- Volume 130, Issue 9, 1984

The phosphoenolpyruvate-dependent glucose phosphotransferase system (PTS) of Clostridium pasteurianum was studied in toluene-treated cells and cell-free extracts. Toluene-treated cells phosphorylated the non-metabolizable glucose analogue methyl α-glucoside in a PEP-dependent manner, and exhibited apparent K m values of 93 μm and 12 μm, respectively, for the substrates PEP and methyl α-glucoside. Using cell-free extracts, it was shown that the system consists of both soluble and membrane-bound components. The rate of methyl α-glucoside phosphorylation was dependent on the concentration of both the soluble and membrane fractions, and was independent of the concentration of PEP. Saturation kinetics for PEP were restored in the presence of a partially purified soluble fraction, which appeared to contain a single phosphotransferase component. This was similar to HPr of Escherichia coli by the following criteria:(i) it eluted in the same position from a Sephadex G-100 gel filtration column; (ii)it was relatively resistant to exposure to temperatures up to 100 °C; (iii) it was involved in phosphorylation of a number of sugars which enter the cell via different phosphotransferase systems; (iv) the kinetics of methyl α-glucoside phosphorylation required the soluble protein in C. pasteurianum to be a substrate rather than an enzyme.

The o-type cytochrome oxidase from methanol-grown Methylophilus methylotrophus has been solubilized and purified (15-fold) to homogeneity. The pure, active oxidase consisted of equal amounts of b-type and c-type cytochromes, corresponding to the two types of protein subunit seen on SDS-PAGE; these had molecular weights of 31 500 and 23800, respectively. The active oxidase probably has two cytochrome c subunits and two cytochrome b subunits, both cytochrome types reacting with CO. The cytochrome c subunit did not correspond to either of the two soluble cytochromes c from M. methylotrophus. The pure oxidase complex oxidized TMPD very rapidly, having a V max of 36μmol O2 min−1 (mg protein)−1. It was inhibited noncompetitively by azide (K i, 1.1μM) and KCN (K i, 0.2 μM), the K i values being similar to thoemeasured during respiration by whole bacteria.

Of the two soluble cytochromes c from M. methylotrophus, cytochrome c H was oxidized at 50 times the rate of cytochrome c 1, the turnover number with cytochrome c H as substrate being 21 s−1. The cytochrome c component of the oxidase was unable to act as electron acceptor from pure methanol dehydrogenase (soluble, or after solubilization from membranes). A complete ‘methanol oxidase’ electron transport chain was reconstituted from completely pure proteins: methanol dehydrogenase, cytochrome c 1 and c H and the cytochrome c oxidase complex. This ‘methanol oxidase’ showed the same sensitivity to inhibition as observed during methanol oxidation by whole bacteria.

N-Acetylglucosaminidase was induced by either N-acetylglucosamine or N-acetylmannosamine in several strains of Candida albicans. Enzyme activity was not induced in a N-acetylglucosamine non-utilizing mutant which is unable to express the first three steps in the N-acetylglucosamine catabolic pathway. The enzyme, purified 500-fold, had a specific activity of 36·8 units (mg protein)−1 and catalysed the hydrolysis of p-nitrophenyl-β-n-acetylglucosamine, N,N′-diacetylchitobiose and N,NN′,N”-triacetylchitotriose. No activity was observed toward colloidal chitin, hyaluronic acid or mucin. The cellular distribution of N-acetylglucosaminidase was determined by measuring in situ enzyme activity before and after acid treatment of intact cells. N-Acetylglucosaminidase (80–88% of the total cellular activity) was rapidly secreted to the periplasm when the enzyme was induced either during yeast growth at 28 °C or germ-tube formation at 37 °C. Export of the enzyme from the periplasm into the medium was fourfold greater during germ-tube formation, and after 6 h incubation the amount of enzyme released into the medium represented 70% of cell-associated enzyme activity.

Screening of non-phage group II Staphylococcus aureus strains for antagonistic substances revealed one particular strain, S. aureus D91, to excrete a substance with a wide spectrum of activity; both Gram-positive and Gram-negative bacteria were susceptible. The staphylococcin-like substance D91 produced by this strain was partially purified by column chromatography on Sephadex G-50, DEAE-cellulose, Phenyl Sepharose CL-4B and Sephadex G-200. A molecular weight of 76000 was estimated by gel filtration. The activity was heat sensitive but was not affected by hydrolytic enzymes except for pronase. The protein character of substance D91 was confirmed by gel electrophoresis and subsequent staining with Coomassie blue. The action exerted on sensitive bacteria was bacteriostatic rather than bactericidal. Biosynthesis of DNA, RNA, protein and polysaccharides were inhibited simultaneously in both Escherichia coli and Staphylococcus aureus. Active transport of glutamic acid was stopped in both S. cohnii and E. coli, whereas glucose uptake was inhibited in E. coli only. The substance induced a slow efflux of 86Rb+ from proloaded cells of S. cohnii and E. coli. The antagonistic activity of S. aureus D91 was eliminated by ethidium bromide at a rate of 47.6% suggesting that plasmids may be involved in its production.

