- Volume 133, Issue 10, 1987

SUMMARY: Vibrio parahaemolyotcis utilized ATP, ADP or AMP as the sole source of carbon. About three times higher activity of membrane-bound 5'-nucleotidase was observed in cells grown in the presence of these nucleotides than in their absence: and therefore the enzyme seems to be inducible. Since the 5'-nucleotidase activity could be measured with whole cells, the active site of this enzyme appears to be outwardly oriented. Both Mg2+ and CL− were required for activity. Among the divalent cations tested, Mn2+ and Co2+ could replace Mg2+ to some extent, whereas Zn2+ strongly inhibited activity. Among the anions tested, Br−, I− and NO3, could replace C1−, but SO2+ 4 and CH3COO− could not. When cells were grown with ATP, Cl− was indispensable and Zn2+ strongly inhibited growth. Therefore, it is concluded that extracellular ATP and other 5'-nucleotides are cleaved by the membrane-bound 5'-nucleotidase outside the cells and that the adenosine produced is then utilized.

SUMMARY: The ascomycete yeast Hansenula wingei was able to grow with nitrate, ammonium, glutamate, glutamine or hypoxanthine as sole nitrogen source. Conditions for assay of nitrate reductase (EC 1.6.6.2; NR)and nitritereductase (EC 1.6.6.4; NiR) wereoptimized incell-freeextracts from nitrate-grown cells. NR utilized NADH and NADPH as electron donor and required FAD for maximum activity; NiR was NADPH-specific. Glutamate-grown cells possessed low levels of both enzyme activities and, of the nitrogen sources tested only nitrate was able to induce both enzyme activities above this low basal level of constitutive expression. Ammonium, glutamate and glutamine each prevented nitrate induction of the activities in glutamate-grown cells. Addition of ammonium, glutamate or glutamine to nitrate-grown cells which possessed appreciable levels of NR and NiR caused loss of activity, even if added with nitrate. Loss of both activities under these conditions occurred faster than in cells in which activity loss occurred solely due to nitrate depletion. Both increases and decreases in NR and NiR activity were dependent on protein synthesis.

SUMMARY: Regulation of the dual coenzyme-specific glutamate dehydrogenase (GDH; EC 1.4.1.3) was studied in the anaerobic bacterium Bacteroides fragilis. Cells grown at a low concentration of ammonia had a specific activity for the enzyme 10-fold higher than that for cells grown with excess ammonia. Immunochemical determination with a GDH-specific antiserum showed that the content of immuno-precipitated protein was about 8% of the total protein in the former cells and was 4% in the latter cells. When cells grown on 50 mM-NH4CL were transferred to a fresh medium containing 0.5 mM-NH4Cl, an increase in the molecular activity of the enzyme occurred, and synthesis of immuno-reactive protein started. Rapid inactivation of the GDH occurred when cells grown on 1 mM-NH4, Cl were exposed to 50mM-NH4C1. However, the amount of immuno-precipitated protein was not decreased. The inactivation was specifically induced by ammonia and was reversed by transferring the cells to an ammonia-limited medium even in the presence of chloramphenicol. These findings suggest that the synthesis of the GDH is stimulated under low ammonia conditions and that the enzyme activity is controlled by means of a reversible activation/inactivation mechanism which is regulated by ammonia. However, no phosphorylation of GDH was observed before and after exposure of cells to high concentrations of ammonia.

SUMMARY: Proteolysis of endogenous and exogenous substrates in cell-free extracts of the psychrotrophic bacterium Arthrobacter sp. S155 has been compared. Endogenous proteins were degraded only after treatment with cyanogen bromide. The hydrolysis of exogenous proteins of high Mr, (i.e. casein) was optimum at alkaline pH and was stimulated by Ca2+, Mg2+, Mn2+ and ATP. The serine protease inhibitor phenylmethylsulphonyl fluoride had no effect on ATP stimulation. Small peptides (i.e. insulin) were degraded at very high rates. This activity was optimum at slightly acidic pH and was stimulated by Ca2+, strongly inhibited by Mn2+, but not affected by ATP. Degradation of cyanogen bromide-treated cellular proteins displayed two pH optima which corresponded to the optimum pH for the degradation of insulin and casein. The characteristics of these acidic and alkaline activities were identical to those active against insulin and casein respectively. The proteases which degraded casein were much more heat resistant than those which degraded insulin.

