- Volume 132, Issue 3, 1986
Volume 132, Issue 3, 1986
- Biochemistry
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The Outer Membrane of Methylobacterium organophilum
More LessSUMMARY: Inner and outer membranes were isolated from Methylobacterium organophilum by sucrose density centrifugation after disruption of bacteria by shaking with glass beads. The outer membrane (OM) contained all the pink oxocarotenoid pigment of the cell and unusually small amounts of phospholipid and lipopolysaccharide. An unidentified glucan was present in both membrane fractions. Several major OM proteins had molecular sizes in the range 49 kDa to 80 kDa and most of the OM proteins remained insoluble when OM or cell wall was treated with 2% (w/v) sodium dodecyl sulphate (SDS) at temperatures up to 50°C. Neither polysaccharide nor phospholipid was solubilized under the same conditions. Increasing the concentration of methanol in the growth medium led to an increase in the bacterial phospholipid content and to increased solubility of the OM in 2% SDS. It is suggested that the resistance of the OM to solubilization by the detergent is due in part to the presence of large amounts of three unidentified polar, phosphate-free lipids that might be related to hopane polyols. Phospholipids in isolated walls and OM were rapidly degraded by endogenous phospholipase when incubated in Tris buffer at pH 8 but the unidentified lipids were retained in the particulate fraction.
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Analysis of the Pectin-degrading Enzymes Secreted by Three Strains of Erwinia chrysanthemi
More LessSUMMARY: The protein content of culture supernatants of three Erwinia chrysanthemi strains, B374, 3937j and 3665, grown on different carbon sources was compared. After growth in presence of polygalacturonate, four new polypeptides, identified as pectinases, were synthesized. These induced proteins, and the pattern of pectate lyase induction, differed among the strains. The proteins present in the supernatants of some mutants known or suspected to be affected in pectinase production (secretion-defective mutants and mutants in the degradative pathway of galacturonate and ketodeoxygluconate) were also analysed.
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Lack of Effect of Leader Peptidase Overproduction on the Processing in vivo of Exported Proteins in Escherichia coli
More LessSUMMARY: The kinetics of maturation of certain exported proteins were analysed in Escherichia coli strains that also concomitantly overproduce either a periplasmic protein or the leader peptidase. The results led to three conclusions. (a) Overproduction of leader peptidase has no effect on the rate of maturation of at least two exported proteins, one periplasmic (TEM β-lactamase), one outer membrane (PhoE); therefore, the quantity of leader peptidase is not rate-limiting for normal export. (b) Overproduction of PhoS reduces the rate of maturation of two other periplasmic proteins (β-lactamase and PhoA) and itself, presumably by competing for the rate-limiting component of the export apparatus. (c) Overproduction of leader peptidase in a strain overproducing PhoS has no effect on the retarded maturation of PhoS. Therefore even in these conditions, leader peptidase is not rate limiting.
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Purification and Some Properties of the 2-Hydroxy-6-oxohepta-2,4-dienoate Hydrolase (2-Hydroxymuconic Semialdehyde Hydrolase) Encoded by the TOL Plasmid pWW0 from Pseudomonas putida mt-2
More LessSUMMARY: The 2-hydroxy-6-oxohepta-2,4-dienoate (HOD) hydrolase encoded by the TOL plasmid pWW0 from Pseudomonas putida mt-2 (PaWl) was purified to homogeneity. It has an M r of 65000 and is dissociated by SDS into two subunits of equal size. Alanine was the only N-terminal residue detected, and each subunit contained one cysteine thiol group. The pH optimum for activity and for enzyme stability was around 7·5, whereas the isoelectric point was 4·7. Only the products of catechol 2,3-oxygenase action on linear chain alkylcatechols and 4-chlorocatechol served as substrates. Kinetic measurements showed that the higher activity against the ketone substrates (from 3-substituted catechols) over aldehyde substrates (from 4-substituted catechols) was the result of higher V max values rather than lower K m values. Antisera prepared against this purified HOD hydrolase were shown by Ouchterlony double diffusion and inhibition studies to be related to the HOD hydrolase from phenol-grown P. putida strain U (NCIB 10015) in which the enzyme is chromosomally encoded.
