- Volume 133, Issue 5, 1987
Volume 133, Issue 5, 1987
- Biochemistry
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The Relationship between Chemiosmotic Parameters and Sensitivity to Anions and Organic Acids in the Acidophile Thiobacillus Ferrooxidans
More LessWe investigated the relationships between the trans-cytoplasmic-membrane chemiosmotic parameters, viz. the membrane potential and the pH difference, and the toxicity of anions and organic acids in the acidophile Thiobacillus ferrooxidans. Organic acids accumulated in the cytoplasm in response to the transmembrane pH difference and inorganic anions could be caused to accumulate in response to the membrane potential. The distribution of the organic acids was unaffected by the membrane potential and that of the anions was not influenced by the pH difference. These accumulations may be toxic because of a direct effect of a high concentration of the anion in the cytoplasm or by acidification of the cytoplasm. The point of inhibition of respiration was at the level of the respiratory chain cytochrome oxidase when Fe(II) was the respiratory substrate.
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The Effects of Oxygen on Fermentation in Tritrichomonas Foetus KV1 and its Variant 1MR-100 with Defective Hydrogenosomes
More LessThe effects of low concentrations of O2 on fermentation in the cattle parasite Tritrichomonas foetus KV1 and its variant 1MR-100 were compared using membrane inlet mass spectrometry to measure simultaneously and continuously ethanol, CO2 and H2. In strain KV1 glucose-supported H2 and CO2 production were stimulated by O2 concentrations < 1·4 μm but were inhibited at higher concentrations. Damped oscillatory responses in H2 production indicated the operation of a feedback control system. Measurement of the O2-dependence of O2 consumption rates confirmed the presence of a high-affinity terminal oxidase (apparent K m = 1·6 μm-O2 at 37 °C) and substrate inhibition by O2 at > 8 μm-O2. Successive periods of exposure to O2 resulted in decreased O2 scavenging capacity, as indicated by increasing apparent K m values for O2. The variant strain 1MR-100 which lacks pyruvate: ferredoxin oxidoreductase and hydrogenase showed quite different characteristics: H2 production was not detectable, ethanol formation was inhibited by O2 (K i = 1 μm) and O2-dependence of O2 consumption indicated that no high-affinity oxidase was present (apparent K m = 33 μm-O2). Progressive increases in respiration rates on repeated exposure to low O2 concentrations indicated a capacity for adaptation to aerobiosis.
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Peptidoglycan and Arabinogalactan of Mycobacterium Leprae
More LessThe arabinogalactan and peptidoglycan of armadillo-grown Mycobacterium leprae were examined. Within the limits defined by the small amount of material available, the resemblance of these polymers to those of other mycobacteria was confirmed. The polymers were linked by a highly acid-labile bond and the arabinogalactan was itself acid-labile; free arabinose and a variety of oligosaccharides containing both arabinose and galactose, as well as polysaccharide and peptidoglycan, were released by dilute acid. The resonances from anomeric protons in the proton NMR spectrum of the arabinogalactan were similar to those from the arabinogalactan of M. tuberculosis. The composition and structure of the peptidoglycan resembled those of other mycobacteria. The only major difference was the specific replacement of l-alanine by glycine in the peptide of the peptidoglycan.
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Regulation of Phosphofructokinase from Aspergillus Niger: Effect of Fructose 2,6-Bisphosphate on the Action of Citrate, Ammonium Ions and AMP
More LessThe role of fructose 2,6-bisphosphate in the regulation of glycolysis in the citric acid accumulating fungus Aspergillus niger was investigated. Fructose 2,6-bisphosphate stimulated the activity of partially purified phosphofructokinase by increasing the affinity of the enzyme for fructose 6-phosphate and relieving inhibition by ATP. Fructose 2,6-bisphosphate acted synergistically with AMP, but not with NH+ 4 ions, which otherwise also activate phosphofructokinase. Fructose 2,6-bisphosphate also partially antagonized citrate inhibition of phosphofructokinase; complete deinhibition against high (5 mm) concentrations of citrate (as occur during citric acid accumulation), however, required the simultaneous presence of fructose 2,6-bisphosphate (0·1 μm). AMP (0·1 μm) and NH+ 4 ions (20 mm).