SUMMARY: Thermostable lactate dehydrogenases (EC 1.1.1.27) were purified to homogeneity from Clostridium thermohydrosulfuricum cells grown on starch and producing mainly ethanol (LDHE) and from cells grown on sucrose and producing mainly lactic acid (LDHJ, and were found to be distinct isoenzymes. The two enzymes both had native Mr, values close to 145 × 103, but slightly different subunits with Mr, values about 37 × 103. LDHL dissociated into subunits more readily. The isoelectric points were 5.0 for LDHL and 5.2 for LDHE. The catalytic activity of LDHE had an almost absolute requirement for fructose 1,6-bisphosphate (FBP) at all temperatures (22-fold activation with K1/2 12 μM-FBP at 65°C, pH 6.0). LDHL was activated by FBP only at temperatures over 40°C (5-fold activation with K1/2 80 μM-FBP at 65°C, pH 6.0). For both enzymes the optimum temperature for pyruvate reduction in the presence of 1 mM-FBP was 70°C and the pH optimum at 65°C was sharp and at 5.5-6.0. FBP lowered the apparent Km of LDHL for pyruvate. At 50μM-FBP both enzymes showed a positive co-operative dependence on NADH.

SUMMARY: Eight yeast species (the ascomycetes Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Hansenula wingei, Saccharomycopsis lipolytica, Schizosaccharomyces pombe and Pichia scolyti, and the imperfect yeasts Candida albicans and Rhodotorula glutinis) have been compared with respect to the proteins solubilized by glucanase treatment of amino-acid-labelled, purified walls. Except for R. glutinis, a significant quantity of radioactively labelled protein material was liberated by this treatment. Among the major protein components solubilized was a material larger than 100 kDa (heterogeneous in size in some species) and a molecule ranging in size from 31.5 to 34 kDa depending on the yeast species. The large material was of glycoprotein nature, with the sugar portion N-glycosidically linked to the protein moiety; in some species, this material did not bind concanavalin A (ConA), indicating the presence of terminal sugar residues different from mannose, glucose or glucosamine. The form of 31.5-34 kDa was present in all the species studied except R. glutinis; it was a mannoprotein, except in Schiz. pombe, which possessed a 31.5 kDa form not sensitive to tunicamycin or endoglycosidase H and not recognized by ConA. Antigenic cross-reactivity was observed between the protein moieties of the 33 kDa form of Sacch. cerevisiae and the species of equivalent size in C. albicans and H. wingei. Similar partial proteolysis patterns were obtained for the 33 kDa form of Sacch. cerevisiae and the 34 kDa forms of C. albicans and P. scolyti.

SUMMARY: A region located at around 52' on the Escherichia coli chromosome was cloned by use of mini-Mu-lac containing a plasmid replicon and recloned into pBR322. Enzyme assays on transformants carrying the cloned fragments indicated the presence in the latter of the cysA and cysM genes coding for sulphate permease and O-acetylserine sulphydrylase B, respectively.

SUMMARY: Procedures for the introduction of plasmid DNA into Gram-negative bacteria have been adapted and optimized to permit transformation of the plant pathogen Xanthomonas campestris pathovar campestris with the cloning vector pKT230 and other broad-host-range plasmids. The technique involves CaC12-induced competence and heat shock and is similar to that routinely used for Escherichia coli. Wild-type X. c. campestris strains appear to restrict incoming unmodified DNA, so that plasmid DNA for transformation must be prepared from X. c. campestris (into which it has previously been introduced by conjugation). To overcome this disadvantage a restriction-deficient mutant has been isolated.

SUMMARY: The transformation frequency of Escherichia coli C-600, continuously cultivated in a chemostat operated at constant dilution rate, increased with increase in agitation rate (impeller speed). Cell counts at each impeller speed remained approximately constant. The phenomenon correlated with changes in mean cell volume associated with the changes in agitation rate.

SUMMARY: Two mutations associated with antibiotic supersusceptibility in Pseudomonas aeruginosa strain Z61 were transferred separately into strain PA0222, using R68.45-mediated conjugation and phage F116L transduction. One mutation (absA) was 40% contransducible with pro-82 at 26 min on the P. aeruginosu chromosome and was associated with increased susceptibility to β-lactams, gentamicin and hydrophobic agents. Strains carrying the absA mutation also displayed enhanced uptake of a hydrophobic fluorescent probe, 1-N-phenylnaphthylamine, and were found, by SDS-PAGE, to be altered in the pattern of lipopolysaccharide O-antigen distribution. The other mutation (absB), associated with increased susceptibility to β-lactams and gentamicin but not to hydrophobic agents, was cotransducible with met-28 and proC at 20 min on the chromosome. The absB mutation caused a structurally undefined alteration in the physical interaction of EDTA and gentamicin with the outer membrane.

SUMMARY: A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine β-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces liuidans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of β-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by β-iodopenicillanate) the overproduced S. lividans ML 1 β-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of β-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the β-lactamase structural gene. The β-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more β-lactamase than the original S. cacaoi.

SUMMARY: Using a gene probe derived from the cloned var. sotto insecticidal crystal protein (ICP) gene, we have cloned a Bacillus thuringiensis var. aizawai HD-133 ICP gene in Escherichia coli. The gene encodes a polypeptide that is toxic to Lepidoptera in vivo and in vitro. The protein is expressed at a level sufficient to produce phase-bright inclusions in recombinant E. coli strains, and these inclusions can be partially purified using discontinuous sucrose density gradients. Immunoblot- ting shows that the inclusions contain a 135 kDa polypeptide which reacts strongly with antiserum raised against the B. thuringiensis var. kurstaki HD-I P1 polypeptide.

