- Volume 134, Issue 10, 1988

The utilization of exogenous nicotinamide adenine dinucleotide (NAD) by Haemophilus parainfluenzae was studied in suspensions of whole cells using radiolabelled NAD, nicotinamide mononucleotide (NMN), and nicotinamide ribonucleoside (NR). The utilization of these compounds by H. parainfluenzae has the following characteristics. (1) NAD is not taken up intact, but rather is degraded to NMN or NR prior to internalization. (2) Uptake is carrier-mediated and energy-dependent with saturation kinetics. (3) There is specificity for the β-configuration of the glycopyridine linkage. (4) An intact carboxamide group is required on the pyridine ring. The intracellular metabolism of NAD was studied in crude cell extracts and in whole cells using carbonyl-14C-labelled NR, NMN, NAD, nicotinamide, and nicotinic acid as substrates in separate experiments. A synthetic pathway from NR through NMN to NAD that requires Mg2+ and ATP was demonstrated. Nicotinamide was found as an end-product of NAD degradation. Nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide were not found as intermediates. The NAD synthetic pathway in H. parainfluenzae differs from the Preiss-Handler pathway and the pyridine nucleotide cycles described in other bacteria.

Summary: The effects of l- and d-methionine on production of the gaseous secondary metabolite chloromethane (CH3C1) by the white-rot fungus Phellinus pomaceus were investigated. Although d-methionine stimulated CH3C1 biosynthesis, the l-isomer either failed to affect or depressed production depending on the concentration. Nevertheless, very high levels of incorporation of label (~80%) from l-[methyl-2H3]methionine into CH3C1 could be achieved demonstrating that the amino acid is a highly effective precursor of CH3C1. Experiments using labelled d-methionine showed substantial but lower incorporation. Patterns of incorporation of label from both isomers into methyl benzoate, another important volatile fungal metabolite, were very similar to those observed with CH3C1. This behaviour is only explicable in terms of either (a) the utilization of the same enzyme system requiring an identical methyl donor for methylation of both chloride and benzoate ions or (b) the presence of a transmethylation system. The incorporation of label from l-[methyl-2H3]methionine into methyl 2-furoate was similar to that of methyl benzoate implying that the same methylating system was used for esterification of both 2-furoic and benzoic acids. However, the incorporation pattern for methyl salicylate was different and suggests that methyl salicylate is probably formed by o-hydroxylation of methyl benzoate.

We screened 63 clinical isolates of Bacteroides gingivalis from eight different laboratories for the presence of fimbriae by negative staining and by immunological methods. Techniques used were bacterial agglutination, Ouchterlony immunodiffusion and Western immunoblotting analysis using rabbit anti-fimbriae and anti-fimbrilin sera raised against fimbriae and fimbrilin (a constituent protein of B. gingivalis fimbriae) from B. gingivalis strain 381. In 49 of the 51 strains tested, fimbriae were clearly detected by negative staining, and 30 (60%) of the fimbriate strains were positive in all three of the immunological assays. A total of 37 strains (75%) were positive by immunoblotting analysis, which was the most reliable of the serological methods used in this study. The study shows that the majority of B. gingivalis strains are fimbriate, and that these fimbriae are immunologically related to the fimbriae of B. gingivalis strain 381. Molecular heterogeneity of fimbrilin was discovered by the immunoblotting analysis, when different strains were compared. With most of the strains, including strain 381, the antifimbrilin serum reacted with a protein of apparent molecular mass 43 kDa, but with 15 strains the immuno-reactive protein had an apparent molecular mass of 46 kDa.

The ability of 13 amino-group-modified and 12 carboxyl-group-modified l-alanine derivatives to initiate germination of Bacillus subtilis spores was examined. The ‘relative affinity’, defined as the ratio of the concentration of l-alanine required to give 20% of the maximal germination rate to the concentration of analogue required to give this same rate, was used to estimate the effectiveness of the analogues as germinants. l-Ala-l-Ala had a relative affinity of only 1/4200. For most of the l-alanyl dipeptides studied (with the exception of l-Ala-l-Pro and l-Pro-l-Ala), their effectiveness as germinants was lower if the second amino acid was substituted in the carboxyl group rather than the amino group of l-alanine. N-(2-Methylsulphonyl)ethyloxy-carbonyl-l-Ala was a good germinant (relative affinity 1/100), indicating that there may be an additional complementary site for this group in the germinant receptor field on the spore. Compounds with a shorter chain, N-t-butoxycarbonyl-l-Ala and N-acetyl-l-Ala, showed lower affinities, and compounds with an aromatic ring were very poor germinants. Replacement of the carboxyl group of l-alanine by an alcohol group caused a total loss of germinant activity, and its modification by conversion to an amide group or by substitution of amino acids resulted in low affinity and germination rates. Esterified l-alanine derivatives retained high activity, suggesting that retaining an electronegative group was important. The stimulatory effect of glucose on germination was very marked for compounds with a hydrophobic substituent in either the amino or the carboxyl group, such as l-Leu-l-Ala, l-Ala-l-Leu, N-carbobenzoxy-l-Ala and N-dinitrophenyl-l-Ala. Overall, it can be deduced that both -NH- and -COO- groups, separated by 1 or 2 interatomic distances, are important for germinant activity. Substituents in the amino group were tolerated better than those in the carboxyl group.