Leishmania mexicana mexicana amastigote and promastigote soluble proteinases were purified using gel filtration and ion exchange chromatography. For the amastigotes, two main proteinase activity peaks were separated with both methods. These accounted for approximately 10% and 90% of the total activity. Characterization of the two activities for substrate specificity and sensitivity to inhibitors indicated that the major peak from both column methods contained enzymes with the characteristics of cysteine proteinases. SDS-polyacrylamide gel electrophoresis of the enzyme from the major peak purified by gel filtration revealed one polypeptide with a molecular weight in the region of 31000. In contrast, the activity of the minor peak eluted from the columns was of higher molecular weight (67000) and was similar to metalloproteinases. Purification of the soluble proteinases in the promastigote of L. m. mexicana produced only one activity peak from both column techniques. This activity (mol. wt 67000) corresponded to the high molecular weight proteinase of the amastigote. The purified proteinases were active on 4-nitroanilide and 7-amino-4-methylcoumarin derivatives of various small peptides. The high molecular weight proteinases of both amastigotes and promastigotes were similarly active against most of the peptides, suggesting a low specificity of the enzymes. In contrast, the low molecular weight amastigote proteinases were particularly active against two of the substrates, namely BZ-Pro-Phe-Arg-Nan and Z-Phe-Arg-MCA. These results indicate that a highly active, substrate-specific, soluble proteinase, with characteristics of a cysteine proteinase, is produced upon transformation of the L. m. mexicana promastigote to amastigote. The discovery and characterization of this enzyme offers opportunities for the development of new antileishmanial agents.

Glutamate dehydrogenase (l-glutamate: NAD+ oxidoreductase (deaminating); EC 1.4.1.2) has been purified from Peptostreptococcus asaccharolyticus in a single step using dye-ligand chromatography. The enzyme (GDH) was present in high yields and was stabilized in crude extracts. A subunit molecular weight of 49000 ±500 was determined by SDS polyacrylamide gel electrophoresis and six bands were obtained after cross-linking the subunits with dimethyl suberimidate. This bacterial GDH was predominantly NAD+-linked, but was able to utilize both NADP+ and NADPH at 4% of the rates with NAD+ and NADH, respectively. An investigation of the amino acid specificity revealed some similarities with GDH from mammalian sources and some clear differences. The values of apparent K m for the substrates ammonia, 2-oxoglutarate, NADH, NAD+ and glutamate were 18·4, 0·82, 0·066, 0·031 and 6 mm, respectively. The P. asaccharolyticus GDH was not regulated by purine nucleotides, but was subject to strong inhibition with increasing ionic strength.

β-N-Acetylglucosaminidase has been purified from the walls of Bacillus subtilis 168 and compared with the other known autolysin, N-acetylmuramyl-l-alanine amidase (amidase). The β-N-acetylglucosaminidase was a dimer in LiCl buffers with a sub-unit molecular weight of 90000 and a pH optimum of about 5·0. It was very sensitive to proteolytic enzymes and was critically activated by 0·1 to 0·2 m-LiCl. It was insoluble in concentrations of LiCl lower than 0·05 to 0·1 m. It was less strongly bound to walls than was the amidase, which was a monomer of molecular weight 30000 to 40000 in LiCl buffers. The βN-acetylglucosaminidase is an endoenzyme and showed no exo-activity. Lysozyme-like enzyme (muramidase) activity was undetectable in the wall extracts examined.

Small (3.5 1) laboratory microcosms containing water, sediment and Elodea canadensis were treated with the herbicides diquat and terbutryne. After treatment total numbers of planktonic bacteria increased significantly by 3-11-fold and heterotroph counts also rose significantly, by 3-23-fold but the population increases of exoenzyme-producing bacteria (3-113-fold) were not always significant. As the plants stopped photosynthesizing and decomposed, pH and dissolved oxygen concentrations declined, whilst alkalinity and free CO2 concentrations increased. All these changes were very similar to results from analogous experiments in natural waters and microcosms are recommended as substitutes for field experiments with aquatic herbicides.

A cryptic plasmid, pBL1 of 4·3 kb, has been found in lysine-producing Brevibacterium lactofermentum strains BL0, BL70, BL74 and BL77. pBL1 had single restriction sites for BalI, BclI, HaeII, HindIII and HpaI. It had four sites for AvaI, seven for HaeIII, eight for MboI and a very large number for AluI, but no sites were found for PstI, EcoRI or BamHI. The estimated copy number was 30. Three different pBL1-pBR322 hybrids named pUL1, pUL10 and pUL20 were constructed. Transposon Tn5 was inserted by transposition into either the pBR322 or the pBL1 components of plasmid pUL1, pUL10 and pUL20. A shuttle vector able to replicate in Escherichia coli, Streptomyces lividans and B. lactofermentum was constructed by cloning pBL1 into the plasmid pIJ860, a bifunctional E. coli-S. lividans vector carrying the tsr, bla and kan genes. A polyethylene glycol-assisted transformation system for B. lactofermentum protoplasts was developed. Transformation frequencies of 102 transformants (µg DNA)−1 were obtained. The kan resistance gene from Tn5 was expressed very efficiently in B. lactofermentum (up to 200 µg ml−1). A smaller plasmid, pUL62, was constructed in which the tsr (thiostrepton resistance) gene of pUL61 was deleted.