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Molecular Size Diversity of Citrate Synthases from Pseudomonas Species
More LessSUMMARY: Two forms of citrate synthase (EC 4.1.3.7) have been found in several species of Pseudomonas, a ‘large’ form (M r ≃ 250000) which is generally inhibited by NADH and reactivated by AMP, and a ‘small’ form (M r ≃ 100000) which is insensitive to these nucleotide effectors. Other species of Pseudomonas were found to contain either the ‘large’ or the ‘small’ form. Gel filtration and ion-exchange with the technique of fast protein liquid chromatography were used to resolve the enzymes. Where both citrate synthases were present, there did not appear to be an equilibrium between the two forms. The results reveal a new and complex diversity of citrate synthase within the genus Pseudomonas.
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Adsorption of Bacterial Surface Polymers to Attachment Substrata
More LessSUMMARY: Extracellular polysaccharide (EP) and lipopolysaccharide (LPS) were isolated from three strains of Pseudomonas fluorescens (a wild-type and two mutants) and the adsorption isotherms (relationship between amount of polymer adsorbed and bulk liquid concentration of polymer in solution) of these polymers to hydrophobic, tissue culture treated and sulphonated polystyrene surfaces were measured. The adsorption properties of the polymers were then related to the ability of the three bacterial strains to attach to the polystyrene surfaces in an attempt to elucidate the attachment mechanisms. A Langmuir adsorption isotherm equation was applied to the data, and the mathematical constants thus derived indicated if and at what concentration each surface became polymer-saturated and whether multilayer adsorption occurred. EP isolated from a crenated mutant (strain with the greatest attachment ability) adsorbed at higher concentrations than EP from wild-type and mucoid strains, and the isotherm indicated multilayer adsorption. EP from the mucoid strain (strain with little attachment ability) showed comparatively little adsorption. The isotherm of wild-type LPS was very similar to that of EP from the mucoid strain. Polymer adsorption to the three surface types was different and was generally consistent with the different degrees of bacterial attachment to the surfaces.
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Pyruvate Metabolism and the Phosphorylation State of Isocitrate Dehydrogenase in Escherichia coli
More LessSUMMARY: During growth of Escherichia coli on acetate, isocitrate dehydrogenase (ICDH) is partially inactivated by phosphorylation and is thus rendered rate-limiting in the Krebs cycle so that the intracellular concentration of isocitrate rises which, in turn, permits an increased flux of carbon through the anaplerotic sequence of the glyoxylate bypass. A large number of metabolites stimulate ICDH phosphatase and inhibit ICDH kinase in the wild-type (E. coli ML 308) and thus regulate the utilization of isocitrate by the two competing enzymes, ICDH and isocitrate lyase. Addition of pyruvate to acetate grown cultures triggers a rapid dephosphorylation and threefold activation of ICDH, both in the wild-type (ML308) and in mutants lacking pyruvate dehydrogenase (ML308/Pdh-), PEP synthase (ML308/Pps-) or both enzymes (ML308/Pdh- Pps-). Pyruvate stimulates the growth on acetate of those strains with an active PEP synthase but inhibits the growth of those strains that lack this enzyme. When pyruvate is exhausted, ICDH is again inactivated and the growth rate reverts to that characteristic of growth on acetate. Because pyruvate stimulates dephosphorylation of ICDH in strains with differing capabilities for pyruvate metabolism, it seems likely that pyruvate itself is a sufficient signal to activate the dephosphorylation mechanism, but this does not discount the importance of other signals under other circumstances.
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Effects of Divalent Cations and of Phospholipase A Activity on Excretion of Cloacin DF13 and Lysis of Host Cells
More LessSUMMARY: Induction of cloacin DF13 synthesis in Escherichia coli harbouring plasmid CloDF13 results in the release of cloacin DF13, inhibition of growth and ultimately in lysis of the host cells. Expression of the pCloDF13-encoded protein H is essential for both the release of cloacin DF13 and the lysis of the cells. The divalent cations Mg2+ and Ca2+ interfered with the mitomycin C-induced, protein H-dependent lysis, but hardly affected the release of cloacin DF13. Essentially all of the bacteriocin was released from the cells before a detectable degradation of the peptidoglycan occurred, independent of the presence of mitomycin C. Experiments with phospholipase A mutants revealed that activation of detergent-resistant phospholipase A was essential for the export of cloacin DF13 across the outer membrane and the lysis of induced cells. Transport of cloacin DF13 across the cytoplasmic membrane was mainly dependent on protein H. A revised model for the excretion of cloacin DF13 is presented.