The genetic diversity of symbiosis (Sym) plasmids was investigated in samples from two field populations of Rhizobium leguminosarum biovar viceae that had previously been characterized for chromosomally-encoded enzyme electrophoretic polymorphism. Five overlapping cloned DNA fragments from the Sym plasmid pRL1JI were used as hybridization probes to identify restriction fragment variation in the homologous genes of the isolates. In addition, a clone of the β-galactosidase gene region was used as a probe, extending the data on chromosomal relatedness. The plasmid-encoded Sym region was very polymorphic (11·4% average DNA sequence divergence). Some isolates had the same Sym fragment pattern as pRL1JI, but most were very different. One was closely similar to pRL6JI, another widely-studied viceae plasmid. The distribution of plasmids across chromosomal backgrounds was far from random, as though the species were subdivided into compartments with largely separate plasmid pools. Nevertheless, indistinguishable plasmids were found in quite different genetic backgrounds, implying that plasmid transfer must occur in the field. This has implications for the population genetics and evolution of bacteria and for the release of genetically engineered strains.

The Enzyme IIfru of the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system (PTS), which catalyses the uptake of fructose and its concomitant phosphorylation to fructose 1-phosphate by Escherichia coli, is specified by a gene designated fruA. The nucleotide sequence of a 2·5 kb Pvull restriction fragment spanning fruA +, cloned on a plasmid, was determined. This fragment contained three open reading frames (ORFs) but only one complete ORF, 1689 base pairs long, which was preceded by a well-defined Shine-Dalgarno sequence and ended with a rho-independent transcription terminator. The amino acid sequence deduced from this DNA corresponds to that of a protein of 563 amino acids (57·5 kDa), which has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0·40) and which is characterized by a number of well-marked hydrophobic loops that may correspond to membrane-spanning regions. There is relatively little overall homology between this protein and those of other Enzymes II of the PTS but there is considerable correspondence between the region surrounding one of the six histidine residues (His381) of Enzyme IIfru and those surrounding the particular histidines of other Enzymes II, and of HPr, known to be involved in phosphorylation. A plasmid carrying the complete fruA + nucleotide sequence, but not that of any other functional protein, fully restored the ability of fruA mutants to grow on fructose and of extracts of fruA mutants to phosphorylate fructose, which confirms that the nucleotide sequence determined specifies Enzyme IIfru.

The regulated meta pathway operon for the catabolism of salicylate on the naphthalene plasmid pWW60-22 was cloned into the broad-host-range vector pKT230 on a 17·5 kbp BamHI fragment. The recombinant plasmid conferred the ability to grow on salicylate when mobilized into plasmid-free Pseudomonas putida PaW130. A detailed restriction map of the insert was derived and the locations of some of the genes were determined by subcloning and assaying for their gene products in Escherichia coli and P. putida hosts. The existence of a regulatory gene was demonstrated by the induction of enzyme activities in the presence of salicylate. DNA-DNA hybridization indicated a high degree of structural homology between the pWW60-22 operon and the analogous meta pathway operon on TOL plasmid pWW53-4. The data are consistent with the structural genes being arranged in an identical linear array and suggest an evolutionary link between the two catabolic systems.

Transposon mutagenesis was used to isolate two Escherichia coli mutants which express very large amounts of haemolysin when carrying the multicopy plasmid pANN202-312. E. coli strain Hha-2 was isolated by Mud1 mutagenesis, and strain Hha-3 by Tn5 mutagenesis. The transposon insertion was chromosomal in both mutants and could be demonstrated to be unrelated to the haemolytic region of the plasmid. The substantial increase in both extracellular and intracellular haemolysin production was dependent upon plasmid copy number and was drastically reduced when either mutant carried the low-copy-number haemolytic plasmid pHly152. In both mutants, the marked increase in extracellular production was dependent upon the specific haemolysin transport genes, hlyB and hlyD. The lack of either gene function resulted in no external haemolysin production. SDS-PAGE analysis showed no change in the pattern of outer-membrane proteins of the mutants, although changes (differing between the two mutants) were seen in their periplasmic proteins. The mutations of both strains (termed hha-2 and hha-3) were mapped at minute 10·5 of the E. coli chromosome. No relation to any known gene affecting gene regulation in E. coli could be found.

