- Volume 128, Issue 8, 1982
Volume 128, Issue 8, 1982
- Biochemistry
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A Study of the Proteinase Activity Released by Dictyostelium discoideum during Starvation
More LessThe release of proteinase activity from myxamoebae of Dictyostelium discoideum after suspension in non-nutrient buffer has been confirmed. Between 15% and 30% of the total activity against Hide Powder Azure was released, the bulk of this appearing in the buffer within 30 min. Activity against the cathepsin B substrate, α-benzoyl-dl-arginine 2-naphthylamide, was also released. The release of proteinase activity was more rapid than that of other secreted lysosomal enzymes and was insensitive to cyanide. The extracellular proteinases catalysed both limited and extensive proteolysis. Gel electrophoresis showed similar patterns of released and intracellular proteinases, and their sensitivities to inhibitors were also similar. However, there was evidence for an increased proportion of thiol proteinases in the released enzymes. The results suggest that proteinases were released by a non-secretory process but one possible mechanism, cell lysis, has not been clearly demonstrated. A specific developmental role for extracellular proteinases seems unlikely.
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The Bacterial Biogenesis of Isobutyraldoxime O-Methyl Ether, a Novel Volatile Secondary Metabolite
More LessProduction of the volatile metabolite, isobutyraldoxime O-methyl ether (IBME) by a Moraxella-like organism NCIB 11650 was investigated under a variety of environmental conditions using gas chromatography. Under aerobic conditions up to 10 μg IBME ml−1 was produced on mineral salts media containing 0.5% (w/v) glucose or succinate as sole C source with 0·1% (w/v) NH4Cl as sole N source. Exogenous l-valine further stimulated IBME formation up to 25 μg ml−1 but supplementation of the medium with d-isomer or other amino acids had little effect on IBME production and did not lead to the appearance of analogues of IBME. Trapping experiments using [14C]valine confirmed that IBME was derived from this amino acid. Several other bacterial species examined, e.g. Alcaligenes sp. NCIB 11652, another Moraxella-like organism NCIB 11651 and Pseudomonas sp. NCIB 11653 also produced IBME under similar conditions. The Alcaligenes strain synthesized up to 20 μg ml−1 in the absence of valine and up to 90 μg ml−1 in its presence.
The production of IBME exhibited many features characteristic of the formation of a secondary metabolite. Thus biosynthesis was confined to a narrower range of temperature than cell division, was almost completely suppressed by 300 mm-phosphate and was inhibited by high concentrations of readily utilizable C sources. Although IBME synthesis in the Moraxella-like organism NCIB 11650 appeared to be growth-related, its formation by both the Alcaligenes sp. and the Moraxella-like organism NCIB 11651 was delayed until the late-exponential and early-stationary phases of growth. The biological significance of this novel class of secondary metabolite is discussed and a possible biosynthetic route proposed.
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Partial Purification and Characterization of Glutamate Synthase from a Thermophilic Bacillus
More LessGlutamate synthase (GOGAT), glutamine synthetase (GS), NAD+-dependent glutamate dehydrogenase (GDH) and NAD+-dependent alanine dehydrogenase (AlaDH) activities were detected in cell extracts of Bacillus stearothermophilus PH24, a strain deficient in NADP+-dependent GDH. GDH and GOGAT activities were low under most growth conditions and GOGAT was not detectable in extracts of cells grown with amino acids as carbon and nitrogen source. AlaDH and GS activities were more variable, the former being high in cells grown on l-alanine as carbon and nitrogen source. GS was repressed during growth with high concentrations of NH4C1 as nitrogen source but a corresponding increase in AlaDH activities suggests that this enzyme may replace NADP+-dependent GDH as the main enzyme for ammonia assimilation under these conditions.
GOGAT was purified 40-fold using affinity chromatography on NADPH-Sepharose. The molecular weight of the partially purified enzyme was estimated to be 160000 and K m values for NADPH, 2-oxoglutarate and l-glutamine were 22, 15 and 29 μm, respectively. Glutamine could be replaced by NH4C1 as nitrogen donor (K m 44 mm) but the rate was only 10% to 15% that of the l-glutamine-dependent reaction. The pH optimum for glutamine-dependent activity was 8·0 and the temperature optimum 75 C: the enzyme displayed a discontinuous Arrhenius plot over the range 30 °C to 75 °C. Azaserine, l-methionine sulphone and Cibacron Blue 3GA were all inhibitors and the enzyme was rapidly inactivated in the presence of NADPH when l-glutamine and 2-oxoglutarate were absent.
