- Volume 136, Issue 12, 1990

Cytoplasmic membranes of Acinetobacter calcoaceticus cells, cultured under acetate-limiting conditions, contain cytochrome b554 and a cytochrome o-containing oxidase. Both have been purified to homogeneity and characterized. Cytochrome b554 is a monomeric protein (molecular mass 70 kDa) with a midpoint potential of +100 mV (in the membrane it has most probably a value of +50 mV). The cytochrome o-containing oxidase seems to be an αβγδ protein since the molecular mass of the native protein was estimated to be 150 kDa and the molecular masses of the subunits, determined by SDS-polyacrylamide-gel electrophoresis, are 55, 41, 33 and 17 kDa. Redox spectroscopy of the purified complex shows the presence of a cytochrome b 555/563 having a midpoint potential of approximately +160 mV (both in purified form and in the membrane). CO difference spectroscopy shows the presence of a second b-type cytochrome, viz. cytochrome, o. Cytoplasmic membranes of A. calcoaceticus cells grown under oxygen-limiting conditions also show the presence of the cytochrome b 554 and the cytochrome o-containing oxidase. In addition a protein has been solubilized with the spectral characteristics of a cytochrome d-containing oxidase. The cytochrome o- and d-containing oxidases appear to be similar to those reported for Escherichia coli and Proteus mirabilis. On the other hand, cytochrome b554 has no counterpart in these organisms since the cytochrome b556 described for E. coli is quite dissimilar.

The in situ location of phytoene desaturease, an enzyme in the carotenoid biosynthetic pathway, has been investigated in the photosynthetic organisms Rhodobacter capsulatus SB 1003, Synechocystis PCC 6714, and Anabaena variabilis ATCC 29413. A post-embedding immunogold electyron microscopy procedure using a polyclonal antibody against a phytoene desaturase fusion protein was employed. For all three species most of the gold particles (about 85% in the case of Anabaena and Synechocystis) were associated with the photosynthetic membreanes. Other parts of the were only randomly and not significanlty labelled. When the anti-phytoene desatuase antibody was omitted, or when pre-immune serum was used, no gold deposition was observed. In vitro phytoene desaturase reactions, especially with purified thylakoid membranes from Synechocystis, confirmed the nature of phytoene as an integral protein of the thylakoid membrane.

Tetrahymanol, a rare triterpene from the gammacerane series, previously detected only in a few scattered eukaryotic taxa, has been isolated for the first time from the phototrophic bacterium Rhodopseudomonas palustris. This extends the significance of geochemical markers from this series, which are abundant in marine sediments and palaeohypersaline environments.

A combined physical and genetic map of Pseudomonas aeruginosa PAO was constructed by pulsed-field gel electrophoresis and Southern hybridization using cosmid clones from a genomic library carrying known genes. A total of 37 SpeI restriction fragments have been mapped on the 5862 kb genome, and fragment contiguity demonstrated by hybridization with clones from a SpeI junction fragment library and fragments obtained by partial SpeI digestion, both derived from the P. aeruginosa PAO chromosome.

The Saccharomyces cerevisiae pyruvate kinase gene (PYK1) was transformed into yeast using the multicopy vector pJDB207. Growth rates and PYK1 gene expression levels varied considerably amongst the transformants. Yeast transformants expressing the PYK1 gene at high levels formed small colonies compared with those expressing the gene at relatively low levels. Slow-growing transformants reverted at high frequency to more rapid growth, and this correlated with decreases in PYK1 gene copy number and PYK1 mRNA abundance. This apparent selection against PYK1 over-expression was disrupted by the introduction of a stop codon at the 5′-end of the PYK1 coding region, thus confirming that the growth effects were mediated by the PYK1 gene. However, massive overproduction of pyruvate kinase in yeast, using multiple copies of a PGK:PYK gene fusion, had no significant effect upon cell growth. This suggests that the deleterious effect upon the host yeast cell is mediated by abnormally high levels of the wild-type gene or PYK1 mRNA, rather than by increased pyruvate kinase levels.

The glpPKD region of the Bacillus subtilis chromosome was cloned in its natural host in plasmid pHP13. The glpPKD region contains genes required for glycerol catabolism: glpK coding for glycerol kinase, glpD coding for glycerol-3-phosphate (G3P) dehydrogenase and glpP, proposed to code for a positively acting regulatory protein. The cloned 7 kb fragment carries wild-type alleles of glpK, glpD and glpP. It can also complement a strain deleted for the entire glpPKD region. The wild-type alleles were mapped to different subfragments, establishing the gene order glpP-glpK-glpD. The nucleotide sequence of glpK and glpD was determined. Immediately upstream of glpK, an additional open reading frame was found, possibly being part of the same operon. Putative transcription terminators were found in the region between glpK and glpD and downstream of glpD. In a coupled in vitro transcription/translation system, two proteins were found, corresponding in size to those predicted from the deduced amino acid sequences of glycerol kinase and G3P dehydrogenase (54 kDa and 63 kDa, respectively).

