfold less NADP+-depcndent glutamate dehydrogenase (GDH) activity than the wild-type, although its glutamine synthetase (GS) activity was still inducible. When this mutant was incubated with glutamate as the sole nitrogen source, the 2-oxoglutarate content rose, and the NADP+-dependent GDH activity increased. Furthermore, carbon-limited cultures showed a direct relation between NADP+-dependent GDH activity and the intracellular 2-oxoglutarate content. We propose that the low NADP+-dependent GDH activity found in the mutant was due to the lack of 2-oxoglutarate or some other intermediate of the tricarboxylic acid cycle.
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Respiration Rate, Growth Rate and the Accumulation of Streptomycin in Escherichia coli
More LessSummary: Using chemostat cultures of Escherichia coli it was possible to vary respiration rates while maintaining a constant growth rate. This allowed the effect of variations in respiration rates on the accumulation of streptomycin to be studied in cultures at constant growth rates. At a particular dilution rate cultures exhibited higher respiration rates when phosphate limited growth than when carbon limited growth. A ubiquinone-deficient strain had a lower rate of respiration at a particular dilution rate than a related ubiquinone-sufficient strain. In spite of these differences in respiratory activity, the accumulation of streptomycin was identical in carbon-and in phosphate-limited chemostat cultures of ubiquinone-deficient and ubiquinone-sufficient strains. Moreover, accumulation of streptomycin in an anaerobic chemostat culture occurred at the same rate as that in an aerobic chemostat. There was however a lag of 1.5 h before accumulation commenced in the anaerobic culture, a feature that was not apparent in the aerobic culture. These results indicate that the lower rates of respiration in slow-growing bacteria are not responsible for the decreased accumulation of streptomycin in slow-growing compared to fast-growing cultures. Moreover, it seems unlikely that quinones are involved directly (e.g. as carriers) in streptomycin accumulation, since removal of 90% of cellular ubiquinone, or replacement of ubiquinone with a structural analogue, did not affect accumulation as long as mutant and parent cultures grew at the same rate.
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Effects of Imidazole-and Triazole-derivative Antifungal Compounds on the Growth and Morphological Development of Candida albicans Hyphae
More LessSummary: Six azole-derivative antifungal compounds affected several aspects of Candida albicans hyphal development with only a relatively small degree of inhibition of growth rate, measured in terms of ATP concentration, whereas amphotericin B and 5-fluorocytosine affected morphology only when they also substantially inhibited fungal growth rate. At 10−8M, all the azoles tested inhibited branch formation by C. albicans hyphae. At 10−7 M and higher concentrations, clotrimazole and miconazole strongly suppressed emergence of new hyphal outgrowths from parent yeast cells, whereas ICI 153066 and itraconazole had little effect on this phenomenon and ketoconazole and tioconazole had intermediate effects. At the highest concentrations tested (10−5 M) hyphal development was ultimately arrested by the azole compounds and the fungus grew predominantly in the form of budding yeast cells; however, none of the azole antifungals prevented initial emergence of an apparently normal germ tube. The antifungals only exerted their morphological effects when they were present in the culture medium: removal of the compounds after exposure of C. albicans to them led to reversion to normal growth.
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Stimulation of Sexual Reproduction of Phytophthora cactorum by Phospholipids
More LessSummary: In a chemically defined liquid medium, soybean lecithins (99% pure) were much more effective than, β-sitosterol and cholesterol in stimulating oospore formation by isolate Pct N of Phytophthora cactorum at 20 and 24 °C. Assuming that the 1% impurity is all sterols, such sterol impurity would account for less than 2% of the oospores produced in the presence of lecithins. Lecithins were also stimulatory to sexual reproduction of Phytophthora capsici and Pythium aphanidermatum but notPythium vexans, whereas sterols were stimulatory to sexual reproduction of Pythium aphanidermatum and Pythium vexans but not Phytophthora capsici, Lecithins from soybean were more effective in inducing sexual reproduction by isolate 121F of Phytophthora cactorum than were those from egg yolk. L-α-Phosphatidylethanolamine from soybean was as effective as lecithins in inducing sexual reproduction, whereas L-α-Phosphatidylinositol from soybean and L-α-Phosphatidyl-L-serine from bovine brain were ineffective. Of four synthetic lecithins tested, only dioleoyl-L-α-Phosphatidylcholine was active. The triglycerides and fatty acids tested were inactive. The results suggest that the activity of lecithins is determined by the type and position of fatty acids in the molecular structures.