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Stereospecificity of 2-Monochloropropionate Dehalogenation by the Two Dehalogenases of Pseudomonas putida PP3: Evidence for Two Different Dehalogenation Mechanisms
More LessPseudomonas putida PP3 grew on dl-2-monochloropropionate (2MCPA) with a release of chloride ions consistent with the dechlorination of both isomers. The organism grew on either d-or l-2MCPA. Dehalogenase activity in cell-free extracts showed that both d-and l-2MCPA were dehalogenated. Pseudomonas putida PP3 contains two dehalogenases, and studies with the separated enzymes revealed that the fraction I enzyme used both d-and l-2MCPA, the rate of dechlorination of l-2MCPA being 80% of the rate of d-2MCPA dechlorination. The product of the reaction, lactate, retained the same optical configuration as the substrate provided. The fraction II dehalogenase also dechlorinated d-and l-2MCPA, with the same difference in rates as for the fraction I dehalogenase, but the lactates produced were of the opposite configuration to their precursors. The two dehalogenases showed further differences with respect to inhibition by two sulphydryl-blocking agents, N-ethylmaleimide and p-chloromercuribenzoate. Fraction I dehalogenase was considerably more sensitive to these two reagents compared with the fraction II dehalogenase. Dithiothreitol partially protected the fraction I dehalogenase from N-ethylmaleimide inhibition. The results are discussed in terms of the possible evolutionary relationships of the two dehalogenases.
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Glycerol Utilization by Schizosaccharomyces pombe: Phosphorylation of Dihydroxyacetone by a Specific Kinase as the Second Step
More LessCrude cell extracts of the fission yeast Schizosaccharomyces pombe (strains NCYC 132 and 972h−) can phosphorylate dihydroxyacetone but not glycerol; activity for dl-glyceraldehyde is very low. This suggests that a specific dihydroxyacetone kinase is present and catalyses the second step in a pathway for glycerol utilization, in which the initial step is an oxidation by an NAD+-linked glycerol dehydrogenase. In support of this pathway (glycerol→dihydroxyacetone→dihydroxyacetone phosphate), both strains can utilize glycerol or dihydroxyacetone. but not dl-glyceraldehyde, as growth substrates. Both enzymes are subject to catabolite repression and may also be inducible but are not co-ordinately regulated.
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Kinetic Characterization of Mitochondrial Malate Dehydrogenase from Dictyostelium discoideum
More LessMalate dehydrogenase (MDH; EC 1.1.1.37) from Dictyostelium discoideum was purified and characterized. MDH activity from whole cells was purified 275-fold. The mitochondrial cytoplasmic MDH present co-purified through three ion exchange and affinity chromatography steps. The isoenzymes were barely separable by either disc gel electrophoresis or isoelectric focusing. The purified preparation containing both isoenzymes had a single pH optimum (9·3–9·5) and an apparent molecular weight of 70000. It exhibited linear kinetics and responded to known inhibitors of MDH, i.e. thyroxine and hydroxymalonate. Michaelis and dissociation constants obtained with this preparation were similar to those obtained with a 10-fold purified mitochondrial MDH.
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Assimilation of Inorganic Nitrogen Compounds by Nitrobacter agilis
More LessNitrobacter agilis, a chemolithotrophic bacterium, utilizes ammonia as well as nitrite as a nitrogen source for growth. The growth yields were increased about twofold by growing the bacterium in a nitrite medium supplemented with 2 mm-ammonium chloride. Higher concentrations of ammonium chloride, however, competitively inhibited nitrite oxidation and growth of the bacterium. Washed cells readily incorporated 15NH4 +, 15NH2OH, 15NO2 − and 15NO3 − respectively (in decreasing order) into cell nitrogen. Enzyme activities in cell extracts of ammonia-supplemented cultures, compared to those without ammonia, were greater for glutamate dehydrogenase (EC 1.4.1.2) and similar for glutamine synthetase (EC 6.3.1.2), whereas glutamate synthase (EC 1.4.1.14) was barely detectable. Inhibitors of glutamine synthetase (l-methionine dl-sulphoximine) and glutamate synthase (azaserine) did not affect the incorporation of 15NH4 − and 15NO2 − into cell nitrogen of washed cells. The results indicate that glutamate dehydrogenase is a key enzyme in N. agilis for the assimilation of either nitrite or ammonia. Glutamine synthetase, which was also active in cell extracts, is probably required for the production of glutamine. Glutamate synthase, however, does not appear to be an important enzyme for ammonia assimilation.
