- Volume 135, Issue 11, 1989

The cell walls of Brevibacterium casei NCDO 2048, B. epidermidis NCDO 2286 and 14 B. linens isolates from cheese were shown to contain teichoic acids as the dominant non-peptidoglycan polymers. The variation in teichoic acid composition was similar to that described earlier for B. linens strains and for B. iodinum. In B. casei NCDO 2048 a glycerol teichoic acid was found, whereas in B. epidermidis NCDO 2286 glycerol teichoic acid existed together with mannitol teichoic acid. The occurrence of teichoic acids in the cell walls of all Brevibacterium strains investigated constitutes a biochemical characteristic of the genus and is of chemotaxonomic relevance. The co-existence of glycerol and mannitol within the cell walls of the B. linens strains AC480 and AC577 was investigated in detail. The polyols belonged to separate polymers: a glycerol teichoic acid and a mannitol teichoic acid.

The presence of glutathione transferase (GST; EC 2.5.1.18) in Escherichia coli ATCC 25922, E. coli ATCC 25422, Proteus vulgaris ATCC 8427, Pseudomonas aeruginosa ATCC 27853, Klebsiella oxytoca CIP 666, K. oxytoca AF 101, Enterobacter cloacae CIP 6085, Serratia marcescens CIP 6755, and Proteus mirabilis AF 2924 was investigated. Using 1-chloro-2,4-dinitrobenzene as substrate, GST activity was found in the glutathione- (GSH-) affinity-purified fraction of all strains tested. SDS-PAGE analysis of GSH-affinity-purified enzyme indicated that the GSTs of all these bacteria are dimers of two identical subunits of M r about 22500. Rabbit antiserum directed against the major isoenzyme present in Proteus mirabilis AF 2924, Pm-GST-6·0, was used to investigate the antigenic properties of bacterial GSTs. Western blot analysis indicated that a GST antigenically identical to Pm-GST-6·0 is present in Enterobacter cloacae CIP 6085, Escherichia coli ATCC 25422 and Proteus vulgaris ATCC 8427, but absent in Escherichia coli ATCC 25922, Klebsiella oxytoca CIP 666, K. oxytoca AF 101 and Serratia marcescens CIP 6755. The presence of Pm-GST-6·0, but not mammalian GST, increased the MIC values of amikacin, ampicillin, cefotaxime, cephalothin and nalidixic acid for E. coli ATCC 25922. It is suggested that bacterial GST may represent a defence against the effects of antibiotics.

Two new lectins were isolated from mycelium of Rhizoctonia crocorum and Athelia rolfsii by affinity chromatography on mucin-Sepharose. The Rhizoctonia crocorum lectin (RCL) was a tetramer of 11 kDa subunits whereas the Athelia rolfsii lectin (ARL) was a dimer of two 17 kDa subunits. Both lectins were rich in asparagine/aspartic acid, glutamine/glutamic acid, glycine, lysine and serine. In contrast to the previously described Rhizoctonia solani agglutinin (RSA), which exhibits specificity towards N-acetylgalactosamine, RCL and ARL had a complex specificity. Despite obvious differences between the three lectins, they are serologically related to each other.

Coat proteins from mature spores of two enterotoxin-positive (Ent+) and two enterotoxin-negative (Ent+) strains of Clostridium perfringens were solubilized using 50 mm-dithiothreitol and 1% sodium dodecyl sulphate at pH 9·7, and alkylated using 110 mm-iodoacetamide to prevent aggregation. The coat proteins and C. perfringens type A enterotoxin (CPE) were separated by SDS-PAGE and analysed by Western blotting using anti-CPE antibody. As previously reported, CPE aggregated in the presence of SDS, but no aggregation occurred at concentrations below 15 μg CPE ml−. Two CPE-related proteins (34 and 48 kDa) were found in the solubilized spore coat protein of Ent+ strains while only the 48 kDa CPE-related protein was found in the spore coat fraction of Ent+ strains. CPE-related proteins comprised 2·7% and 0·8% of the total solubilized coat protein of Ent+ and Ent+ strains respectively. CPE-related proteins could be extracted from the spores with 1 % SDS alone. They could also be released by disruption of whole spores, indicating that the CPE-related proteins may be in the spore core or trapped between the core and coat layers. The results suggest that CPE is not a major structural component of the coat fraction of C. perfringens spores.

Tn502mer differs in size and restriction map from the well-characterized Tn501mer. It also differs in its preferential and high-frequency insertion into the 6 kb PstI-C region of RP1. The affinity for this region is perpetuated in pVS76, a clone of RP1 PstI-C in pBR322. Restriction mapping of independent pVS76::Tn502 derivatives revealed that Tn502 inserted at the same site (or small region) in PstI-C corresponding to the 35 kb coordinate in RP1. Insertion occurred in both orientations, but one was preferred. When PstI-C was deleted from RP1, acquisition of Tn502 was reduced and the sites of insertion randomized.