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- Development And Structure
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Cytology of Self Fusions in Hyphae of Phanerochaete velutina
More LessSUMMARY: Hyphal fusions forming in secondary mycelia of Phanerochaete velutina were examined using combined light and electron microscopy. Only ‘self’ fusions, occurring between hyphal compartments derived from the same colony, were studied. At points of contact, fusions formed from a single opening which expanded radially by highly localized lysis of the surrounding cell wall. Fusion pore enlargement failed to reach the full hyphal diameter and a rim of undissolved cell wall remained marking the original point of contact. Within 2 h of this, the compartments became re-partitioned by septum formation, synthesis being associated with mitotic division and beginning precisely at the site of fusion pore expansion. Septa formed at fusions showed identical structure and development to those occurring in unfused compartments.
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Cytology of Non-self Hyphal Fusions and Somatic Incompatibility in Phanerochaete velutina
More LessSUMMARY: The somatic incompatibility reaction occurring at sites of fusion between hyphae of genetically different secondary mycelia of Phanerochaete velutina has been examined using combined light and electron microscopy. Hyphal compartments affected by incompatibility rapidly showed increased vacuolation and the development of autophagic bodies throughout the cytoplasm. Dense osmiophilic spherical bodies that developed within the vacuole lumen characterized the early and highly regulated phase of the reaction. Eventually, as expansion of the vacuolar system proceeded, more widespread degeneration began in the remaining cytoplasm, nuclei and mitochondria. The large microtubule bundles present within this species showed variable behaviour during degeneration of the cell compartments, either breaking down early on in the process or persisting intact during breakdown of the other cell components. The incompatibility finally caused extensive disruption and death of the compartments engaged in fusion and often contiguous cells. Plugging of dolipore septa apparently restricted spread of the incompatibility response along the fused hyphae.
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Asexual Spore Release from Saprolegniaceous Water Moulds: Involvement of Calmodulin
More LessSUMMARY: The localized cell wall lysis associated with asexual spore release from sporangia and spore cysts of saprolegniaceous water moulds appears, based on immunocytochemical and inhibitor studies, to be dependent on calmodulin (CaM). Distinct bands of CaM surround the exit pores of sporangia and cysts produced by Achlya ambisexualis, Dictyuchus monosporus and Saprolegnia ferax. In differentiating sporangia and cysts, CaM becomes concentrated in the apical papillae, at the tips of which exit pores are formed. While all stages of sporulation can be inhibited to an extent by micromolar concentrations of trifluoperazine, an anti-CaM drug, spore release is the most sensitive.
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Ultracytochemical Localization of ATP-hydrolysing Activity in Vegetative Cells, Spores and Isolated Cytoplasmic Membranes of Bacillus subtilis 168
More LessSUMMARY: The localization of ATP-hydrolysing activity in vegetative cells, spores and isolated membranes of Bacillus subtilis 168 was studied by a cytochemical method combined with electron microscopy. The activity was located mainly in the cytoplasmic membrane and the mesosomes, and was also found in the inner layer of the cell wall facing the cytoplasmic membrane. Activity was also detected in the cross-membranes of dividing cells and in spore coats. The product of the reaction was observed either as fine electron-dense granules incorporated into the membranes, or as high-contrast lead precipitates on the surfaces of the membranes.
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- Ecology
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Biology of Rhizobium trifolii Bearing the Hairy Root Plasmid
More LessSUMMARY: We have transferred the hairy root plasmid (pRi) from Agrobacterium rhizogenes into Rhizobium trifolii. The plasmid fully expressed its root-inducing activity in the R. trifolii (pRi) transconjugant. Generally, the R. trifolii (pRi) transconjugant was unable to nodulate clover. However, occasionally a spontaneous mutant(s) of the R. trifolii (pRi) transconjugant arose which was capable of nodulating clover and of inducing root proliferation in a number of plant species. The mutant(s) induced significantly more nodules than the wild-type bacterium, but significantly less N2 was fixed by these nodules.