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Anaerobic Growth, Nitrate Reduction and Denitrification in 46 Rhizobium Strains
More LessA total of 46 rhizobial strains were assessed for anaerobic growth in the presence of nitrate, and, using the criteria of nitrate utilization and nitrous oxide and nitrogen production, for their ability to denitrify. Nitrite production was also measured. Half of the strains were denitrifiers: these included all five strains of R. meliloti tested which produced N2 from nitrate and most of the slow-growing rhizobia, but none of the 14 strains of R. trifolii.
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Cyclic (1→2)-β-d-Glucan and the Octasaccharide Repeating-unit of Succinoglycan Produced by Agrobacterium
More LessNine strains of Agrobacterium produced extracellular cyclic (1→2)-β-d-Glucan. Most of the strains produced an octasaccharide repeating-unit of succinoglycan. Mutant strains of Agrobacterium sp. and mutant strain 10C3K derived from Alcaligenes faecalis var. myxogenes, producing curdlan without succinoglycan or with a slight amount of succinoglycan, produced only cyclic (1→2)-β-d-Glucan. Cyclic (1→2)-β-d-Glucan was shown by paper chromatography and methylation analysis to be composed of two components without other glucosidic linkages. These were confirmed to be heptadecaose and octadecaose by field desorption mass spectrometric analysis.
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- Development And Structure
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Microelectrophoretic Studies of Coat-defective Spores of Bacillus megaterium
More LessMicroelectrophoretic studies of lysozyme-resistant spores of Bacillus megaterium QM B1551 suggested that carboxyl groups were the only ionized species on the spore surface. Spores of B. megaterium NCIB 8291 have defective coats, allowing lysozyme to attack the underlying cortical peptidoglycan and initiate germination-like changes. The surface of such spores was characterized by the presence of ionized carboxyl and amino groups, suggesting that the amino groups were present on the cortical surface. Spores of B. megaterium QM B1551 rendered defective by extraction of coat protein with sodium dodecyl sulphate and dithiothreitol at pH 10 were also lysozyme sensitive and had similar electrophoretic behaviour to the naturally coat-defective spores. Since the electrophoretic behaviour of coat defective spores is qualitatively similar to that of germinated spores, holes or channels may appear in the spore coat during the early stages of germination, exposing the cortical peptidoglycan.
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Permeability of Gas Vesicles to Perfluorocyclobutane
More LessThe permeability of intact gas vesicles, isolated from the cyanobacterium Anabaena flos-aquae, to percyclofluorobutane (C4F8), has been investigated by two methods. Warburg manometry showed that a suspension of intact gas vesicles exchanges more C4F8 than an equivalent suspension of collapsed ones. Pressure nephelometry showed that increasing the partial pressure of C4F8 in solution caused a corresponding increase in apparent critical collapse pressure of the gas vesicles. From these observations it is concluded that C4F8 is able to permeate freely through the gas vesicle wall. If it permeates via pores, these must be at least as large as the gas molecules, which have a collision diameter of 0·63 nm. Warburg manometry showed that the volume of C4F8 exchanged exceeds the volume of gas-vesicle gas space, and this indicates that, in addition, there is specific absorption of the gas, probably on the inner, gas-facing surface of the gas vesicle wall.