Nonbactericidal monoclonal antibodies (MAbs) directed against gonococcal surface antigens were examined for their effect on complement-mediated bactericidal killing by other MAbs and normal human serum. One MAb, SM73, directed against the H.8 antigen activated complement only moderately well and had little influence on bactericidal antibodies. Two antibodies directed against an epitope on protein III had very different effects. Antibody SM51 activated complement poorly and had no effect on bactericidal killing, whereas antibody SM50, although itself nonbactericidal, activated complement and blocked the bactericidal effect of other antibodies. The extent of the blocking ability of MAb SM50 was studied using MAbs of different specificities as well as polyclonal antisera raised aginst gonococcal surface antigens. Antibody SM50 blocked IgG MAbs of all specificities, but several MAbs of the IgM class retained their bactericidal effect. Each of these IgM MAbs reacted with lipopolysaccharide, but with different epitopes.

Microcycle conidiation in shaken cultures of Penicillium cyclopium M 79 was induced at 24°C without any shock treatment. The occurrence of a microcycle depended on the presence of an organic acid (especially glutamic acid) in combination with glucose, low phosphate concentration, light and sufficient aeration. Absence of glucose and/or lowered aeration evoked vegetative growth. A synchronous and homogeneous microcycle required a certain relationship between the number of inoculated conidia and the concentration of the organic acid in the medium; the optimum was at 0·08 nmol acid per conidium. Higher values stimulated normal vegetative growth. A shortage or absence of the organic acid led to an atypical growth. The effect of organic acid can be partially replaced by addition of 2% (w/v) CaCO3. Addition of NH+ 4 in concentrations higher than 6 mm to cultures with glutamate or glutamine evoked vegetative growth.

Chemotaxis of Rhizobium leguminosarum biovar phaseoli RP8002 towards a range of carbohydrates, phenolic compounds and flavonoids was assayed. Xylose (peak response 10−4 M), sucrose (peak response 10−6 M) and raffinose (peak response 10−5 M) were strong chemoattractants amongst the carbohydrates, whilst glucose, fructose, galactose and maltose produced little or no detectable response. Of the monocyclic phenolic compounds, vanillyl alcohol, p-hydroxybenzoic acid (both peak responses 10−6 M) and 3,4-dihydroxybenzoic acid (peak response 10−4 M) all evoked strong chemotactic responses. Amongst the nod-inducing flavonoids, apigenin and luteolin were both strong chemoattractants (peaks at 10−5 M) while naringenin produced a very low response. Competition experiments suggest that apigenin and luteolin are recognized by a common receptor, but that there exists a separate receptor for luteolin alone. The inhibitors of nod-induction, umbelliferone and acetosyringone, both produced strong chemotactic responses, with peaks at 10−3 M and 10−2 M respectively. This evidence is indicative of a role for chemotaxis towards nod-inducing flavonoids in the initiation of root nodule formation by rhizobia, and also suggests that chemotaxis may influence the host range of the interaction.

Sulphite inhibited growth of all four yeasts studied, Zygosaccharomyces bailii NCYC 563 being most sensitive and Saccharomyces cerevisiae NCYC 431 the least. Vertical Woolf-Eadie plots were obtained for initial velocities of 35S accumulation by all four yeasts suspended in high concentrations of sulphite. Equilibrium levels of 35S accumulation were reached somewhat faster with strains of S. cerevisiae than with those of Z. bailii. With all four yeasts, the greater the extent of 35S accumulation, the larger was the decline in internal pH value. Growth of S. cerevisiae TC8 and Z. bailii NCYC 563, but to a lesser extent of S. cerevisiae NCYC 431 and Z. bailii NCYC 1427, was inhibited when mid exponential-phase cultures were supplemented with 1·0 or 2·0 mm-sulphite, the decrease in growth being accompanied by a decline in ethanol production. Unless growth was completely inhibited, the sulphite-induced decline in growth was accompanied by production of acetaldehyde and additional glycerol.

The biochemical properties of 39 strains of Haemophilus avium from chickens were determined. All the strains produced acid from fructose, galactose, glucose and mannose but not from lactose. Variable reactions were found for arabinose, maltose, mannitol, sorbitol, trehalose and xylose. No strains showed urease activity or produced indole, while β-galactosidase and/or ornithine decarboxylase activity was present in some strains. This variability allowed the recognition of 15 biochemical biovars including some not previously recognized in H. avium. Only 25 (64%) of the H. avium strains could be assigned to the three species (Pasteurella avium, P. volantium and Pasteurella species A) recently proposed to replace H. avium.