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A Major Outer-membrane Protein Functions as a Porin in Haemophilus Influenzae
More LessPorins are pore-forming outer-membrane proteins which serve as a non-specific pathway for the entry of hydrophilic molecules into Gram-negative bacteria. We studied four strains of Haemophilus influenzae that had decreased permeability to chloramphenicol associated with diminished quantities of a 40 kDa major outer-membrane protein. Isogenic pairs of organisms containing and lacking this protein were compared. The latter strains grew more slowly and were less permeable to sucrose and raffinose. They were also more resistant to multiple hydrophilic antibiotics than an isogenic strain containing the 40 kDa protein and were less permeable to penicillin G and chloramphenicol. We conclude that the 40 kDa outer-membrane protein functions as a porin in H. influenzae.
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Characterization of a Ca2+ Uniporter from Bacillus Subtilis by Partial Purification and Reconstitution into Phospholipid Vesicles
More LessCa2+ was accumulated by right-side-out membrane vesicles of Bacillus subtilis following imposition of a diffusion potential, inside-negative, owing to K+-efflux via valinomycin. Uptake was dependent on the magnitude of the membrane potential. This voltage-dependent Ca2+ uptake was inhibited by Ca2+ channel blockers such as nitrendipine, verapamil and LaCl3, and was competitively inhibited by Ba2+ and Sr2+. The system showed saturation kinetics with an apparent K m for Ca2+ of about 250 μm. Proteins responsible for the voltage-dependent Ca2+ uptake were partially purified by preparative isoelectric focusing in a Sepharose bed. A fraction at pH 5·28–5·33 contained the activity. The characteristics of Ca2+ uptake in reconstituted proteoliposomes were the same as those in membrane vesicles (sensitive to Ca2+ channel blockers; inhibited by Ba2+ and Sr2+). In addition, uptake was not influenced by a pH gradient imposed on the vesicles. The apparent K m for Ca2+ in the reconstituted system was about 260 μm. The specific activity was increased about 50-fold by purification with isoelectric focusing.
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Further Characterization of PL-1 Phage-associated N-Acetylmuramidase of Lactobacillus Casei
More LessSUMMARY: The properties of an N-acetylmuramidase previously isolated from PL-1 phage lysates of Lactobacillus casei were studied in more detail. The enzyme differed from hen egg white lysozyme in its substrate specificity spectrum, in its physical and chemical nature and in other properties. The cell wall lytic action showed narrow specificity. The enzyme was most active against the phage's host. It was composed of a single polypeptide chain of M r about 37000 as estimated by gel filtration, SDS-PAGE and amino acid analysis. The circular dichroic spectrum showed that the peptide backbone of the enzyme molecule most probably had a mainly random structure. The protein was rich in neutral amino acids, and the enzyme had an isoelectric point of pH 5·0. The N-terminal sequence was determined up to 27 amino acid residues, showing alanine as the N-terminus; the C-terminus was a tyrosine residue.
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Purification and Characterization of a Third Glucosyltransferase from Streptococcus Mutans Serotype g
More LessSUMMARY: Streptococcus mutans strain AHT (serotype g) secretes at least two glucosyltransferases with different pI values. A novel glucosyltransferase with a pI of 5·8 was purified 244-fold from the ammonium sulphate fraction by DEAE-cellulose chromatography, FPLC (Mono Q column, Pharmacia) and hydrophobic chromatography. The enzyme preparation gave a single protein band on analysis by both PAGE and SDS-PAGE, and did not form multiple protein bands detectable by IEF. The M r was estimated to be about 130000 by SDS-PAGE and about 135000 by ultracentrifugal analysis. The apparent K m value and pH optimum of the enzyme were 3·9 ± 0·2 mm (mean ± sd) and about 4·7, respectively. The enzyme synthesized water-soluble glucan from sucrose, and the glucan consisted of over 90 mol% 1,6-α-d-glucosidic linkages. The enzyme activity was not stimulated by primer dextran. Anti-enzyme serum produced a single precipitin band with the purified enzyme preparation, whereas it did not react with either of the other two known glucosyltransferases.
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- Biotechnology
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Enrichment and Selection of Hydantoinase-producing Micro-organisms
More LessA screening method that allows rapid, efficient and reliable detection and selection of hydantoinase-positive micro-organisms was devised. Out of 76 strains selectively isolated from soil, 15 strains possessing hydantoin-hydrolysing activity were selected using a spot-test based on the direct detection in agar of N-carbamyl amino acids. The results obtained were confirmed by quantitative determination of hydantoinase activity in whole cells and crude extracts of positive strains. Inactivity and/or instability of some hydantoin-hydrolysing activity were observed upon preparation of a few crude extracts. Preliminary experiments indicated that some selected strains had an inducible hydantoinase activity. The reliability and the indicative value (presence of hydantoinase) of the spot-test were confirmed by incubating crude extracts in enzyme membrane reactors for up to 112 h. The six strains that were positive with the spot-test and had high hydantoinase activity continuously produced N-carbamylvaline. Following nitrite treatment of N-carbamylvaline, only d-valine could be detected as confirmed by three different methods (enzymic and chromatographic).
