- Volume 138, Issue 6, 1992

The phototrophic bacterium Rhodobacter sphaeroides strain Si4 induced ribitol dehydrogenase (EC 1.1.1.56) when grown on ribitol- or xylitol-containing medium. This ribitol dehydrogenase was purified to apparent homogeneity by ammonium sulphate precipitation, affinity chromatography on Procion red, and chromatography on Q-Sepharose. For the native enzyme an isoelectric point of pH 6.1 and an apparent M r of 50000 was determined. SDS-PAGE yielded a single peptide band of M r 25000 suggesting a dimeric enzyme structure. The ribitol dehydrogenase was specific for NAD+ but unspecific as to its polyol substrate. In order of decreasing activity ribitol, xylitol, erythritol, D-glucitol and D-arabitol were oxidized. The pH optimum of substrate oxidation was 10, and that of substrate reduction was 6.5. The equilibrium constant of the interconversion of ribitol to D-ribulose was determined to be 0.33 nM at pH 7.0 and 25 °C. The K m-values determined for ribitol, ribulose, xylitol and NAD+ (in the presence of ribitol) were 6.3, 12.5, 77 and 0.077 mM, respectively. Because of the favourable K m for ribitol, a method for quantitative ribitol determination was elaborated.

This paper describes the metabolism, transport and growth inhibition effects of 2-aminoethylarsonic acid (AEA) and 3-aminopropylarsonic acid (APrA). The former compound supported growth of Pseudomonas aeruginosa, as sole nitrogen source. The two arsonates inhibited the growth of this bacterium when 2-aminoethylphosphonic acid (AEP) but not alanine or NH4Cl, was supplied as the only other nitrogen source. The analogy between AEA and the natural compound AEP led us to examine the in vitro and in vivo interaction of AEA with the enzymes of AEP metabolism. The uptake system for AEP (K m 6 μM) was found to be competitively inhibited by AEA and APrA (K i 18 μM for each). AEP-aminotransferase was found to act on AEA with a K m of 4 mM (3.85 mM for AEP). Alanine and 2-arsonoacetaldehyde were generated concomitantly, in a stoichiometric reaction. In vivo, AEA was catabolized by the AEP-aminotransferase since it was able to first induce this enzyme, then to be an efficient substrate. The lower growth observed may have been due to the slowness with which the permease and the aminotransferase were induced, and hence to a poor supply of alanine by transamination.

Mutants of Actinobacillus actinomycetemcomitans strain Y4 defective in the capsular-like serotype b-specific polysaccharide antigen (SPA) were constructed by inserting the transposon Tn 916. Southern blot analysis suggested that the transposon was inserted into a variety of different sites on the chromosome. Whole cells from two mutants (strains ST1 and ST2) lacked reactivity with a monoclonal antibody to SPA of A. actinomycetemcomitans Y4 (mAb S5) in enzyme-linked immunosorbent assay, but those from another nine mutants (e.g. strains ST3 and ST5) reacted very weakly with mAb S5. Immunodiffusion tests showed that mAb S5 or rabbit antiserum against whole cells of strain Y4 produced a fused precipitin band with purified SPA and autoclaved extract from strain Y4, but no precipitin band with autoclaved extracts from these four mutants. The hydrolysate of autoclaved extract from strain Y4 contained equal amounts of rhamnose and fucose, component sugars of SPA. The hydrolysates of autoclaved extracts from strains ST1 and ST2 contained a trace amount of rhamnose, but not fucose. Those of autoclaved extracts from strains ST3 and ST5 contained a trace amount of fucose, but not rhamnose. All of these SPA-defective mutants reacted with a mAb to lipopolysaccharide of strain Y4. The cell hydrophobicity of SPA-defective mutants was higher than that of the parent strain. These mutant clones will be useful for analysing the gene complex responsible for the synthesis of SPA of A. actinomycetemcomitans and the regulation of expression of the polysaccharide.