SUMMARY: We examined the production of temperate bacteriophages by several strains of Bacillus amyloliquefaciens. Each of the seven strains we induced with mitomycin C produced at least one temperate phage capable of infecting strains of Bacillus subtilis; some of the phages could also infect other B. amyloliquefaciens strains. It is probable that some of the bacteria also produce bacteriocins.

SUMMARY: Bacillus amyloliquefuciens strain H is lysogenic for a large temperate phage we call H2. H2 has a polyhedral head 85 nm in diameter and a tail of about 17 × 434 nm. H2 lysogenizes Bacillus subtilis between the tyrA and metB genes, and gives specialized transduction of metB and, at lower frequencies, of ilvD and ilvA. The phage carries a thymidylate synthase gene and converts thymine auxotrophs of B. subtilis to prototrophy. The H2 genome is a linear DNA molecule about 129 kb in length. DNA extracted from phage particles grown in B. subtilis is not cut by the restriction endonucleases HaeIII, Fnu4H1, Bspl2861, and BamHI; the latter enzyme is produced by B. amyloliquefaciens strain H. The prophage in lysogenic B. subtilis cells can be cut by these enzymes. We have isolated H2 mutants that carry the transposon Tn917, or a mutation resulting in clear-plaque morphology, or both.

SUMMARY: Bacteriophage CG33 was isolated from a strain of Corynebacterium glutumicum that had become contaminated during an industrial fermentation. CG 33 was assigned to Bradley's group B since it had a polyhedral head 40 nm wide and a short non-contractile and striated tail 78 nm long. Adsorption to its host, C. glutamicum ATCC 13287, was enhanced in the presence of Ca2+. The latent period was 18 min at 34°C; the burst size was 16 p.f.u. ml−1. CG33 also formed plaques on C. lilium ATCC 15990 but at a low frequency. Its genome consisted of a linear double stranded DNA molecule of 13.4 kb with cohesive ends. A restriction map of the genome was obtained by using various endonucleases.

SUMMARY: A satellite band in CsCl-bisbenzimide density gradients of Pythium diclinum DNA was found to consist of ribosomal DNA (rDNA) repeating units with a complexity of 10.3 kilobases (kb). Similar satellite bands were also obtained from a morphologically diverse selection of species from the Pythiaceae: Pythium torulosum (10.4 kb), Pythium irregulare (11.8 kb), Pythium anandrum (10.6 kb), Pythium paddicum (10.6 kb) and Phytophthora cryptogea (11.2 kb). Physical maps were constructed using seven endonuclease restriction enzymes and the maps were aligned on the basis of nine conserved restriction sites in a 6 kb region which was shown to be homologous to a DNA plasmid probe containing ribosomal genes from Neurospora crassa. A map of the rDNA region in Apodachlya pyrifera (10.1 kb), a species external to the Pythiaceae (Leptomitales), was also constructed to serve as a taxonomic reference. Percentage sequence divergence values indicated that P. diclinum and P. torulosum are closely related, but not identical. P. anandrum and P. irregulare also appeared closely related in spite of morphological differences. P. paddicum and Phytophthora cryptogea were the most outlying of the six species of Pythiaceae, but all six species formed a tight cluster when considered against A. pyrifera.

SUMMARY: Biochemical reactions, 0 and K serotypes and presence of P-fimbriae were analysed in 116 Escherichia coli strains isolated in blood cultures from patients with bacteraemia and in 99 faecal strains isolated from healthy individuals. By using biochemical typing, the strains could be grouped into six main clusters with similarity index >0.8 (Gower, 1971) and altogether 16 subclusters with similarity index 0.82-0-89. The most discriminating tests between the clusters were fermentation of D-tagatose, saccharose, salicin and sorbose. No single biochemical property could differentiate bacteraemic isolates from faecal strains, although strains isolated from blood were significantly more often found in certain subclusters, whereas other subclusters contained mainly control strains. Bacteraemic strains possessed P-fimbriae more often, especially strains isolated from patients with E. coli in the urine concomitantly with bacteraemia. Equally, no single reaction could separate P-fimbriated from non-P-fimbriated strains. D-Tagatose was fermented more often by the P-fimbriated strains; on the other hand, melibiose and lactose fermentation tests were less often positive. Certain 0 serotypes (01, 04, 06, 07, 018 and 025) were more common among bacteraemic isolates than controls. K serotypes such as K 1, K5 and K52 were also more frequent among blood isolates. We conclude that a combination of biochemical tests, fimbriation and serotyping might be used to identify potentially pathogenic clusters of E. coli.