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Absence of Error-prone Repair in a Vibrio species
More LessSummary: The effect of various DNA-damaging agents on a Vibrio species was investigated. The organism was readily mutable by N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C but not by UV light. No Weigle reactivation of UV-irradiated α3a phage was detected. These results suggest that an error-prone repair mechanism is lacking in this species.
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Chemotaxis of a Cyanobacterium on Concentration Gradients of Carbon Dioxide, Bicarbonate and Oxygen
G. Malin and A. E. WalsbySummary: Trichomes of an Oscillatoria sp. suspended in soft agar in Petri dishes showed active movement away from areas where gaseous exchange had been prevented by placing glass coverslips on the surface. This clearance response occurred only in the light and it was related to gradients of inorganic carbon that formed in the agar layer. In plates incubated in the dark trichomes accumulated in the central areas below glass coverslips but the substance eliciting this response could not be identified. Gradients of diffusible substances were established within lawns of cyanobacteria suspended in soft agar and it was demonstrated that trichomes of Oscillatoria sp. moved towards CO2. HCO3 and O2 in light-dependent chemotactic reactions. No chemotaxis occurred in response to these substances in the dark. The chemotactic responses were detected after 2 h but became increasingly distinct up to 8 h. The chemotactically active concentration of CO2 was greater than the atmospheric concentration since trichomes moved from air towards a source of CO2. Using diffusivity coefficients it was calculated that trichomes of Oscillatoria sp. accumulated at a CO2 partial pressure of 0.02 bar (equivalent to 0.83 mm). With O2 as the chemoattractant the value was 0.35 bar (14.56 mm). These results are discussed with reference to the roles of inorganic carbon and O2 in cyanobacterial metabolism and it is concluded that chemotactic behaviour may be important in movements within the photic zone of sediment environments.
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Amino Acid Transport by Prosthecae of Asticcacaulis biprosthecum: Evidence for a Broad-range Transport System
Eric Tam and Jack L. PateSummary: Prosthecae purified from cells of Asticcacaulis biprosthecum possess active transport systems that transport all 20 amino acids tested. Using ascorbate-reduced phenazine methosulphate in the presence of oxygen, all 20 amino acids are accumulated against a concentration gradient by isolated prosthecae. Results of experiments testing the inhibition of transport of one amino acid by another, and of experiments testing the exchange of exogenous amino acids with those preloaded in prosthecae, along with characteristics of mutants defective in amino acid transport, suggest the presence in prosthecae of three amino acid transport systems. One, the general or G system, transports at least 18 of the 20 amino acids tested. Another system, referred to as the proline or P system, transports seven amino acids (including proline) that are also transported by the G system. The third system transports only glutamate and aspartate, and is referred to as the acidic amino acid transport system or A system.
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Inhibition and Inactivation of Glucose-phosphorylating Enzymes from Saccharomyces cerevisiae by D-Xylose
More LessSummary: Three glucose-phosphorylating enzymes were separated from cell-free extracts of Saccharomyces cerevisiae by hydroxylapatite chromatography. Variations in the amounts of these enzymes in cells growing on glucose and on ethanol showed that hexokinase PI was a constitutive enzyme, whereas synthesis of hexokinase PII and glucokinase were regulated by the carbon source used. Glucokinase proved to be a glucomannokinase with K m values of 0.04 mm for both glucose and mannose. D-Xylose produced an irreversible inactivation of the three glucose-phosphorylating enzymes depending on the presence or absence of ATP. Hexokinase PI inactivation required ATP, while hexokinase PII was inactivated by D-xylose without ATP in the reaction mixture. Glucokinase was protected by ATP from this inactivation. D-Xylose acted as a competitive inhibitor of hexokinase PI and glucokinase and as a non-competitive inhibitor of hexokinase PII.