The biochemical, immunological, and biological properties of an S layer purified from an Aeromonas hydrophila strain (AH-342) involved in a case of bacteraemia were investigated. The S layer selectively removed from the cell surface was composed of a single acidic (pI 4·65) protein subunit (surface array protein, SAP) with a molecular mass of approximately 52 kDa. Amino acid analysis of this 52 kDa protein indicated a molecule composed of 498 amino acids with 46% hydrophobic residues. No cysteine residues were detected. The first 35 residues of the N-terminus were sequenced by Edman degradation; only 4–24% homology was noted between this sequence and those previously published for SAPs of Aeromonas salmonicida (A450) and a strain of A. hydrophila (TF7) originally isolated from a moribund fish. Polyclonal antibodies raised against AH-342 SAP were genospecific, reacting only against S layers produced by A. hydrophila strains and not those from Aeromonas veronii. Acute serum from the bacteraemic patient from whom AH-342 was isolated reacted strongly with the SAP of AH-342 in immunoblot studies. Purified SAP, when intraperitoneally co-inoculated with SAP− strains of A. hydrophila into Swiss-Webster mice, could reduce the 50% lethal dose by approximately 30–70 fold. The results suggest that the SAP of A. hydrophila strains may play an important role in systemic dissemination after invasion through the gastrointestinal mucosa.

Determination of the cell-surface hydrophobicity of group B streptococci by hydrophobic interaction chromatography on phenyl-Sepharose revealed that human and bovine group B streptococcal isolates with protein surface antigens, either alone or in combination with polysaccharide antigens, were mainly hydrophobic, whereas those with polysaccharide antigens alone were mainly hydrophilic. Removal of capsular neuraminic acid enhanced, and pronase treatment reduced, surface hydrophobicity. The hydrophobic surface proteins, solubilized by mutanolysin treatment of the bacteria and isolated by hydrophobic interaction chromatography, appeared in SDS-PAGE as numerous protein bands. Staphylococcal carrier cells loaded with antibodies produced against hydrophobic surface proteins agglutinated specifically with hydrophobic group B streptococci. No agglutination reaction was observed with hydrophilic cultures. Hydrophobic group B streptococci adhered to buccal epithelial cells in significantly higher numbers than did hydrophilic cultures. The adherence of group B streptococci to epithelial cells was inhibited in the presence of isolated hydrophobic proteins and in the presence of specific antibodies produced against hydrophobic proteins. The results of this study demonstrate a close relation between the occurrence of type-specific antigens, surface hydrophobicity and the adherence of group B streptococci to epithelial cells.

In addition to GvpA, the main structural protein, an SDS-soluble protein has been found in gas vesicles isolated from six different genera of cyanobacteria. N-terminal sequence analysis of the first 30 to 60 residues of the gel-purified proteins showed that they were homologous to GvpC, a protein that strengthens the gas vesicle in Anabaena flos-aquae. The proteins from some of the organisms showed rather low homology, however, and this may explain why the genes that encode them have not been found by Southern hybridization studies. The gas vesicles of another cyanobacterium, Dactylococcopsis salina, contained two SDS-soluble proteins (M r 17000 and 35000) that were identical in sequence for the first 24 residues but not thereafter; these two proteins showed no clear homology to GvpC. The sequence of GvpA, the main structural gas vesicle protein, was very similar in each of the organisms investigated. GvpA from the purple bacterium Amoebobacter pendens was different for the first 8 residues but 51 of the next 56 residues were identical to those of the cyanobacterial GvpA. Analysis of the GvpA and GvpC sequences provides support for the idea that the low diversity of GvpA reflects a high degree of conservation rather than a recent origin followed by lateral gene transfer between different bacteria.

Laccase activity accumulated during rhizomorph development in malt extract cultures of Armillaria mellea. The activity was readily separated into two bands by PAGE under non-denaturing conditions. The less rapidly migrating activity (on PAGE) was purified by ammonium sulphate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme (laccase I) showed a main polypeptide of M r 59000. Activity of the purified enzyme was greatest with 2,6-dimethoxyphenol (DMOP) as substrate. Syringaldazine, p-phenylenediamine and other typical laccase substrates were readily oxidized. No oxidation of tyrosine was detected. The K m for DMOP was 0·178 mM and for p-phenylenediamine was 1·69 mM. The pH optimum for p-phenylenediamine oxidation was 3·5. Electrophoretically purified main polypeptide of laccase I was used to raise a specific antibody. Immunoblot analysis showed that whilst the antibody bound strongly to laccase I, no binding to laccase II was detectable. Antibody raised against pure laccase from Agaricus bisporus reacted with laccase I from Armillaria mellea but not with laccase II. The isolectric pH was 4.1 for laccase I and 3·3 for laccase II.