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- Development And Structure
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An Endogenous Inducer of Sexual Development in Aspergillus Nidulans
More LessSUMMARY: During development of the homothallic ascomycete Aspergillus nidulans, asexual sporulation is followed by sexual sporulation. We report here the detection of a solvent-extractable activity which inhibits asexual sporulation and stimulates premature sexual sporulation. This activity, called precocious sexual inducer (psi), is overproduced by certain mutants that are blocked in both modes of sporulation. Using partially purified preparations of psi, biological response could be elicited with as little as 50 ng of material. We suggest that psi is a hormone-precursor which is converted to a hormone by normal sporulating strains that respond to psi, but not by the asporogenous mutants that overproduce psi. The stability of psi activity gives promise that the compound can be purified and identified.
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Isolation and Characterization of the Facultative Methylotroph Mycobacterium ID-Y
More LessSUMMARY: A facultatively methylotrophic Mycobacterium was isolated from Cleveland Harbor, Ohio, USA. The isolate, designated ID-Y, used a wide range of carbon and energy sources including methane and several other hydrocarbons. It displayed a growth cycle from rod-shaped exponential-phase cells, with many cell pairs exhibiting V-formation, to cocco-bacillary stationary-phase cells. A fixation technique involving glutaraldehyde/alcian blue resulted in the observation of a three-layered cell wall. Isolate ID-Y has an ultrastructure similar to that of other mycobacteria, particularly Mycobacterium phlei and Mycobacterium flavum, which is presently classified as a Xanthobacter species.
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- Genetics And Molecular Biology
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Molecular Analysis of Regulatory and Structural xyl Genes of the TOL Plasmid pWW53-4
More LesspWW53-4 is a cointegrate between RP4 and the catabolic plasmid pWW53 from Pseudomonas putida MT53, which contains 36 kbp of pWW53 DNA inserted close to the oriV gene of RP4; it encodes the ability to grow on toluene and the xylenes, characteristic of pWW53, as well as resistance to tetracycline, kanamycin and carbenicillin, characteristic of RP4. A physical map of the 36 kbp insert of pWW53 DNA for 11 restriction enzymes is presented, showing that the relative positions of the two xyl operons are different from those on the archetypal TOL plasmid pWW0. The location of the genes for 4-oxalocrotonate decarboxylase (xylI) and 4-oxalocrotonate tautomerase (xylH) were shown by subcloning and enzyme assay to lie at the distal end of the meta pathway operon. Although 2-oxopent-4-enoate hydratase (xylJ) and 4-hydroxy-2-oxovalerate aldolase (xylK) could be detected on a large cloned HindIII fragment, they could not be accurately located on smaller subcloned DNA, but the only credible position for them is between xylF and xylI. The gene order in the meta pathway operon is therefore xylDLEGF(J,K)IH. The regulatory genes xylS and xylR were located close to and downstream of the meta pathway operon, and the restriction map of the DNA in this region, as has previously been shown for the two operons carrying the structural genes, shows similarities with the corresponding region on pWW0. Evidence is also presented for the existence of two promoters, termed P3 and P4, internal to the meta pathway operon which support low constitutive expression of the structural genes downstream in Pseudomonas hosts but not in E. coli.
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Thermosensitive Bacillus Subtilis Mutants Which Lyse at the Non-permissive Temperature
C. Brandt and D. KaramataA collection of 655 thermosensitive mutants of Bacillus subtilis 168, obtained by indirect selection, was screened for those lysing at the non-permissive temperature. Thirty-three mutations thus identified were distributed by transformation into eight linkage groups designated lssA to lssH. The distribution was non-random. With the exception of group A, all groups were small, suggesting that mutations identified in each of them may map in one gene only. Linkage groups identified here were mapped in four different regions of the B. subtilis chromosome and their positions relative to reference markers were the following: (i) aroI-lssA-dal-purB; (ii) metC-lssB-lssC-furA-pyrE-cysC-lssD; (iii) lssF-gtaA-lssG-hisA-lssH-cysB; and (iv) cysA-lssE-dnaC-purA. Kinetics of N-acetyl-D-[1-14C]glucosamine incorporation revealed that groups A, B, C, D and F are deficient in peptidoglycan synthesis at the restrictive temperature. In group G, anomalies at the cell wall level were suggested by incorporation and growth curves. It appears that in almost all known cases, thermosensitive lysis mutations in B. subtilis either affect genes involved in peptidoglycan synthesis or lead, more or less directly, to induction of prophages.