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- Ecology
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The Acquisition of Streptococcus mutans by Infant Monkeys (Macaca fascicularis) and its Relationship to the Initiation of Dental Caries
More LessThe acquisition and transmission of Streptococcus mutans by 16 consecutively born infant monkeys (Macaca fascicularis) was studied. The 16 infant monkeys were weaned in four groups and caged together to form a commune. Transmission from mother to infant occurred infrequently. Streptococcus mutans was isolated from nine of the mothers but only from the dental plaque of two infant monkeys at weaning at which time the predominant streptococci were S. sanguis and S. mitior. One further animal was colonized by S. mutans during the formation of the commune, but only after it was caged with an infant harbouring the same organism. For 46 d after the completion of the commune, the monkeys were fed a starch-based diet during which time S. mutans of serotypes c, e or h were isolated from the faeces of all 16 animals and from the dental plaque taken from the developmental groove of the first deciduous molar of 11 animals. Faecal transmission appeared to be an important factor in the spread of S. mutans between monkeys in the commune. The monkeys were then fed a caries-promoting high sucrose diet resulting in a rapid increase in the proportion of S. mutans in the plaque and in the faeces. Streptococcus mutans serotype e was more frequently isolated from both plaque and faeces and its predominance may in part be due to the production of a bacteriocin active in vitro against S. mutans serotype h and other species of oral streptococci isolated from monkey dental plaque. The proportion of S. mutans in the developmental groove 8 d after the introduction of the high sucrose diet was significantly related to both the caries status of the groove and the total caries score 6 months later. The results suggest that, in this model of human dental caries, S. mutans is the major bacterial factor in the initiation of tooth decay.
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- Genetics And Molecular Biology
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Genetic Analysis of the Slug Stage of Dictyostelium discoideum
More LessDuring the course of development, cells of Dictyostelium discoideum are able to produce a multicellular body (a ‘slug’) which is capable of movement over the substratum. This phase, which is induced by production of ammonia by the starving cells, may last for hours or days depending on environmental stimuli. In order to probe the regulatory system controlling formation and duration of the slug phase, mutants were isolated that remained in the slug phase for an extended period. Thirty-two such ‘slugger’ mutants were analysed by parasexual genetic techniques and placed into 10 complementation groups (slgA-slgJ). The linkage groups bearing representatives of these complementation groups were determined by segregation of diploids formed between mutants and tester strains. Phenotypic studies of mutants indicated that members of slgD, slgE and slgG were over-sensitive to the ammonia slug-inducing stimulus.
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Methods for Selection of Mutants and In Vitro Culture of Cochliobolus heterostrophus
More LessAuxotrophic, morphological and drug-resistant mutants of Cochliobolus heterostrophus (= Bipolaris maydis = Helminthosporium maydis = Drechslera maydis) were isolated after mutagenesis with ethyl methanesulphonate. Exposure of the fungus to other mutagens (N-methyl-N′-nitro-N-nitrosoguanidine, nitrous acid, and UV light) resulted in recovery of morphological but not auxotrophic mutants. Antibiotic enrichment or filtration enrichment in medium containing high levels of sorbose increased the proportion of auxotrophs in the surviving population. All sexually fertile mutants were found to have single gene lesions; those with the same phenotype were allelic in some cases but not in others. Defined minimal and complete media, designed to facilitate genetic and molecular biological manipulations, supported maximal growth rate at 30 °C and maximal conidiation of the fungus at 23 °C with a 16 h long-wave UV light photoperiod. Under all in vitro growth conditions tested, near-isogenic lines of two known races of the fungus were essentially identical. Sexual fertility was improved by a programme of breeding and selection, but not by physical or chemical changes in the medium used for crossing. The fungus can be stored conveniently on silica gel.
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Characterization of a Temperature-sensitive DNA Replication Mutant of Staphylococcus aureus
More LessA temperature-sensitive DNA replication mutant of Staphylococcus aureus NCTC 8325 has been isolated and characterized. After transfer to the non-permissive temperature (42 °C), DNA synthesis continued for 30 min and the mean DNA content increased by 56%. The amount of residual DNA synthesis was not reduced when the non-permissive temperature was raised, nor when chloramphenicol was added at the time of the temperature shift. During incubation at 42 °C, mutant bacteria accumulated the capacity to synthesize DNA after return to the permissive temperature (30 °C) in the presence of chloramphenicol. This capacity was lost when chloramphenicol was present at 42 °C. The properties of the mutant are consistent with a defect in the initiation of DNA replication at 42 °C.
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Isolation of a UV Endonuclease from the Cyanobacterium Synechocystis PCC 6308
More LessA UV endonuclease with activity towards UV-irradiated lambda DNA but no activity towards non-irradiated DNA was isolated from a crude lysate of the unicellular cyanobacterium Synechocystis PCC 6308. The enzyme has a molecular weight between 15000 and 18000. ATP was essential for maximum enzyme activity and the optimum pH was 7·5–7·8.