This paper describes the metabolism, transport and growth inhibition effects of 2-aminoethylarsonic acid (AEA) and 3-aminopropylarsonic acid (APrA). The former compound supported growth of Pseudomonas aeruginosa, as sole nitrogen source. The two arsonates inhibited the growth of this bacterium when 2-aminoethylphosphonic acid (AEP) but not alanine or NH4Cl, was supplied as the only other nitrogen source. The analogy between AEA and the natural compound AEP led us to examine the in vitro and in vivo interaction of AEA with the enzymes of AEP metabolism. The uptake system for AEP (K m 6 μM) was found to be competitively inhibited by AEA and APrA (K i 18 μM for each). AEP-aminotransferase was found to act on AEA with a K m of 4 mM (3.85 mM for AEP). Alanine and 2-arsonoacetaldehyde were generated concomitantly, in a stoichiometric reaction. In vivo, AEA was catabolized by the AEP-aminotransferase since it was able to first induce this enzyme, then to be an efficient substrate. The lower growth observed may have been due to the slowness with which the permease and the aminotransferase were induced, and hence to a poor supply of alanine by transamination.

The role of the major conidial-bound cellulase — cellobiohydrolase II (CBH II) — in the triggering of cellulase formation in the fungus Trichoderma reesei was investigated by comparing the mutant strain QM 9414 with a recombinant strain unable to produce CBH II. For this purpose, the cbh2 gene was isolated from a chromosomal gene bank of T. reesei, cloned into pGEM-7Zf(+), and disrupted by insertion of the homologous pyr4 gene in its coding region to yield the plasmid vector pSB3. Transformation of the auxotrophic, pyr4-negative strain T. reesei TU-6 with pSB3 yielded 23 stable prototrophs, of which three were unable to produce CBH II — assessed by means of a monoclonal antibody — during growth on lactose or in the presence of sophorose. However, they formed cellobiohydrolase I (CBH I) at a rate comparable to strain QM 9414 under these conditions. Southern analysis of DNA of some CBH II− and CBH II+ transformants confirmed that pSB3 had integrated at the cbh2 locus in the CBH II− strains. The latter displayed normal growth on glucose or maltose as carbon source. They showed retarded growth on cellulose as sole carbon source, however, and exhibited a lag in the time course of CBH I and EG I formation, although producing roughly the same final cellulase activities. It is concluded from these results that CBH II is not essential for induction of cellulase formation by cellulose, but that it contributes significantly to the formation of lower molecular mass inducers in the early phase of growth of the fungus on cellulose.

The phototrophic bacterium Rhodobacter sphaeroides strain Si4 induced ribitol dehydrogenase (EC 1.1.1.56) when grown on ribitol- or xylitol-containing medium. This ribitol dehydrogenase was purified to apparent homogeneity by ammonium sulphate precipitation, affinity chromatography on Procion red, and chromatography on Q-Sepharose. For the native enzyme an isoelectric point of pH 6·1 and an apparent M r of 50000 was determined. SDS-PAGE yielded a single peptide band of M r 25000 suggesting a dimeric enzyme structure. The ribitol dehydrogenase was specific for NAD+ but unspecific as to its polyol substrate. In order of decreasing activity ribitol, xylitol, erythritol, D-glucitol and D-arabitol were oxidized. The pH optimum of substrate oxidation was 10, and that of substrate reduction was 6·5. The equilibrium constant of the interconversion of ribitol to D-ribulose was determined to be 0·33 nM at pH 7·0 and 25 °C. The K m-values determined for ribitol, ribulose, xylitol and NAD+ (in the presence of ribitol) were 6·3, 12·5, 77 and 0·077 mM, respectively. Because of the favourable K m for ribitol, a method for quantitative ribitol determination was elaborated.

Spleen cells from mice infected with the rough Brucella melitensis strain B115 were fused with NSO myeloma cells. Hybridoma supernatants were screened in ELISA with cell walls (CW), sonicated cell extracts (CE) and rough lipopolysaccharide (R-LPS) of B. melitensis strain B115 and whole B. melitensis B115 cells. Surprisingly, 22 monoclonal antibodies (mAbs) reacting in ELISA with both CW and CE but not with R-LPS and bacterial cells were shown by immunoblot analysis and ELISA to react with smooth lipopolysaccharide (S-LPS). These mAbs also reacted in ELISA with O polysaccharides (OPS) from the smooth Brucella abortus strain 99 and the smooth B. melitensis strain 16M and thus recognize epitopes present on the O-chain. Proteinase K LPS preparations from B. melitensis B115 analysed by immunoblotting with one mAb (12G12) recognizing S-LPS of both A and M specificity displayed the typical S-LPS high-molecular-mass ladder pattern but no S-LPS was detected in the phenol/water/chloroform/light petroleum LPS preparation of the same strain. mAb 12G12, specific for S-LPS, and a mAb (A68/03F03/D05) specific for R-LPS were used to localize the O-chain and R-LPS expressed in B. melitensis strain B115 by immunoelectron microscopy. Immunogold labelling was observed at the surface of B. melitensis B115 cells with the anti-R-LPS mAb but not with the anti-S-LPS mAb. In ultrathin sections, immunogold labelling with the S-LPS specific mAb was observed in the cytoplasm and in the periphery of the cytoplasm, probably at the cytoplasmic membrane. Immunogold labelling with the R-LPS specific mAb was observed at the outer membrane, outside the cells and on R-LPS vesicles. These results indicate that O-chain expressed in the rough B. melitensis strain B115 is immunogenic in mice, not exposed at the cell surface but present in the cytoplasm and most probably at the cytoplasmic membrane.