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The Nucleotide Sequence of a Streptomycin 6-Phosphotransferase Gene from a Streptomycin Producer
More LessThe nucleotide sequence of the DNA fragment containing the streptomycin 6-phosphotransferase (streptomycin 6-kinase) gene from the streptomycin-producer Streptomyces griseus strain HUT 6037 was determined. Analysis of the sequence revealed an open reading frame which could encode 325 amino acid residues. A biased codon usage pattern, reflecting the high G + C composition (approximately 74%) of Streptomyces DNA, was observed in the gene.
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Molecular Cloning of Clostridium Thermocellum DNA and the Expression of Further Novel Endo-β,4-glucanase Genes in Escherichia Coli
More LessSau3A fragments of Clostridium thermocellum (NCIB 10682) DNA were ligated into the BamHI site of pBR322 and expressed in Escherichia coli HB101 and a Lac− mutant thereof. Twenty-eight clones with carboxymethylcellulase (CMCase) activity were selected from two libraries by means of the Congo Red plate assay. Restriction enzyme analysis indicated that the CMCase+ clones contained a total of 13 unique DNA inserts. Hybridization of recombinant plasmids with chromosomal DNA confirmed the physical maps in all but one case and was further used to demonstrate the absence of homology between the HindIII restriction fragments of similar size which occurred in many of the clones. Without exception, CMCase+ E. coli clones expressed endoglucanase activity, but differed with respect to the amount and nature of the enzyme activity produced; additionally, some clones had exoglucanase activty which, in at least one case, was not attributable to the production of a second enzyme. For a few selected clones, the partially purified CMCase was analysed by electrophoresis. A temperature profile characteristic of a thermostable enzyme was demonstrated for the endoglucanase of one of the most active clones. Based on the evidence presented here, it is probable that the 13 unique DNA fragments described do not contain any of the C. thermocellum endoglucanase genes previously cloned.
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Heterogeneity of Escherichia Coli Phages Encoding Vero Cytotoxins: Comparison of Cloned Sequences Determining VT1 and VT2 and Development of Specific Gene Probes
More LessPhages coding for production of Vero cytotoxins VT1 or VT2 in strains of Escherichia coli serotype O157. H7 or O157. H− were morphologically indistinguishable. Their genome size and restriction enzyme digests of the phage DNA were similar. These phages were clearly different in these respects from a VT1-encoding phage isolated from a strain of E. coli O26. H11 (H19). However the VT1 region cloned from the phage originating in the E. coli O157. H7 strain was identical to the VT1 region previously cloned from the phage carried by H19. Sequences encoding VT2 that were cloned from the phage in E. coli O157. H− have been mapped and the VT2 region identified by transposon insertion. The cloned regions coding for VT1 or VT2 production had no similarities in the presence of restriction enzyme sites over a distance of about 2 kb, and two VT1-specific probes spanning a region of about 1·4 kb did not hybridize under stringent conditions with cloned VT2 DNA. A 2 kb HincII fragment contained the VT2 genes but hybridized to VT1-encoding phages and recombinant plasmids via flanking phage DNA. A 0·85 kb AvaI-PstI fragment was a specific probe for VT2 sequences and did not hybridize under stringent conditions to phages or plasmid recombinants encoding VT1.
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- Immunology
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Purification, Characterization and Serological Properties of a Glycolipid Antigen Reactive with a Serovar-specific Monoclonal Antibody against Leptospira Interrogans serovar Canicola
E. Ono, H. Takase, M. Naiki and R. YanagawaA glycolipid antigen possessing a serovar-specific antigenic determinant of Leptospira interrogans serovar canicola was purified from a chloroform/methanol extract of the organism. The purification procedures included silicic acid column chromatography and preparative thin-layer chromatography (TLC). Antigenic activity was detected by a TLC-enzyme immunostaining technique using monoclonal antibody CT3, which specifically agglutinates serovar canicola and only weakly serovar sumneri but no other serovars of Leptospira. The purified glycolipid reacted with CT3 antibody, indicating that the glycolipid possessed a serovar-specific antigenic determinant. Infrared spectrum and proton nuclear magnetic resonance analyses showed that the glycolipid contained sugar and lipid moieties, which possessed amide linkages and an acetyl group. Gas-liquid chromatography-mass spectrometry analysis showed that the glycolipid contained two unknown sugars, one of which (unknown sugar II) appeared to be associated with the antigenic determinant specific for canicola. The serovar-specific antigenic determinant was destroyed by mild alkali treatment of the glycolipid. These findings suggested that the antigenic determinant was an alkali-labile moiety which may be related to the unknown sugar II.