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A New Class of Mutants of the cysB Regulatory Gene for Cysteine Biosynthesis in Salmonella typhimurium
More LessA new class of regulatory mutants in the cysB locus has been isolated by plating cysM strains, under anaerobic conditions, on medium containing 1,2,4-triazole. The isolated cysB mutants are cysteine prototrophs and triazole-resistant, although the levels of cysteine and O-acetyl-l-serine sulphydrylase are not changed. In contrast to the constitutive cysB mutants identified previously, the expression of the cysteine biosynthetic enzymes in the newly isolated mutants is regulated by the same factors as in wild-type strains. In the double mutant cysE cysB2971, the cysteine biosynthetic enzymes are absent with the exception of O-acetyl-l-serine sulphydrylase.
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Delayed Inducibility of Sulphite Reductase in cysM Mutants of Salmonella typhimurium Under Anaerobic Conditions
More LessSalmonella typhimurium cysM mutants grow at a normal rate under aerobic conditions, but only after a lag period under anaerobic conditions. No difference in the induction of two cysteine biosynthetic enzymes sulphite reductase and O-acetyl-l-serine sulphydrylase in wild-type, cysK and cysM strains was observed under aerobic conditions. Under anaerobic conditions, however, the cysM strain differed from the others in showing a long delay in the induction of sulphite reductase. These observations are consistent with the assumption that the observed growth delay of the cysM mutant under anaerobic conditions is the result of abnormalities in the regulation of sulphite reductase.
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Recombinant Nodulation Plasmids in Rhizobium leguminosarum
More LessPlasmids pRL1JI and pRL6JI, which carry determinants essential for symbiosis in Rhizobium leguminosarum feld isolates 248 and 128C53, respectively, were both incompatible with two other transmissible plasmids that did not carry symbiotic determinants. When derivatives of these two plasmids (pRL3JI or pRL4JI) were introduced into a strain which already contained pRL1JI or pRL6JI, recombinant replicons were often obtained: these were of uniform size for each pair of incompatible plasmids. Recombinant nodulation plasmids were also observed following infrequent interactions with pRL10JI, a natural nodulation plasmid that does not belong to the same incompatibility group as pRL3JI or pRL4JI.
By the formation of a recombinant nodulation plasmid, it has been possible to transfer the determinants for nodulation ability (Nod+), nitrogen fixation (Fix+) and hydrogen uptake (Hup+) from pRL6JI, a plasmid that was not self-transmissible, on to a replicon that was transmissible at high frequency and carried a selectable drug resistance marker. In the case of pRL10JI, which is also a non-transmissible nodulation plasmid, the formation of a recombinant nodulation plasmid transferred the symbiotic determinants to a transmissible replicon belonging to a different incompatibility group from pRL10JI itself.
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Improvement of Symbiotic Properties in Rhizobium leguminosarum by Plasmid Transfer
More LessTransfer of plasmid pIJ1008, a recombinant of two indigenous Rhizobium leguminosarum plasmids into R. leguminosarum strain 300 produced strain 3960, which reduced significantly more N2 in pea root nodules than did strain 300 itself. Strain 3960 was superior to the field isolate 128C53, from which the symbiotic determinants of pIJ1008 were derived, and transfer of plasmid pIJ1008 into two other genetic backgrounds also improved symbiotic performance compared to the introduction of other nodulation plasmids.
As plasmid pIJ1008 carries genetic determinants for an uptake hydrogenase activity (Hup+) as well as nodulation capability (Nod+) and other determinants for symbiotic nitrogen fixation (Fix+), the increased effectiveness of strains carrying pIJ1008 may result from their capacity to conserve energy by recovering H2 evolved by nitrogenase.
Intact plant studies with 15N showed that the superior N2 fixation capability associated with pIJ1008 was enhanced by 2 mm-NO3 −, a common concentration of soil N. It was also shown that, as plants grew older, the hydrogenase determined by pIJ1008 was not able to recycle all the hydrogen evolved by pea root nodules.
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Only One of the Large Plasmids in Rhizobium leguminosarum Strain PRE is Strongly Expressed in the Endosymbiotic State
More LessPlasmid DNA from six strains of Rhizobium leguminosarum was separated by agarose gel electrophoresis and showed a large variation in the number and size of the plasmids. Blotting on to nitrocellulose and hybridization with a probe containing nif (nitrogen fixation) genes from Rhizobium meliloti demonstrated that all strains contain only one plasmid carrying nif genes. The molecular weights of the nif plasmids ranged between approximately 130 × 106 and 550 × 106. Using DNA-DNA hybridization no homologous sequences were detected between the nif plasmid of strain PRE and the other plasmid present in this strain. Hybridizations with RNA from bacteroids showed that only the nif plasmid is strongly expressed in the endosymbiotic state. No selective amplification of plasmid DNA occurs during the transition of free-living bacteria into bacteroids.