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- Genetics And Molecular Biology
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Three New Temperate Phages of Bacillus subtilis
More LessSUMMARY: Three temperate bacteriophages of Bacillus subtilis were isolated from soil samples and analysed, together with all the other known temperate phages of this organism, with respect to their host range, immunity, serology and DNA restriction pattern, and by other tests. The results show that the newly isolated phages are new members of the immunity sub-group I of the group III of B. subtilis temperate bacteriophages. We named these new phages IG1, IG3 and IG4.
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Synthesis of ompA Protein of Escherichia coli K12 in Bacillus subtilis
More LessSUMMARY: We have inserted a C-terminally truncated gene of the major outer membrane protein ompA of Escherichia coli downstream from the promoter and signal sequence of the secretory α-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the α-amylase derived signal peptide was not removed; this was verified by N-terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of ompA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested.
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A DNA Sequence Containing the Control Sites for the uxaB Gene of Escherichia coli
More LessSUMMARY: The nucleotide sequence of a 286 bp fragment containing the uxaB control region of Escherichia coli has been determined. The transcriptional start of the uxaB gene has been located and the promoter signals identified. Various fragments of the uxaB promoter-proximal region were fused in vitro with the lacZ gene. Results obtained with these fusions indicate that the operator-promoter sites are located on a 110 bp restriction fragment. The determination of the amino acid sequence of the NH2-terminus of the uxaB gene product revealed that the uxaB gene is not initiated with the AUG codon but with the unusual GTG codon. CRP, the cyclic AMP receptor protein, does not bind to the uxaB control region DNA even though expression of the uxaB gene is sensitive to catabolite repression.
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Molecular Cloning into Tn5 and Integration in the Pseudomonas aeruginosa chromosome: a Tool for Heterologous Gene Expression
More LessSUMMARY: The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in Escherichia coli. The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to Pseudomonas aeruginosa strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the P. aeruginosa chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed P. aeruginosa strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from P. aeruginosa to P. stutzeri or to Acinetobacter calcoaceticus due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria.
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Biochemical Analysis of the Role of Cytoplasmic Ribosomes of Coprinus cinereus in Cycloheximide Resistance
More LessSUMMARY: The development of an optimized in vitro polyuridylic acid-dependent polyphenylalanine-synthesizing system using cell-free extracts of the basidiomycete fungus Coprinus cinereus is described. The in vitro assay has been used to show that cycloheximide-resistant strains CY8.2, CY9.23 and Sp98, all mutant at the cy-2 locus, have cytoplasmic ribosomes which are more resistant to the drug than the corresponding sensitive strains, CY8, CY9 and CY3. Cycloheximide concentrations and molar ratios of cycloheximide to ribosomes required for 50% inhibition in vitro under standard assay conditions are presented for these strains. The molar ratio required for 50% inhibition in vitro is dependent on the concentration of ribosomes in the assay.
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Plasmid Curing and Generation of Mutations Induced with Ethidium Bromide in Streptomycetes
More LessSUMMARY: The DNA-intercalating agent ethidium bromide was used to eliminate plasmid DNA from streptomycetes. Other mutational events associated with chromosome changes occurred at high frequency; they resulted in phenotypic changes such as loss of enzyme activity, antibiotic resistance or ability to sporulate.
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Bacteriophages Mediating Somatic Antigenic Conversion in Salmonella cholerae-suis: Their Isolation from Sewage and Other Salmonella Serotypes Possessing the Somatic 6 Antigen
More LessSUMMARY: Bacteriophages which mediate the conversion of the O somatic antigen of Salmonella choleraesuis from the 627 to the 617 phenotype have been isolated from two strains of S. newport and one of S. muenchen, and also from sewage collected from two areas where there have been no reports of S. cholerae-suis infection for several years. The phages differed from each other by cross-resistance tests.
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- Pathogenicity And Medical Microbiology
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Immunoelectrophoretic Analysis of Streptococcus agalactiae Serotype Ia Antigens
More LessSUMMARY: Rabbit antibodies to heat-killed whole cells of Streptococcus agalactiae serotype Ia were used to establish an antigen map using Triton X-100 sonicates of homologous cells and crossed immunoelectrophoresis. A total of 11 antigens were identified but the density of immunoprecipitates was varied and only seven could be reliably detected, one of which dominated the immunoprecipitate pattern by its intensity. The antigens were partially characterized by immunological, chemical and cell-location methods. Five of the antigens contained carbohydrate and two of those were sensitive to trypsin and probably represent cell-wall compounds. Of the three most prominent antigens, one was surface located and represented the type and shared type antigens (Iabc), one was a cell-wall carbohydrate and very sensitive to periodate, and one was a protein/carbohydrate complex which was heat-labile and trypsin sensitive. Group B epitopes were detected in three immunoprecipitates. Cross-reactions between type Ia and other serotypes and streptococci were recorded.