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Isolation and Mapping of Escherichia coli K12 Mutants Defective in Phenylacetate Degradation
More LessSummary: Mutants of Escherichia coli K12 unable to grow on phenylacetate have been isolated and mapped. The mutations were located in the relatively ‘silent’ region of the E. coli K12 chromosome at min 30.4 on the genetic map, with the gene order rac pac-1 pac-2 trg.
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- Systematics
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A Numerical Taxonomic Study of Actinobacillus, Pasterella and Yersinia
More LessSummary: A numerical taxonomic study of strains of Actinobacillus, Pasteurella and Yersinia, with some allied bacteria, showed 23 reasonably distinct groups. These fell into three major areas. Area A contained species of Actinobacillus and Pasteurella: A. suis, A. equuli, A. lignieresii, P. haemolytica biovar A. P. haemolytica biovar T, P. multocida, A. actinomycetemcomitans, ‘P. bettii’. ‘A. seminis’ P. ureae and P. aerogenes. Also included in A was a composite group of Pasteurella pneumotropica and P. gallinarum, together with unnamed groups referred to as ‘BLG’ ‘Mair’ ‘Ross’ and ‘aer-2’. Area B contained species of Yersinia: Y. enterocolitica, Y. pseudotuberculosis. Y. restis and a group ‘ent–b’ similar to Y. enterocolitica. Area C contained non–fermenting strains: Y. philomiragia, Moraxella anatipestifer and a miscellaneous group ‘past’. There were also a small number of unnamed single strains.
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Numerical Taxonomy and Emended Description of Renibacterium salmoninarum
More LessSummary: Forty-four strains of Renibacterium salmoninarum and 12 representative cultures of Actinomyces, Arthrobacter, Lactobacillus, Microbacterium, Micrococcus, Planococcus, Rothia and Listeria denitrifcans were compared using numerical taxonomic techniques based upon 86 unit characters. Data were examined using the simple matching (SSM ) and Jaccard (Sj ) coefficients, and clustering was achieved using the unweighted pair group method with arithmetic averages (UPGMA) technique. Cluster composition was barely affected by the statistics used, or by the test error, which was estimated at 0·36%. The renibacteria formed a distinct and homogeneous cluster and were distinguished from the marker cultures by a characteristic API-ZYM profile. An emended description of R. salmoninarum is presented, based on the numerical data and the results of recent chemical and microbiological studies.
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- Short Communication
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Antigenic Alteration of Contaminating Lipopolysaccharide during Extraction of Escherichia coli Outer-membrane Proteins from Polyacrylamide Gels
More LessSummary: An antiserum raised to the ferric enterobactin receptor protein of Escherichia coli, isolated from SDS-polyacrylamide gels, contained high-titre antibodies to the lipopolysaccharide (LPS) of E.coli 0111. This antiserum was used to show that proteins dissected from polyacrylamide gels can be contaminated with comigrating LPS at levels belowthose detectable by very sensitive silver staining methods. Using this antiserum it was also shown that the procedures used to extract proteins from polyacrylamide gels can alter the molecular structure and, consequently,the antigenic properties of the contaminating LPS.
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Characterization of a Soluble Cytochrome Oxidase/Nitrite Reductase from Nitrosomonas europaea
More LessSummary: The soluble cytochrome oxidase/nitrite reductase of Nitrosomonas europaea, induced under low O2 tensions, has a high affinity for O2. EPR spectroscopy indicates that this enzyme contains 'type I' ('blue') and ('type II') Cu2+-sites, and UV/visible spectroscopy suggests the presence of a ('type III') Cu2+-pair. The enzyme binds exogenous copper in an unusual way, resulting in modified EPR spectra and a very high εM for the 'blue' 598 nm absorption band when low levels of Cu2+ (10 μM) are present during purification.
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Volumes and issues
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Volume 170 (2024)
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