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Mutant Strains of Salmonella typhimurium with Defective Phosphoribosylpyrophosphate Synthetase Activity
More Lessmutant of Salmonella typhimurium with undetectable phosphoribosylpyrophosphate (PRPP) synthetase activity in vitro and abnormally low PRPP pools in vivo was identified by screening temperature-sensitive isolates by an autoradiographic procedure. The lack of PRPP synthetase activity in vitro and temperature-sensitive growth were shown to result from separate, but closely linked mutations mapping at 47 units on the Salmonella chromosome. Mutant cell extracts prepared by a variety of methods did not show any detectable PRPP synthetase activity, but material that was immunochemically cross-reactive with PRPP synthetase was detected by complement fixation analysis. A second mutant, isolated by localized mutagenesis, contained about half the PRPP synthetase and cross-reacting material of the parental strain.
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Determination of Methylenomycin A Synthesis by the pSV1 Plasmid from Streptomyces violaceus-ruber SANK 95570
More LessA plasmid (pSV1) of 110 × 106 daltons from a methylenomycin A producing strain of Streptomyces violaceus-ruber was detected on and isolated from agarose gels. Elimination of this plasmid by protoplasting and regeneration resulted in the simultaneous loss of methylenomycin A production and resistance. pSV1 hybridized with pBR322 containing a cloned fragment of 1.7 × 106 daltons from S. coelicolor A3(2) which codes for methylenomycin A resistance. The pSV1 plasmid could be transferred to S. lividans by conjugation and by transformation and plasmid DNA identical in size to pSV1 could be isolated from the recipient strains. These experiments show that pSV1 codes for methylenomycin A production and resistance, in close analogy to the SCP1 plasmid from S. coelicolor A3(2).
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A Mutant of Bacillus subtilis Secreting a DNAase Inhibitor During Sporulation
More LessUnder sporulation conditions, Bacillus subtilis mutant SE63 secreted into the medium an inhibitor of the extracellular DNAase that is specifically associated with stage II to III of sporulation. The inhibitor was a heat-labile protein with a molecular weight of 18000–20000. A cell-bound, presumably intracellular, inhibitor with the same properties was found in extracts of wild-type cells and of the mutant. The mechanisms by which the mutation, designated din, might cause the secretion of a protein that is normally intracellular are discussed. The mutation was mapped by transduction with phage PBS1 and it lies between met and fru. It was also introduced into a variety of sporulation mutants including some blocked at the earliest known stages of the process. In all of these it caused secretion of the inhibitor. It is concluded that the secretion is therefore not integrally associated with sporulation but is, instead, part of the response of the cells to the nutritional step-down conditions in which spore formation is induced.
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- Physiology And Growth
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A Re-examination of the Cytochromes of Escherichia coli using Fourth-order Finite Difference Analysis: Their Characterization under Different Growth Conditions and Accumulation during the Cell Cycle
More LessThe cytochromes of Escherichia coli K12 have been studied using low-temperature (77 K) difference spectrophotometry. Numerical (i.e. fourth-order finite difference) analysis resolved the α band of reduced minus oxidized spectra of whole cells from aerobically grown cultures into five components, with absorption maxima at 548, 551·5, 555·5, 560 and 563 nm. Using the same differencing intervals, numerical analysis of cells grown under oxygen-limited conditions revealed only two components, at 555·5 and 559 nm. Similar analysis of cells grown anaerobically with fumarate as electron acceptor revealed four absorption maxima, at 548, 550·5, 555·5 and 559 nm. Membrane particles from aerobically grown cells showed the same absorption bands as intact cells; preliminary evidence was obtained for a component of the 555·5 nm band that could be relatively easily washed from the membrane. The contribution of cytochrome o or its CO-liganded form to the α region could not be determined by numerical analysis. We conclude, in contrast to a previous application of numerical analysis to the cytochromes of E. coli, that growth under anaerobic or oxygen-limited conditions results in the appearance of cytochromes spectrally distinct from those in aerobically grown cells. An attempt has been made to reconcile the presence of multiple components detected in the α region by numerical analysis of aerobically grown cells with the diverse components described by others.