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Multilocus Genotypes Determined by Enzyme Electrophoresis of Neisseria meningitidis Isolated from Patients with Systemic Disease and from Healthy Carriers
SUMMARY: Variation in nine enzymes in 152 isolates of Neisseria meningitidis from Norway (118 from blood or cerebrospinal fluid of patients with systemic disease and 34 from the pharynx of healthy carriers) was analysed by starch-gel electrophoresis. All nine enzymes were polymorphic and the number of allozymes (electromorphs) identified per locus ranged from 3 to 12, with a mean of 6.1. Among the 152 isolates, 55 unique combinations of electromorphs (electrophoretic types, ETs) were distinguished. Twenty ETs were represented among the carrier isolates and 37 among the systemic isolates; hence, only two ETs were found in both groups of isolates. ET-5 was identified 67 times among the 118 systemic isolates (58%), indicating an association of this ET with invasiveness; ET-5 was also the most common type among the carrier isolates (18%). Genetic similarity between ETs was analysed by pairwise comparison of all 55 ETs with respect to the number of electromorphs by which they differed. No evidence of a general genetic difference between carrier and case isolates was found. Two well-defined clusters of ETs were observed, each including one of the two most common ETs identified among the systemic isolates (ET-5 and ET-37), together with isolates differing from them only at one or two loci. All isolates of ET-5 and ET-37, as well as their closely related variants defined by the similarity matrix, were resistant to sulphonamide, independent of their antigenic characteristics and isolation site. The extensive allozyme variation among isolates of the same serogroup demonstrated the limited value of serogrouping as an epidemiological tool. All but one isolate of serotype 15:P1.16 were electrophoretically similar, as were all the 2a:P1.2 isolates. The 15:P1.15 isolates, however, were genetically heterogeneous. The distribution of alleles in genotypes identified among the systemic isolates indicated that genetic recombination may occur in natural populations of N. meningitidis.
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- Physiology And Growth
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In Streptococcus faecium Penicillin-binding Protein 5 Alone Is Sufficient for Growth at Sub-maximal But Not at Maximal Rate
More LessSUMMARY: In Streptococcus faecium inhibition by both benzylpenicillin and cefotaxime of cells growing at maximal and at reduced rates was associated with saturation of different penicillin-binding proteins. Cells growing at reduced rates were not inhibited by benzylpenicillin concentrations that saturated all penicillin-binding proteins except penicillin-binding protein 5, but did stop growing when this protein was saturated.
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Concerted Induction of the S3 Alkylsulphatase of Pseudomonas C12B by Combinations of Alkyl Sulphates and Alcohols
More LessSUMMARY: Growth of the detergent-degrading bacterium Pseudomonas C12B on pyruvate supplemented with pure tetradecan-2-sulphate and tetradecan-2-ol together, led to induction of the S3 alkylsulphatase active towards symmetrical or near-symmetrical secondary alkyl sulphates. Induction was accompanied by disappearance of the tetradecan-2-sulphate surfactant and by the appearance of some of the S3 activity (up to 45% of the total) in the culture fluid. Induction did not occur when one or other or both of the C14 compounds was omitted from the minimal-pyruvate growth medium, nor with resting cell suspensions even with both compounds present. Provided that the pyruvate medium contained tetradecan-2-sulphate, good induction of S3 enzyme could also be achieved by replacing tetradecan-2-ol with any one of most secondary alcohols tested, including alkan-2-ols (C4 to C16), symmetrical alcohols (C5 to C13) and decanol isomers with the hydroxyl at C-3, C-4 or C-5. Primary alcohols were ineffective. Requirements for the ester component of the inducer combination were more closely defined; maintaining tetradecan-2-ol and pyruvate as constant components, no activity was detected with C3, C4 or C6 alkan-2-sulphates, very feeble activity with C8 and C10 homologues, and increasing activity from C12 to C16. Symmetrical and near-symmetrical alkyl sulphates (substrates of the S3 enzyme) and primary alkyl sulphates, all tested in combination with tetradecan-2-ol plus pyruvate, failed to induce significant amounts of S3 enzyme even at elevated concentrations. Critical micelle concentrations were measured for alkan-2-sulphates and correlated with co-inducer capacity and with the concentration dependence of induction by tetradecan-2-sulphate, which followed a hyperbolic saturation curve with Kco-inducer = 0.29 mm (inducer concentration giving half-maximum induction). Successful induction was also achieved with tetradecan-2-sulphate alone (no alcohol added), but only in the absence of pyruvate, under which circumstances hydrolysis of tetradecan-2-sulphate to the parent alcohol occurred as a prerequisite to bacterial growth. The collective results suggested that induction required not only a combination of secondary alcohol and long-chain alkan-2-sulphate in appropriate amounts (concerted induction), but also that their presence was required at a particular time, when the cells were actively growing and dividing.