After EMS mutagenesis, mutants of Methylobacterium organophilum DSM 760 unable to synthesize pyrroloquinoline quinone (PQQ) were selected among mutants which did not utilize methanol but were still able to use methylamine as growth substrate. Six different pqq genes (pqqA to pqqF) were identified by complementation analysis. The genes pqqA to pqqD, cloned in a single R′ plasmid, were grouped in a 3·9 kb DNA fragment. The genes pqqA and pqqB belonged to a single transcription unit independent from the adjacent gene pqqC. The gene pqqD was contained in a short DNA segment of approximately 0·1 kb, separated from pqqC by a region with no apparent role in PQQ biosynthesis. Two other genes were identified:pqqE, which was closely linked to pqqD; and pqqF, located approximately 19 kb from the other genes. Directed mutagenesis by marker exchange provided chromosomal insertion mutations of these genes in M. organophilum. Attempts to express the pqq genes in two heterologous hosts, Escherichia coli and Pseudomonas testosteroni, were unsuccessful, and no plasmid containing all of the pqq genes was isolated.

The aspartokinase II(ask) operon of Bacillus subtilis consists of two in-phase overlapping genes that encode the two subunits of the lysine-sensitive isoenzyme of aspartokinase (ATP:l-aspartate 4-phosphotransferase, EC 2.7.2.4). Transduction mapping of the ask operon, inactivated by recombinational insertion of a cat marker, indicates a chromosomal location (about 253°) between leuA and aroG. ask is thus remote from aecB, eliminating aecB as a possible locus for the structural gene of aspartokinase II, but close to aecA and uvrB. The nucleotide sequence of a 2 kb DNA fragment just upstream of the ask operon was determined and found to contain two open reading frames. The deduced amino acid sequence of the distal reading frame exhibits extensive homology with Escherichia coli thioredoxin and that of the proximal one, which overlaps with the ask promoter, is homologous to the deduced product of the E. coli uvrC gene. Insertional mutagenesis of the proximal open reading frame led to a mitomycin-sensitive phenotype, consistent with a role in DNA repair. In conjunction with the data of M. Petricek, L. Rutberg & L. Hederstedt [FEMS Microbiology Letters 61, 85–88] our results define the nucleotide sequence of an 8·8 kb segment of the B. subtilis chromosome near 253 ° and the following order of genes: trx-uvrB-ask-orfX-sdhC-sdhA-sdhB-orfY-gerE.

Bacillus thuringiensis strain AF101 possesses a single plasmid (pAF101) with a molecular size of 42 MDa (69 kb). During plasmid curing experiments in strain AF101, we found that a phage (J7W-1) was induced by ethidium bromide treatment. Moreover, the phage genome (48 kb) hybridized only with pAF101 on a Southern blot of the DNA of a cleared lysate prepared from strain AF101. Comparison of the restriction patterns of pAF101 and J7W-1 phage DNA revealed that pAF101 contains not only the entire phage DNA but also a plasmid-specific DNA region. These results indicate that the J7W-1 genome has been stably integrated into pAF101 in strain AF101. Integration of the J7W-1 genome into a plasmid was also observed after phage infection of the type strain of B. thuringiensis subsp. israelensis.

The Escherichia coli entD gene encodes a product necessary for the synthesis of the iron-chelating and transport molecule enterobactin (Ent); cells harbouring entD mutations fail to grow in iron-deficient environments. For unknown reasons, it has not been possible to identify the entD product. The nucleotide sequence of the entD region has now been determined. An open reading frame extending in the same direction as the adjacent fepA gene and capable of encoding an approximately 24 kDa polypeptide was found; it contained a high percentage of rare codons and two possible translational start sites. Complementation data suggested that EntD proteins truncated at the carboxy terminus retain some activity. Two REP sequences were present upstream of entD and an IS186 sequence was observed downstream. RNA dot-blot hybridizations demonstrated that entD is transcribed from the strand predicted by the sequencing results. An entD-lacZ recombinant plasmid was constructed and shown to express low amounts of a fusion protein of the anticipated size (approximately 125 kDa). The evidence suggests a number of possible explanations for difficulties in detecting the entD product. Sequence data indicate that if entD has its own promoter, it is weak; the REP sequences suggest that entD mRNA may be destabilized; and translation may be slow because of the frequency of rare codons and a possible unusual start codon (UUG). The data are also consistent with previous evidence that the entD product is unstable.

An Escherichia coli K12 chromosomal EcoRI-BamHI fragment containing a mutant hsdS locus was cloned into plasmid pBR322. The mcrB gene, closely linked to hsdS, was used for selection of clones with the inserted fragment using T4αgt57βgt14 and λvir. PvuII phages; the phage DNAs contain methylated cytosines and hence can be used to demonstrate McrB restriction. For the efficient expression of the hsdS gene, a BglII fragment of phage λ carrying the pR promoter was inserted into the BamHI site of the hybrid plasmid. Under these conditions a trans-dominant effect of the hsdX ts +d mutation on restriction and modification was detected. Inactivation of the hsdS gene by the insertion of the λ phage BglII fragment into the BglII site within this gene resulted in the disappearance of the trans-dominant effect. When the cloned BamHI-EcoRI fragment was shortened by HpaI and EcoRI restriction enzymes, the trans-dominant effect was fully expressed. The results indicate that the X ts +d mutation is located in the hsdS gene. The effect of gene dosage of the HsdS subunit on the expression of X ts +d mutation was studied. The results of complementation experiments, using F′-merodiploids or plasmid pBR322 with an inserted X ts +d mutation, support the idea that the HsdSts +d product competes with the wild-type HsdS product, and has a quantitatively different effect on restriction and modification.