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- Pathogenicity And Medical Microbiology
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The Control of Experimental Escherichia coli Diarrhoea in Calves by Means of Bacteriophages
More LessSeven phages highly active in vitro and in vivo against one or other of seven bovine enteropathogenic strains of Escherichia coli belonging to six different serotypes were isolated from sewage. Severe experimentally induced E. coli diarrhoea in calves could be cured by a single dose of 105 phage organisms. It could be prevented by doses as low as 102, by spraying the litter in the calf rooms with aqueous phage suspensions or simply by keeping the calves in uncleaned rooms previously occupied by calves whose E. coli infections had been treated with phage. Microbiological examinations of calves used in these experiments revealed that the phage organisms multiplied rapidly and profusely after gaining entry to the E. coli-infected small intestine, quickly reducing the E. coli to numbers that were virtually harmless. The only phage-resistant E. coli that emerged in the studies on calves infected with one or other of the seven E. coli strains were K−. These organisms were much less virulent than the K+ organisms from which they were derived and did not present a serious problem in calves given adequate amounts of colostrum. Infections produced by oral inoculation of a mixture of six strains of the E. coli could be controlled by administration of a pool of the six phages that were active against them but, in general, the control was less complete than that observed in the single-strain infections. K+ phage-resistant bacteria emerged in some of the calves used in these mixed infections and they were as virulent as their parent organisms; evidence from in vitro studies suggested that they might have arisen by genetic transfer between organisms of the different infecting strains. Infections produced by these K+ mutants and their parents could be controlled by the use of mutant phages derived from phages that were active on their parents. During the experiments with mixed E. coli infection, an extraneous phage active against one of the six E. coli strains suddenly appeared in calves kept in the same rooms. Microbiological examinations revealed that this phage was effectively controlling the multiplication of organisms of that particular strain of E. coli in the small intestines of the calves.
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Factors Influencing the Survival and Multiplication of Bacteriophages in Calves and in Their Environment
More LessSUMMARY: Seven phages were fairly susceptible in vitro to the lethal effect of acidified whey, more so than the enteropathogenic Escherichia coli strains on which they were active. The low acidity that prevailed in the abomasum contents of calves shortly after a milk feed had little harmful effect on orally administered organisms of these phages; they flooded into the small intestine. The high acidity that prevailed later was lethal to orally administered phage organisms; few entered the small intestine. The lethal effect could be counteracted by giving CaCO3 in the feed. Low concentrations of phage-neutralizing antibodies were found in some serum samples from human beings, cattle and pigs. Antibodies to one of the seven phages were common in the human samples and antibodies to another, phage B44/1, were common in the cattle and pig samples and in bovine colostrum. Phage B44/1 antibodies in a sample of colostral whey were destroyed at pH 3·5 or less. Giving colostrum containing phage B44/1 antibodies with CaCO3 to a calf greatly reduced the numbers of orally administered phage B44/1 organisms in its alimentary tract. Antibodies to another phage were induced in the serum of a calf suffering from E. coli diarrhoea by treating it with that phage. The phages were as susceptible as the E. coli strains to the lethal action of formaldehyde and sodium hypochlorite. In contrast to the E. coli strains, they were almost completely resistant to phenol and chloroxylenol. The in vitro virulence of 21 phages varied according to the temperature at which tests were performed. The majority were almost avirulent at 20 °C and 43 °C and most virulent at 37 °C. Experiments in calves suggested that their body temperatures were sufficiently high to adversely influence the virulence of some phages.
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Identification of Two Chemical Types of K21 Capsular Polysaccharide from Klebsiellae
More LessSUMMARY: Strains of Klebsiella pneumoniae of serotype K21 are frequently involved in outbreaks of nosocomial infections. The type strain of Klebsiella pneumoniae K21 (which we have renamed K21a) produces capsular polysaccharide that contains mannose, galactose and glucuronic acid in the ratio 2:2:1. In contrast, all eight of the randomly chosen isolates of Klebsiella pneumoniae that were initially typed as K21 were shown by paper chromatography and NMR spectroscopy to produce a different capsular polysaccharide. We have designated this new polysaccharide K21b. The K21b capsular material appears to have a closely similar immunodominant side chain to K21a. However, K21b polysaccharide has two molecules of rhamnose in the polysaccharide backbone replacing the two molecules of mannose found in the K21a capsule. Our results suggest that the K21b Klebsiella serotype may be more frequently distributed than the classical K21a type.