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Mechanism of Dodecane Uptake by Whole Cells of Cladosporium resinae
More LessSUMMARY: Whole cells of Cladosporium resinae took up n-dodecane in two stages. The hydrocarbon was first passively adsorbed to the cell surface, and then hydrocarbon was taken in by a mechanism that obeyed Michaelis–Menten saturation kinetics [K m 1 mm, V max 12∙1 nmol min−1 (mg protein−1)]. Under conditions of poor agitation the initial adsorption limited uptake rates. The organism accumulated unaltered substrate to higher concentrations within the cytosol than in the surrounding medium, but this depended upon high concentrations of dodecane being adsorbed to the cell surface, thus creating a diffusion gradient.
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Regulation of Glycogen Synthase in Sclerotium rolfsii
R. SHAPIRA, M. PINES, I. CHET and Y. HENISSUMMARY: Changes in glycogen, extracellular polysaccharides, glycogen synthase (EC 2.4.1.11) and cAMP were followed during growth of submerged mycelium of Sclerotium rolfsii. While glucose was present in the medium glycogen and extracellular polysaccharides accumulated to maximum levels of 40 μg (mg dry wt)−1 and 0∙3 mg (mg dry wt)−1 respectively; they were degraded following glucose exhaustion. Neither glycogen nor extracellular polysaccharide concentrations were affected by increasing the glucose concentration in the growth medium from 0∙5 to 1∙5% (w/v). Following inoculation, both glucose-6-phosphate-dependent and glucose-6-phosphate-independent glycogen synthase activities increased during exponential growth, remained constant during the stationary phase as long as glucose was present in the medium, and declined rapidly after the exhaustion of glucose. The cAMP content of submerged cultures of S. rolfsii was 5 pmol (mg protein)−1; however, upon glucose exhaustion there was a sharp increase followed by a decrease to the initial basal level. The activity of glycogen synthase in crude extracts was increased by glucose 6-phosphate and inhibited by cAMP.
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Extracellular Pectinolytic Enzymes of Fungi Elicit Phytoalexin Accumulation in Carrot Suspension Culture
More LessSUMMARY: A heat-labile elicitor of phytoalexin accumulation in carrot (Daucus carota L.) was detected in culture filtrate of Chaetomium globosum grown in Czapek-Dox medium. The elicitor activity correlated with the activity of pectinolytic enzymes in the culture filtrates. The culture filtrates induced the release of heat-stable elicitors from carrot cell homogenates, pectic fraction of carrot cell walls and citrus pectin. The coincidence of heat sensitivity and sodium periodate inactivation profiles of elicitor and pectinolytic activity suggested that the activity of pectinolytic enzymes is essential for the release of the heat-stable elicitor. The culture filtrates of Botrytis cinerea, Fusarium moniliforme and Helminthosporium oryzae showed similar elicitation patterns.