A species-specific monoclonal IgM antibody (mAb) 9BF8 directed against the major outer membrane protein (MOMP) of Chlamydia trachomatis neutralized several chlamydial serovars in a complement-independent manner. The presence of Mg2+ ions negated the neutralization in serovars F, L1 and L2, but not in serovars A, B, E, D and K. The ability of monovalent Fab-fragments of this mAb to neutralize chlamydial infectivity in a Mg-independent manner suggested that conformational alterations on the chlamydial surface induced by the cation hindered the IgM but allowed the smaller Fab fragment access to its epitope. In order to determine the chlamydial component that binds Mg, elementary bodies (EB) of serovars E and L1 were treated with EDTA at pHs 8 and 9. The infectivity of the treated EB and the amount of released LPS were determined. Only after EDTA treatment at pH 9, as the LPS release increased, did the binding of the mAb on the chlamydial surface become Mg-independent. The infectivity of the EB was almost completely lost after such a treatment. These results suggest that the chlamydial LPS has the potential to modulate the exposure of antigenic sites on the MOMP, when it is cross-linked by Mg2+. They further imply that serovars protected by Mg and those that are not differ in the surface topology of one particular MOMP epitope, but are antigenically very similar. This difference might be of considerable importance in vivo.

The upstream region of the N-acetylmuramoyl-L-alanine amidase gene (cwlB; a major Bacillus subtilis autolysin) was cloned into Escherichia coli by chromosome walking. Sequencing of the region showed the presence of two open reading frames, one (designated as cwbA) which starts at a UUG codon and encodes a polypeptide of 705 amino acids with an M r of 76725, and the other (designated as lppX), upstream of cwbA, comprising 102 amino acids and having a signal sequence characteristic of a lipoprotein. Purification of the CwbA protein and determination of its N-terminal amino acid sequence revealed that it contains a presumed signal peptide which is processed after Ala at position 25 from the N-terminal, and that the M r of the mature form is 75000. The amino acid sequences of the N-terminal and C-terminal regions of CwbA were found to be highly homologous with those of the cell wall binding domain of CwlB and the spoIID gene product, respectively. CwbA stimulated the major autolysin activity approximately threefold in vitro. These data indicate that CwbA is the modifier protein of the major autolysin reported by Herbold, D. R. & Glaser, L. (1975; Journal of Biological Chemistry 250, 1676–1682). In-frame fusion between the lppX and lacZ genes demonstrated that lppX is translated in vivo and expressed during the exponential growth phase.

To investigate the relationship between DNA content and cell volume, we have attempted to repeat the construction of stable Bacillus subtilis diploid cells through protoplast fusion. Colonies with a biparental phenotype and those with a prototrophic phenotype were identified among exfusants of a cross between two polyauxotrophic strains. The ploidy of cells constituting such colonies was assessed by protoplast self-fusion, determination of the DNA to dry weight ratio of exponentially growing cells, and by quantitative DNA-DNA hybridization. Within the precision of these methods, all colonies were found to consist of haploid cells. A previously described non-complementing diploid was also found to be haploid. Therefore, the genetic evidence in favour of diploidy, based on continuing segregation of cells with a parental or recombinant phenotype, cannot be accounted for except by the maintenance of such cells as a minority population in mixed colonies through cross-feeding. Reconstruction experiments with mixtures of whole parental cells confirm that biparental colonies are indeed mixed colonies which arise either by sticking of parental cells or through coincidence, i.e. their plating within a distance of about 0·4 mm. The previously reported experimental results can be accounted for in the light of our results.