Tetracycline (Tc) resistance was found in nine out of ten clinical isolates of Enterococcus faecium. Conjugative transposons, designated Tn5031, Tn5032 and Tn5033, were present in the chromosome of three isolates. The transposons were similar both structurally and functionally to Tn916 containing the tetM determinant. A large non-conjugative plasmid found in a fourth isolate contained an element homologous to Tn916. The four isolates containing the element showing homology to Tn916 exhibited a substantially higher level of Tc resistance than the remaining five Tc-resistant isolates. Tc-resistance genes which have not been identified are apparently responsible for the low-level Tc resistance in five clinical isolates.

A 4·3 kb XbaI restriction fragment of DNA from Clostridium sordellii G12 hybridized with a synthetic oligonucleotide representing the N-terminus of the sialidase protein secreted by C. sordellii. This cloned fragment was shown to encode only part of the sialidase protein. The sialidase gene of C. sordellii was completed by a 0·7 kb RsaI restriction fragment overlapping one end of the XbaI fragment. After combining the two fragments and transformation of Escherichia coli, a clone that expressed sialidase was obtained. The nucleotide sequence of the sialidase gene of C. sordellii G12 was determined. The sequence of the 18 N-terminal amino acids of the purified extracellular enzyme perfectly matched the predicted amino acid sequence near the beginning of the structural gene. The amino acid sequence derived from the complete gene corresponds to a protein with a molecular mass of 44735 Da. Upstream from the putative ATG initiation codon, ribosomal-binding site and promoter-like consensus sequences were found. The encoded protein has a leader sequence of 27 amino acids. The enzyme expressed in E. coli has similar properties to the enzyme isolated from C. sordellii, except for small differences in size and isoelectric point. Significant homology (70%) was found with a sialidase gene from C. perfringens.

Interspecific complementation of an Escherichia coli recA mutant with a Legionella pneumophila genomic library was used to identify a recombinant plasmid encoding the L. pneumophila recA gene. Recombinant E. coli strains harbouring the L. pneumophila recA gene were isolated by replica-plating bacterial colonies on medium containing methyl methanesulphonate (MMS). MMS-resistant clones were identified as encoding the L. pneumophila recA analogue by their ability to protect E. coli HB101 from UV exposure and promote homologous recombination. Subcloning of selected restriction fragments and Tn5 mutagenesis localized the recA gene to a 1·7 kb BglII-EcoRI fragment. Analysis of minicell preparations harbouring a 1·9 kb EcoRI fragment containing the recA coding segment revealed a single 37·5 kDa protein. Insertional inactivation of the cloned recA gene by Tn5 resulted in the disappearance of the 37·5 kDa protein, concomitant with the loss of RecA function. The L. pneumophila recA gene product did not promote induction of a λ lysogen; instead, the presence of the heterologous recA gene caused a significant reduction in spontaneous and mitomycin-C-induced prophage induction in recA + and recA E. coli backgrounds. Despite the lack of significant genetic homology between the L. pneumophila recA gene and the E. coli counterpart, the L. pneumophila RecA protein was nearly identical to that of E. coli in molecular mass, and the two proteins showed antigenic cross-reactivity. Western blot analysis of UV-treated L. pneumophila revealed a significant increase in RecA antigen in irradiated versus control cells, suggesting that the L. pneumophila recA gene is regulated in a manner similar to that of E. coli recA.

Summary: Extracellular hydrogen peroxide has in the past been implicated in the degradation of the crystalline cellulose component of plant cell walls, particularly by brown-rot fungi. Using assays sensitive to 1 μg peroxide ml−1 no evidence could be found of extracellular hydrogen peroxide in culture media of several basidiomycete fungi examined, although some free radicals may have been produced on the surface of the fungal hyphae. Analysis of the products of enzymic degradation of crystalline cellulose by gas chromatography/mass spectroscopy revealed a range of oxidized sugars which most probably resulted from the action of peroxide free radicals derived from hydrogen peroxide.

Summary: Carbon catabolite repression was studied in secreted (invertase) and mitochondrial (isocitrate dehydrogenase) enzymes of Phycomyces blakesleeanus. Both enzymes were subject to repression by glucose when the fungus was grown at pH 4·5. Repression of invertase but not of isocitrate dehydrogenase was overcome when xylose was added to the growth medium together with glucose. Derepression of invertase activity was obtained when the fungus was grown in a medium with glucose and at pH 7·3. This pH-dependent derepression was not due to enzyme redistribution or stimulation of activity. In contrast, isocitrate dehydrogenase activity was not derepressed at pH 7·3, but was significantly reduced either in the presence or absence of glucose.