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The Energetics and Kinetics of Extracellular Polysaccharide Production From Methanol by Micro-organisms Possessing Different Pathways of C1 Assimilation
More LessSUMMARY: Product excretion by Methylophilus sp. NCIB 12047, Pseudomonas extorquens NCIB 9399 and Pichia pastoris during growth on methanol was examined. These organisms possess the ribulose monophosphate pathway, the serine pathway and the dihydroxyacetone pathway of C1 assimilation, respectively. Only Methylophilus sp. NCIB 12047 produced significant amounts of extracellular product from methanol under conditions of nitrogen limitation in chemostat culture. This was a low-viscosity extracellular polysaccharide containing glucose and mannose in the ratio 3:1. Maximum polysaccharide production occurred under nitrogen limitation at a methanol/ammonium sulphate ratio > 10. The other two organisms responded to nitrogen limitation by increasing the rate of methanol oxidation to CO2. The maximum yield for polysaccharide production by Methylophilus sp. was 0·34 g (g oxygen)-1 and 0·30 g (g methanol)-1. The maximum specific rate of polysaccharide production was 0·18 g (g protein)-1 h-1. Methylophilus sp. grew readily under oxygen limitation and excreted an extracellular polysaccharide under these conditions. Examination of the biochemical pathways for polysaccharide production via the various C1 fixation routes indicates that the ribulose monophosphate pathway is energetically the most favourable. Polymer production by Methylophilus sp. is energetically neutral in terms of net ATP demand; however, the rate of ATP utilization for polymer production is equivalent to 65 to 80% of that required for cell production at the same growth rate. The results reported suggest that the energetic constraints imposed by the various pathways of C1 assimilation strongly influence both the rate of synthesis and the composition of exopolysaccharides produced by methylotrophs.
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Nitrogen Fixation by Gloeothece sp. PCC 6909: Respiration and Not Photosynthesis Supports Nitrogenase Activity in the Light
More LessSUMMARY: Nitrogenase activity of suspensions of the unicellular cyanobacterium Gloeothece sp. PCC 6909 plotted against the concentration of dissolved O2 (dO2) resulted in a bell-shaped curve, both in the light and in the dark, with optima of 25 or 80 μm-O2 depending on the age of the culture. At the optimum dO2, nitrogenase activity [typically 4 to 6 nmol C2H4 (mg protein)−1 min−1] was similar in the light or in the dark. Alteration of light intensity from zero to 2 klx, or addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), had no effect on nitrogenase activity. At 1 klx the ADP/ATP ratio was 0∙2 and showed only a marginal increase as the dO2 was increased. However, a high level of illumination (30 klx) stimulated or inhibited nitrogenase activity, depending on the external dO2, presumably as a consequence of changes in the intracellular O2 concentration; in the presence of DCMU, activity increased twofold, independent of dO2.
In the dark, the dependence of the rate of respiration on O2 concentration suggested the presence of three O2-uptake systems with apparent K m values of 1 μm, 5 μm and 25 μm. The ADP/ATP ratio under anaerobic conditions was 0∙47 and showed a marked decrease as dO2 was increased to 25 μm. A CN-insensitive respiratory activity, which neither supported nitrogenase activity nor was coupled to ATP synthesis, was associated with the system with the apparent K m of 5 μm. The dependence of the specific activity of nitrogenase on dO2 indicated that both the high affinity (K m 1 μm) and low affinity (K m 25 μm) O2-uptake systems contributed ATP or reductant for N2-fixation. KCN (2∙5 mm) completely inhibited nitrogenase activity in the dark and at moderate levels of illumination and dO2. We conclude that respiration is the major source of reductant and ATP for nitrogenase activity both in the dark and in the light, but that photosystem I can contribute ATP at very high levels of illumination.
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Defining the Metabolic and Growth Responses of Porcine Haemophili to Exogenous Pyridine Nucleotides and Precursors
More LessSUMMARY: A variety of biologically important pyridine nucleotides and precursors were examined for their capacities to satisfy the V-factor requirement of 30 strains of porcine haemophili. Of the compounds tested, only NAD, NMN and nicotinamide riboside (NR) supported the growth of all strains; NADP supported the growth of only the type strain of Haemophilus parasuis. Further studies with the H. parasuis type strain and the neotype strain of H. pleuropneumoniae demonstrated that, during growth, these organisms exhibited affinities for NMN that were greater than those for NAD; the affinity of H. pleuropneumoniae for NR was similar to that for NMN, whereas H. parasuis exhibited relatively low affinity for NR. With either organism, equimolar amounts of NAD and NMN supported the production of approximately equal amounts of biomass whereas growth yields were substantially lower when NR was the pyridine nucleotide source. When either organism was grown in the presence of excess exogenous [carbonyl-14C]NAD, cessation of growth was accompanied by the apparent exhaustion of the NAD supply. Approximately 80% of the radioactivity added as [14C]NAD could be recovered as extracellular [14C]nicotinamide and the majority of the assimilated radioactive material was present intracellularly in the form of a [14C]NAD(P) pool. The results are discussed in terms of the structural features required of a pyridine compound for it to support the growth of porcine haemophili, the capacity of these organisms to compete for pyridine nucleotide sources in vivo, and possible mechanisms involved in the assimilation of such compounds.