Treatment of Escherichia coli with bleomycin induced a dramatic increase in ATP concentration in the first 30 min. Afterwards, in RecA+ strains, ATP dropped quickly to values similar to those of untreated cells. Mutants of E. coli defective in either RecA protein or RecA protease activity did not show this decrease, indicating that it was due to the action of RecA protease. The increase in ATP in the first 30 min was dependent on RecBC exonuclease activity and must have been due to substrate level phosphorylation, since an uncoupler such as dinitrophenol did not affect it. Nevertheless, mitomycin C did not induce any change in ATP pools of RecA+ strains, at least during 120 min following treatment. The implications of these findings are discussed in relation to the possible pathways of activation of RecA protease.

DNA in the terminal region of the chromosome of Bacillus subtilis was labelled by a procedure in which cells in sporulation inducing conditions were pulse-labelled with [3H]thymidine and then treated with p-hydroxyphenylazouracil, an inhibitor of DNA synthesis. The labelled DNA in isolated spores yielded a small number of restriction fragments. About 14 EcoRI fragments with a total length of 80 kb were labelled in a 2·5 min pulse. A fragment of 4·0 kb had the highest specific radioactivity in terminally labelled DNA from several strains. One of these strains lacked the 120 kb prophage of SPβ that is normally integrated close to the terminus. Loss of the 120 kb prophage did not affect the point of termination which must therefore be regarded as a specific ‘stop’ sequence. Labelled terminus DNA has been used to identify lambda (Charon 4A) clones containing sequences derived from the terminal region. The total length of the restriction fragments present was 150 kb and adds another 90 kb to the 150 kb region mapped previously. Only one group of these sequences was present in a B. subtilis strain (CU1695) carrying a deletion spanning from SPβ to the right of the terminus and gltA. This suggests that the terminator sequence found in the wild-type can be deleted but presumably this strain has an alternative mechanism of termination.

The kinetics of mouse salmonellosis caused by Salmonella typhimurium was studied in mice preinjected with the IgM or IgG fraction prepared from a rabbit anti-Salmonella serum. Compared on the basis of antibody units determined by an enzyme immunoassay, IgM was ten times more effective than IgG in promoting removal of the bacteria from blood after intravenous (IV) injection and their uptake in the reticuloendothelial system (RES). The subsequent killing of the bacteria was, however, only minor, in accord with the negligible protective effect of serum antibodies in IV infection. IgM was over 1000 times more effective than IgG in promoting killing of the bacteria after intraperitoneal (IP) challenge. Neither antibody had an effect on the multiplication of the bacteria in the RES. The protective action of antibody was thus almost entirely mediated by peritoneal-cavity cells acting in the very early phase of infection. The greater effect of IgM is suggested to be a special feature of Salmonella infections, connected with the capacity of these bacteria for intracellular survival and multiplication in the RES.

Two murine monoclonal antibodies of the IgG3 class have been isolated after immunization with Brucella abortus. An indirect immunofluorescence test was used to screen hybridoma supernatants and subsequently to determine the cross-reactivity of the monoclonal antibodies with other bacteria. One monoclonal antibody reacted with all the smooth Brucella biotypes tried and with Yersinia enterocolitica serogroup O:9, though not with rough Br. ovis or with strains of Escherichia, Proteus, Salmonella, Pseudomonas, Francisella and Bordetella. The other monoclonal antibody displayed a high degree of specificity for brucellae carrying the A lipopolysaccharide-protein surface antigen. The implications for the diagnosis of brucellosis are discussed.

The serotype-specific capsular polysaccharide from two strains of Pasteurella haemolytica serotype A1 organisms was purified and characterized by chemical analysis and NMR spectroscopy. The polymer has the structure →3)-O-(2-acetamido-2-deoxy-4-O-acetyl-D-mannopyranosyluronic acid)-(1→4)-O-(2-acetamido-2-deoxy-d-mannopyranose)-(1→. The polysaccharide was immunogenic (able to evoke production of antibodies) for sheep but not for rabbits. Immuno electron-microscopy studies using the Protein A-gold technique showed the polysaccharide to be peripherally located on the bacterial surface. Reduction, oxidation and de-O-acetylation of the polymer did not appear to alter its immunological precipitability with specific antiserum, but all three treatments destroyed its ability to adhere to sheep erythrocytes at neutral pH. De-N-acetylation of the polymer destroyed both immunological precipitability and erythrocyte adherence.

A procedure has been developed for rapidly inoculating large numbers of turnip seedlings with Xanthomonas campestris pv. campestris, the causal agent of black rot of crucifers. Following mutagenesis bacterial mutants were isolated which showed altered pathogenicity. The mutants gave a range of responses on plants and some were impaired in their ability to grow in seedlings.