Quantification of cytochromes revealed by numerical analysis in aerobically grown cells separated into size (and thus age) classes by zonal centrifugation showed that the major components accumulated continuously, probably exponentially, throughout the cell cycle.
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Mutants of Escherichia coli K12 Sensitive to Acidic pH
More LessFour mutants of Escherichia coli K12 were isolated on the basis of their sensitivity to pH 5·4. Under non-permissive conditions their growth was reversibly inhibited. At pH 7·0 these mutants showed a highly pleiotropic phenotype, which included altered phage and detergent sensitivities and leakage of periplasmic proteins. The findings suggest a defect in the outer membrane, perhaps in lipopolysaccharide. Two mutants mapped in or near the rfa locus, while the other two were remote from this region.
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Active Transport of Benzoate in Pseudomonas putida
More LessBenzoate uptake in Pseudomonas putida is mediated by an active transport system capable of accumulating benzoate against a 150-fold concentration gradient when subsequent metabolism is blocked by mutation. Initial benzoate transport rates are inhibited by CCCP, sodium azide, arsenate and DCCD. Uptake is stimulated by including a respirable carbon source during preincubation of the bacteria. The initial uptake rate and the ATP pool levels are not correlated and no periplasmic components were found to bind benzoate. These observations indicate that benzoate uptake is energized by the membrane potential, rather than by ATP hydrolysis.
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The Utilization of Nitriles and Amides by a Rhodococcus Species
More LessA species of Rhodococcus was isolated from garden soil on the basis of its capacity to use acetonitrile as sole C and N source. Acetonitrile-grown cells hydrolysed a number of amides and nitriles to ammonia. The substrate nitriles, listed in order of decreasing hydrolysis rates, were acetonitrile, acrylonitrile, propionitrile and n-butyronitrile. The corresponding amides were also hydrolysed together with formamide and, to a small extent, nicotinamide. With the exception of acrylonitrile and acrylamide, each compound supported growth as did the non-substrates malonamide, benzamide, α-phenylacetamide and 3-aminopropionitrile. Benzonitrile, phenylacetonitrile (benzyl cyanide), malononitrile and aminoacetonitrile did not support growth. Nicotinamide and benzamide stimulated acetamidase activity but malonamide had no effect. Both the aminonitriles inhibited the acetonitrilase system. Cells grown in succinate (NH4)2SO4 medium did not hydrolyse acetonitrile or acetamide indicating that the enzymes involved in nitrile degradation are subject to induction/repression. Acetamide and acetate appear to be gratuitous inducers of acetonitrilase: acetate also induces the acetamidase.
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Magnesium-limited Growth of the Cyanobacterium Anacystis nidulans
More LessSMg2+-limited growth of the cyanobacterium Anacystis nidulans was investigated in batch and chemostat cultures. In batch cultures the growth rate of the organism depended on the Mg2+ concentration up to 5 μm. Although the maximum growth rate was achieved at this concentration, the organism formed aseptate filaments of three to four times the‘normal’ cell length. About 90 min after increasing the Mg2+ concentration from 5 μm to 1 mm the cell size decreased, followed by an increase in the division rate, which lasted for about 60 min and resulted in a 66% increase in cell number. The rates of DNA, RNA and protein synthesis were not altered during these Mg2+ shift-up experiments, showing that the control by Mg2+ of growth had been separated from its control of cell division, In Mg2+-limited chemostat cultures, the mean cell volume decreased from about 2·0 to 0·6 μm3 when the Mg2+ concentration was increased from 2·5 to 10 μm. This increase in Mg2+ also resulted in an increase in the calculated intracellular Mg2- concentration from 27 to 78 mm, and the amount of cellular Mg2+ bound in chlorophyll increased from 17 to 22%. A comparison of Mg2+-and SO4 2−-limited chemostat cultures showed that the mean cell volume decreased with increasing dilution rate when Mg2+ was the limiting factor, whereas it increased with dilution rate when SO4 2− was limiting. Only small differences in the rates of RNA and protein synthesis were found in the two cultures, although the synthesis of RNA was Mg2+-dependent. The ratio of total RNA to protein, which gives the amount of RNA necessary to synthesize one protein unit (RNA efficiency), was independent of the growth rate in both SO4 2−-and Mg2+-limited chemostat cultures showing that the efficiency of culture RNA was variable in both cases. The efficiency was higher under SO4 2−-than Mg2+-limited conditions.