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Production of Cuticle-degrading Enzymes by the Entomopathogen Metarhizium Anisopliae during Infection of Cuticles from Calliphora Vomitoria and Manduca Sexta
More LessSUMMARY: A biochemical and histochemical investigation with specific substrates and inhibitors was used to visualize protease, esterase and aminopeptidase activities produced in situ during penetration of Calliphora vomitoria and Manduca sexta cuticles by hyphae of the entomopathogenic fungus Metarhizium anisopliae. Two endoproteases, and aminopeptidase and esterase activities, were mainly localized in simple and complex appressoria and germinating conidia. The effect of inhibitors on two characterized proteases (Pr1 and Pr2) and aminopeptidase activity in appressorial plates was quantified by microdensitometric measurement of reaction products. Pr1 and Pr2 activities were differentially inhibited by various protease inhibitors. Pr1, Pr2, esterase, aminopeptidase and N-acetylglucosaminidase (exochitinase) activities were present during penetration as detected directly following desorption from fungal and cuticle components. The proteases produced in situ were fractionated, and were shown by immunological and enzymological criteria to be the same as those produced in culture media. The sequence of enzyme appearance in situ showed that production of proteolytic enzymes precedes exochitinase production. No production of endochitinase was found before or during hyphal penetration of the cuticle.
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- Physiology And Growth
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Sulphur Sources for Growth of Chlorella Fusca and Their Influence on Key Enzymes of Sulphur Metabolism
More LessThe green alga Chlorella fusca strain 211–8b (algal collection of Göttingen) is able to grow on more than 100 different sulphur sources such as mercaptides, disulphides, thioethers, sulphinic and sulphonic acids including ‘Good buffers’, sulphoxides, sulphones and sulphate esters. This suggests that green algae might be of importance for degradation of xenobiotics and natural compounds in the overall sulphur cycle. The data presented describe the growth of C. fusca on such sulphur compounds and the influence of these compounds on enzymes of the assimilatory sulphate reduction sequence.
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Thermotolerance of the Respiratory Chain of Bacillus Coagulans
More LessThe thermostability of the respiratory chain of Bacillus coagulans grown at different temperatures was similar to the thermotolerance of the primary dehydrogenases. NADH was the major electron donor to the respiratory chain in cells grown at both 37 °C and 55 °C. The respiratory chain from cells grown at 55 °C exhibited slightly greater thermostability than that from cells grown at 37 °C. NADH-supported respiration, as well as NADH dehydrogenase activity, was much more thermotolerant than that with succinate in cells grown at both temperatures. Membrane-bound succinate dehydrogenase could be stabilized by the addition of 10% (w/v) NaCl while NADH dehydrogenase exhibited intrinsic thermostability.
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Effect of Osmotic Shock and Shearing Forces on Ferric Enterochelin Transport in Escherichia Coli K12
More LessThe fes mutation in Escherichia coli K12, which inactivates enterochelin esterase, allows the cell to accumulate ferric enterochelin. The ferric complex of enterochelin was released in significant quantities from a fes mutant after osmotic shock. Analysis of the effects of the individual stages of the shock procedure in wild-type cells showed that prior exposure of cells to sucrose and EDTA was not required, careful dilution of cells into a hypo-osmolar medium being sufficient to induce efflux of Fe3+. Prior treatment with EDTA or exposure to shearing forces served either to enhance efflux or to induce efflux in isotonic media. Neither vitamin B12 nor 5′-nucleotidase was released from the periplasm by these procedures. The release observed under mild conditions was stimulated specifically by Co2+, did not occur at 0 °C, and was inhibited by 2,4-dinitrophenol at 37 °C. From these observations, it was concluded that the efflux of Fe3+ represents a physiological response of the cell to exposure to a hypo-osmolar medium. Such changes may enhance survival following physicochemical stressing of the bacterial outer membrane.