Two genes encoding distinct 1,3-β-glucanases have been cloned from Bacillus circulans and expressed in Escherichia coli. A cosmid library of B. circulans WL-12 DNA was constructed in the broad-host-range cosmid pLAFR1 and screened in E. coli for clones which exhibited 1,3-β-glucanase activity. Two 1,3-β-glucanase-positive clones were identified which contained genes encoding two independent 1,3-β-glucanases as shown by biochemical, physical and molecular analyses. The cosmids, designated pMON5401 (27·1 kb insert) and pMON5402 (24·7 kb insert), encoded 68 kDa and 40 kDa 1,3-β-glucanases, respectively. Both 1,3-β-glucanases were purified from their respective E. coli strains, characterized biochemically, and were shown to exhibit lytic activity against purified yeast cell wall preparations.

Mutants of Rhodobacter capsulatus deficient in growth on nitrogen sources other than NH+ 4 were compared with mutants of a similar phenotype isolated from Rhodobacter sphaeroides. In addition to N2 and some amino acids (glutamate, alanine, proline, arginine), mutants of R. sphaeroides and R. capsulatus strain AD2 were unable to utilize NO3 − as sole nitrogen source for growth. Under conditions of nitrogen starvation, mutants of both species lacked the methylammonium (ammonium) uptake system, which was found in the wild-type strains under these conditions. The wild-type (adgA) genes complementing these mutants were isolated from gene banks of the respective species and localized to a 2·9 kb BamHI-SalI fragment in R. sphaeroides and to a 1·7 kb SmaI fragment in R. capsulatus. These two fragments hybridized strongly with each other, showing that they contain homologous sequences. Furthermore, the adgA gene from R. capsulatus fully restored the wild-type phenotype to Adg− mutants of R. sphaeroides and vice versa. Inactivation of the chromosomal adgA gene by insertion of an antibiotic-resistance cassette resulted in a severe inhibition of growth in rich medium and in minimal medium containing NH+ 4. This suggests that the adg A gene is required for normal growth under all growth conditions.

Bacteriophage FP22 has a very broad host range within streptomycetes and appeared to form lysogens of Streptomyces ambofaciens ATCC 15154. FP22 shared strong cross-immunity and antibody cross-reactivity with bacteriophage P23, but not with seven other streptomycete bacteriophages. FP22 particles had a head diameter of 71 nm and a tail length of 307 nm. The FP22 genome was 131 kb, which is the largest bacteriophage genome reported for streptomycetes. The G + C content of the genome was 46 mol% and restriction mapping indicated that FP22 DNA had discrete ends. NaCl- and pyrophosphate-resistant deletion mutants were readily isolated and the extent of the deletions defined at least 23 kb of dispensable DNA in two regions of the genome. The DNA was not cleaved by most restriction endonucleases (or isoschizomers) which have been identified in the streptomycetes, including the tetranucleotide cutter Mbol (GATC).

Cell extracts prepared from Agrobacterium tumefaciens GMI9023 derivatives carrying megaplasmids from Rhizobium meliloti strain SU47 were examined by non-denaturing polyacrylamide gel electrophoresis and subsequent specific enzyme staining. The results demonstrated that an unidentified or nothing dehydrogenase is encoded by the nod-nif megaplasmid pRmeSU47a, and a -galactosidase activity that is required for growth on lactose as sole carbon source is encoded by the exo-thi megaplasmid pRmeSU47b. These results are of particular importance regarding the choice of markers used in bacterial population genetic studies.

Two DNA probes for the detection of insertion sequence IS200 by either Southern blotting or colony hybridization were constructed. One of the probes is a 300 bp EcoRI-HindIII fragment of IS200 cloned onto pBluescript KS(+); the other is a tail-to-tail dimer of the same fragment cloned onto pUC19. A survey of the presence of IS200 among enteric bacteria revealed that more than 90% of the pathogenic or food-poisoning isolates of Salmonella spp. examined contained one or more copies of insertion sequence IS200, with the exception of the subgenus I serovar S. agona in which IS200 is not found. Although insertion sequence IS200 was first considered a Salmonella-specific element, it also exists in many isolates of Shigella sonnei and Shigella flexneri, but not in Shigella dysenteria.