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Mycobactin and the Competition for Iron between Mycobacterium neoaurum and M. vaccae
More LessSUMMARY: Two closely related species of mycobacteria, Mycobacterium vaccae and M. neoaurum, were grown under conditions of iron-deficiency (0·02–0·05 μg Fe ml-1) and iron-sufficiency (2–4 μg Fe ml-1) in a simple glycerol/asparagine medium. The strain of M. vaccae used was a nonmycobactin producer whereas M. neoaurum synthesized between 6–8% of its cell biomass as the lipid-soluble siderophore when grown under iron-limitation. The role of mycobactin for iron-acquisition was examined using both pure and mixed cultures, with cell viability determined following growth at various iron concentrations. M. neoaurum, the mycobactin producer, outgrew M. vaccae when iron was readily available. When grown under conditions where iron was limiting, M. neoaurum showed a decline in viable cell number compared with its competitor, highlighting its increased requirement for the metal. Some recovery was observed following mycobactin biosynthesis, this being greatly enhanced by the addition of an iron supplement to the growing cells.
Mycobactin biosynthesis allowed M. neoaurum to rapidly acquire any additional iron presented to the bacteria when growing under iron-limitation. However, M. vaccae did not synthesize the lipid-soluble siderophore with its iron-requirement satisfied by production of extracellular exochelin.
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Iron Uptake Processes in Mycobacterium vaccae R877R, a Mycobacterium Lacking Mycobactin
More LessSUMMARY: The production of exochelins (MV) was established in Mycobacterium vaccae R877R under iron-deficient conditions in concentrations about five times greater than in Mycobacterium smegmatis. M. vaccae does not produce mycobactin nor is salicylic acid secreted into the medium. A simple method is described using 55Fe-labelled culture filtrates for assessing exochelin production and which would be applicable to other mycobacteria. One of the exochelins produced (MV3) is part of an active iron uptake system and another (MV1) is responsible for a passive uptake system. MV3 exochelin has similar chromatographic properties and biological activity to the major exochelin produced by M. smegmatis: iron uptake from MV3 exochelin was inhibited by dinitrophenol, NaN3 and HgCl2, and was judged to be an active transport process. This process was not inhibited by equimolar amounts of ferri-salicylate or ferri-citrate both of which could be used separately as sources of iron for the organism. Uptake from these latter sources was insensitive to metabolic inhibitors and uncouplers. The multiplicity of pathways for iron uptake in a single organism is discussed.
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- Systematics
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Characterization of Haemophilus parainfluenzae Strains with Low-M r or Ladder-like Lipopolysaccharides
More LessSUMMARY: Twenty-five Haemophilus parainfluenzae strains were characterized for lipopolysaccharide (LPS) profiles, outer membrane protein profiles, serum sensitivity, plasmid profiles and DNA homology. Seventeen strains produced low-M r LPS that did not contain O-sidechains, while the remaining eight strains contained ladder-like LPS suggestive of O-repeated units. This is the first time in the genus Haemophilus that LPS with O-repeated groups has been described. The strains producing the different types of LPS could not be distinguished from each other in outer membrane protein profiles or the other characteristics examined.
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Distribution and Application of Mycobactins for the Characterization of Species within the Genus Rhodococcus
More LessSUMMARY: Representatives of 11 species of Rhodococcus were examined for their ability to synthesize mycobactin, a lipid-soluble siderophore, following iron-limited growth on solidified glycerol/asparagine medium. Rhodococcus bronchialis, R. terrae and R. rubropertinctus formed mycobactins, whereas the remaining species (R. coprophilus, R. equi, R. erythropolis, R. rhodnii, R. rhodochrous, R. ruber, R. maris and R. luteus) failed to synthesize these compounds even under conditions of strictly iron-limited growth. The mycobactins from R. terrae and R. rubropertinctus showed close similarity by thin-layer chromatography and high-performance liquid chromatography and could be easily distinguished from that of R. bronchialis.
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