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The Involvement of Glutamine Synthetase/Glutamate Synthase in Ammonia Assimilation by Aspergillus Nidulans
More LessWild-type Aspergillus nidulans grew equally well on NH4Cl, KNO3 or glutamine as the only nitrogen source. NADP+-dependent glutamate dehydrogenase (EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) activities varied with the type and concentration of nitrogen source supplied. Glutamate synthase (GOGAT) activity (EC 1.4.7.1) was detected but it was almost unaffected by the type and concentration of nitrogen source supplied. Ion exchange chromatography showed that the GOGAT activity was due to a distinct enzyme. Azaserine, an inhibitor of the GOGAT reaction, reduced the glutamate pool by 60%, indicating that GOGAT is involved in ammonia assimilation by metabolizing the glutamine formed by GS.
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Relation between pH, Hydrogenase and Nitrogenase Activity, NH+ 4 Concentration and Hydrogen Production in Cultures of Rhodobacter Sulfidophilus
Y. Peng, P. Stevens, P. De Vos and J. De LeyRhodobacter sulfidophilus, a sulphide-tolerant, salt-tolerant member of the Rhodospirillaceae, was shown to produce molecular hydrogen on a mineral medium with lactate as electron donor and glutamate as nitrogen source. A maximum production of about 1·1 mmol H2 (mmol added lactate)−1 was found at pH 6·75. The in vivo activity of hydrogenase and nitrogenase showed that hydrogenase activity (and thus the H2 recycling system) was greatest at pH 6·5 and 6·75 and thus, at least partially, was responsible for the lack of H2 production below 6·75, but that nitrogenase activity was greatest at between pH 6·5 and 7·0, and decreased to zero at pH 8·0, resulting in reduced H2 production at pH 7·5 and none at pH 8·0. When sufficient NH+ 4 had accumulated in the culture, nitrogenase activity remained below the maximum value and no H2 production occurred.
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Chemolithotrophic and Autotrophic Growth of Thermothrix thiopara and Some Thiobacilli on Thiosulphate and Polythionates, and a Reassessment of the Growth Yields of Thx. thiopara in Chemostat Culture
More LessSeveral thiobacilli and Thermothrix thiopara were grown in chemostat culture with inorganic sulphur compounds as growth-limiting energy substrates for autotrophic growth. Thiobacillus neapolitanus, Thiobacillus thiooxidans and Thiobacillus acidophilus were all able to use thiosulphate, trithionate or tetrathionate as sole energy substrates, as was Thx. thiopara grown at 72 °C. ‘True growth yields’ (Y max) were estimated for the organisms and showed yields of T. neapolitanus to be the same on trithionate and thiosulphate, thereby suggesting that only oxidative, and not substrate-level, phosphorylation is involved in energy conservation in this organism. The yield data suggested that substrate-level phosphorylation could, however, be significant in T. acidophilus. Thiobacillus versutus only grew with thiosulphate, with which it gave yield values similar to those of T. neapolitanus and T. thiooxidans. Thx. thiopara exhibited maximum specific growth rates on thiosulphate, trithionate and tetrathionate of 0·5, 0·23–0·25 and <0·06 h−1 respectively. Its yields on all three were much greater than those of the thiobacilli: the Y max on thiosulphate was in the range 18·0–22·8 g mol−1, but the organisms contained only about 29% carbon and 48% protein. These values do, however, make Thx. thiopara the highest-yielding inorganic-sulphur-oxidizing chemolithoautotroph yet described.
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Oxygen Profiles in, and in the Agar Beneath, Colonies of Bacillus Cereus, Staphylococcus Albus and Escherichia Coli
More LessThis paper reports the use of microelectrodes to measure O2 penetration in different aged colonies of Bacillus cereus, Escherichia coli and Staphylococcus albus. In young (18 h) colonies of B. cereus and E. coli O2 disappeared at depths of 25–30 μm and 35–40 μm respectively. In young S. albus colonies, O2 reached a minimum but was never completely absent. As colonies aged (24–168 h) the depth to which O2 penetrated increased.
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Bacteriolytic Activity of Seminalplasmin
More LessSeminalplasmin, an antimicrobial protein from bovine seminal plasma, lysed both Gram-positive and Gram-negative bacteria but not Candida albicans. The lytic activity was not lysozyme-like and was not affected by inhibitors of RNA or protein synthesis or by azide; it was strongly inhibited by divalent cations like Ca2+, Mn2+ and Mg2+ at millimolar concentrations. Maximum lysis of Escherichia coli was obtained at 37°C; heat treatment of E. coli drastically reduced its susceptibility to lysis by seminalplasmin. E. coli cells in the stationary phase of growth were lysed much less than those in the exponential phase, and those grown in an enriched medium were lysed much more than those grown in a minimal medium. It appears that the lytic activity of seminalplasmin is due to the activation of an autolysin.