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Cell and ATP Yields of Citrobacter freundii Growing with Fumarate and H2 or Formate in Continuous Culture
More LessOut of 19 strains belonging to the family Enterobacteriaceae only Escherichia coli and Citrobacter strains fermented fumarate exclusively to succinate. This fermentation was dependent on the presence of molecular hydrogen or formate. The inability of these micro-organisms to convert fumarate to succinate, acetate and CO2 correlated with their lack, or low activity, of oxaloacetate decarboxylase.
Continuous culture experiments were performed with Citrobacter freundii in minimal or complex medium with fumarate + H2 or formate, and the growth parameters were determined. From the data obtained, a Ymaxfumarate dissimilated max value of 10·5 ± 0·8 g dry wt per mol fumarate dissimilated was calculated. This value demonstrates that, per mol fumarate reduced, at least 0·6 ± 0·05 mol ATP is produced and subsequently used for biosynthetic purposes.
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- Short Communication
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The Determination of Two Morphologically Distinct Types of Pilus by Plasmids of Incompatibility Group I2
More LessElectron microscopy revealed that IncI2 plasmids determined thick pili as well as the thin flexible ones previously detected. Phage PR4, which typifies the broad host-range, plasmid-specific, lipid-containing bacteriophages, formed plaques on bacterial strains carrying derepressed IncI2 plasmids, and adsorbed to the tips of the thick pili rather than the thin ones.
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- Taxonomy
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Nucleic Acid Hybridization Studies on Microbacterium, Curtobacterium, Agromyces and Related Taxa
More LessThirty strains of Agromyces, Arthrobacter, Curtobacterium, Brevibacterium, Corynebacterium and Microbacterium, exhibiting the rare peptidoglycan of group B, were subjected to extensive nucleic acid hybridization studies. The DNA homology values indicate that Corynebacterium insidiosum DSM 20157 is genetically identical with Corynebacterium michiganense DSM 20134. Corynebacterium sepedonicum NCPPB 378 and Corynebacterium nebraskense DSM 20400 are closely related to Corynebacterium michiganense DSM 20134. Corynebacterium betae DSM 20141, Corynebacterium oortii ATCC 25283 and Corynebacterium poinsettiae ATCC 9682 are genetically identical with Corynebacterium flaccumfaciens DSM 20129. In addition, Curtobacterium citreum ATCC 15828, Curtobacterium luteum ATCC 15830 and Curtobacterium pusillum ATCC 19096 share a high degree of relatedness to Corynebacterium flaccumfaciens DSM 20129. All other described species are more distantly related to each other. DNA-rRNA cistron similarity studies reveal that all coryneforms with a peptidoglycan group B are members of one homogeneous cluster for which the rank of a genus is suggested.
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Diaminopimelic Acid Profiles of Alkalophilic and Alkaline-resistant Strains of Actinomycetes
More LessTwenty alkalophilic actinomycetes with optimum growth pH of 9·0 to 9·5 were isolated. Although these new isolates morphologically closely resembled conventional streptomycetes containing ll-diaminopimelic acid, nine out of 20 isolates contained meso-diaminopimelic acid.
Among 420 International Streptomyces Project (ISP) Streptomyces strains tested for their maximum growth pH, only 6 strains grew at pH 11·5. Of these six strains, three strains, S. caeruleus ISP 5103, A. alborubidus ISP 5465 and S. autotrophicus ISP 5011 contained meso-diaminopimelic acid.
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Pyrolysis-Gas Chromatography Combined with SIMCA Pattern Recognition for Classification of Fruit-bodies of Some Ectomycorrhizal Suillus Species
More LessNaturally grown fruit-bodies of Suillus luteus, S. bovinus and S. variegatus were subjected to Py-GC. The pyrograms obtained were statistically analysed and classified with a multivariate classification program (SIMCA). The class describing the S. bovinus pyrograms was significantly separated from both the S. luteus and the S. variegatus classes, while these two classes showed some over-lapping which was due to variation between different fruit-bodies within each species. The taxonomic significance of the statistical distances between the classes representing the species is uncertain.
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