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A Nitrate Non-utilizing Mutant of Gibberella Zeae
More LessA nitrate non-utilizing mutant of Gibberella zeae (Fusarium graminearum) was isolated following UV mutagenesis and filtration enrichment. In preliminary reports, this mutant was designated gdh, but it has now been renamed nnu. This mutant has a complex pleiotropic phenotype which affects most aspects of nitrogen utilization. The wild-type parent could use nitrate, nitrite, ammonium, urea, polyamines, and most amino acids and purines as sole source of nitrogen, but the nnu mutant could use only putrescine, anthranilic acid, and the amino acids asparagine, aspartic acid, glutamic acid, glutamine, ornithine, proline and tyrosine. Most of these compounds could also serve as the sole source of both carbon and nitrogen. Addition of 1 mM-glutamine or 1 mm-proline to the medium resulted in a synergistic growth response if spermidine, arginine, citrulline, histidine, phenylalanine or tryptophan was present as the primary nitrogen source. Addition of 1 mM-ammonium to the medium repressed growth when putrescine, aspartic acid, asparagine, glutamic acid or tyrosine was the primary nitrogen source. The addition of ammonium did not repress growth when anthranilic acid, glutamine, ornithine or proline was the primary nitrogen source. The nnu mutant grew more rapidly than its wild-type parent in race tubes on some complex media. The complexity and diversity of the alterations in nitrogen catabolism caused by the nnu mutation suggest that this locus is important in the regulation of nitrogen catabolism by G. zeae.
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The Role of Exopolysaccharides in Adhesion of Freshwater Bacteria
More LessCultures of two strains of freshwater bacterial isolates adhered readily to inert glass surfaces exposed in the growth medium. The process of microbial film formation could be followed by a new staining technique based on congo red, a dye specific for carbohydrate material. In conjunction with a chemical assay for total carbohydrate, the association of extracellular polysaccharides with attached cells was demonstrated. Under optimal growth conditions, the involvement of exopolysaccharide in the adhesion process appeared to follow the initial attachment of bacterial cells, leading to the formation of microcolonies enmeshed in polysaccharides. A non-polysaccharide-producing mutant attached to glass slides in numbers similar to the wild-type bacteria, but did not form microcolonies. Growth conditions such as glucose or Ca2+ limitation which affected polysaccharide synthesis in the wild-type prevented microcolony formation, but not cell attachment. It is proposed that exopolysaccharide production is involved in the development of the surface films, but possibly not in the initial adhesion-process. In those strains which do produce polysaccharide, the cells which attach develop into microcolonies.
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Protein Synthesis and Degradation during the Differentiation Cycle of Rhodomicrobium Vannielii Swarmer Cells
More LessSUMMARY: Rhodomicrobium vannielii swarmer cells, when synchronized by selective filtration, differentiate synchronously with the loss of motility and synthesis of a prostheca, and subsequently form a daughter cell. Synchronized swarmer cells synthesized an 11·5 kDa protein under dark anaerobic conditions which disappeared from the soluble fraction when the culture was subsequently incubated in the light and the differentiation cycle thus initiated. The protein did not accumulate under dark aerobic conditions and it may play a role in the inhibition of differentiation in the light limited and thus energy limited swarmer cell.
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- Systematics
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SIMCA Pattern Recognition in the Analysis of Streptomycete Fatty Acids
More LessBiomass from representative strains of Streptomyces cyaneus and related taxa was degraded using an alkaline methanolysis procedure. The resultant fatty acid methyl esters were analysed by gas chromatography and the quantitative data examined using the SIMCA statistical package. The S. cyaneus strains were recovered in two major clusters. The larger of these contained sixteen strains which had been previously classified as S. cyaneus in a numerical phenetic survey. The remaining S. cyaneus strains formed a heterogeneous aggregate cluster together with representatives of other streptomycete species. A third cluster encompassed a number of blue-spored streptomycetes from soil. Disjoint principal components analysis in conjunction with cross-validation analyses demonstrated that the S. cyaneus group and the blue-spored soil isolates could be represented by statistically stable principal component models. The fact that the initial, numerically circumscribed, S. cyaneus taxon was only partially recovered by the numerical analysis of fatty acid data supports the view that this taxon contains strains which are not entirely typical of the cluster, despite their high degree of similarity with other members. These preliminary findings suggest that models based on fatty acid data could be used for identifying unknown streptomycetes to established taxa and for the definition of novel streptomycetes.
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