Studies on the enzymes of the cellulase complex of the thermophilic fungus Humicola grisea var. thermoidea are described. A genomic library was constructed in the phage vector EMBL 4, and from this library two clones were isolated using as a probe the cloned cbh-1 (exoglucanase, EC 3.2.1.91) gene of Phanerochaete chrysosporium, a cellulolytic basidiomycete fungus. These clones were analysed by restriction mapping and Southern blotting, and one of them (λ3) was sub-cloned into the M13 phage vectors mp18 and mp19. The gene sequence was determined by the dideoxy chain-termination method. Sequence comparison with the equivalent genes from P. chrysosporium and Trichoderma reesei was made: in terms of primary sequence there is about 60% homology between the three species. Secondary structure prediction of the H. grisea sequence was also computed.

By using pulsed-field gel electrophoresis, we have separated the entire chromosome bands and examined the electrophoretic karyotypes of 27 strains of Candida albicans. The electrophoretic karyotype varied widely among these strains. Their chromosomal DNAs were resolved into 7-12 bands ranging in size from 042 to 30 Mb. Most of the separated chromosomal bands were assigned by eight cloned C. albicans DNA probes. These results suggest that the haploid number of C. albicans chromosomes is eight. Each of the probes hybridized specifically to one or two bands of similar size in most strains. With the exception of the MGL1 probe, when two bands were detected by one probe, the size of one of them was very conserved whilst the other was of fairly variable size. The sizes of the chromosome bands assigned by the MGL1 probe were much more variable. As C. albicans is considered to be a diploid organism, it is inferred that the karyotype polymorphism between strains is mainly derived from wide size heterogeneity in one of the homologous chromosomes. Furthermore, we have confirmed species-specific and strain-specific variation in medically important Candida species (C. stellatoidea, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. kefyr and C. glabrata). Electrophoretic karyotype analysis is thus useful for species assignation. The TUB2 probe, encoding C. albicans beta-tubulin, hybridized to the chromosomal DNA of all the Candida species examined, but four C. albicans probes exhibited cross-species hybridization with C. stellatoidea only. The karyotype of C. stellatoidea seems to be within the range of the intraspecies variation observed in C. albicans.

Renibacterium salmoninarum was shown to possess peritrichous fimbriae. Electron microscopy of strains FMV 84–01 and ATCC 33209T revealed short, flexible fimbriae less than 2 nm in diameter. These surface appendages were isolated from the bacteria by a procedure involving water extraction and urea solubilization. The fimbrin was purified to homogeneity by Fast Pressure Liquid Chromatography, and shown by SDS-PAGE to be a protein of 57 kDa. Isoelectric focusing under non-denaturing conditions indicated a pI of 4·8. The protein had an amino acid composition rich in glycine, Asx (aspartic acid and asparagine), valine and alanine; methionine was absent. Approximately 33% of the amino acid residues were hydrophobic. Immunoblotting using a polyclonal antiserum raised against whole cells showed that the 57 kDa protein was the immunodominant antigen on the cell surface. Immunogold labelling using polyclonal antibodies raised against the fimbrin revealed an alignment of gold particles along the fimbriae. Purified fimbriae caused agglutination of rabbit erythrocytes and antifimbrial serum inhibited this haemagglutination. Altogether the results indicate that the fimbriae on the surface of R. salmoninarum are responsible for the haemagglutinating activity.

Escherichia coli strain 334 is a human enterotoxigenic strain of serotype O15:H11 which had previously been shown to produce “attachment pili”. These fimbriae were compared with other colonization factors. From strain 334 a mannose-resistant haemagglutination positive colony 334A and a mannose-resistant haemagglutination negative variant 334C were isolated. By electron microscopy the fimbriae of strain 334A were shown to have a helical structure resembling coli-surface-associated antigen (CS5) fimbriae. An antiserum was raised to strain 334A and absorbed with a fimbriae-negative variant of that strain, 334C. By immuno-electron microscopy this antiserum was shown to coat fimbriae of strain 334A but not CS5 fimbriae produced by strain E17018A. Conversely, CS5 antiserum did not coat the fimbriae produced by strain 334A. No antigenic cross-reaction was detected between these intact fimbriae when anti-strain 334A serum and CS5 antiserum were used in immunodiffusion tests. By enzyme-linked immunosorbent assays (ELISAs) the fimbriae of strain 334A were shown to be antigenically unrelated to most other human ETEC adhesins, namely colonization factor antigens (CFA/I, CFA/III and CFA/IV), coli-surface-associated antigens (CS1, CS2, CS3, CS4, CS6 and CS17) and putative colonization factors (PCFO159:H4 and PCFO166). However, a heated suspension of strain 334A reacted weakly with CS5 antiserum in an ELISA. By SDS-PAGE the fimbriae of strain 334A were shown to consist of subunits of similar size to CS5 subunits, that is about 21·5 kDa. Western immunoblotting revealed that the subunits of 334A and CS5 fimbriae shared common epitopes. Expression of 334A fimbriae was controlled by a plasmid which also coded for enterotoxin production, a system similar to that reported for a number of other human ETEC attachment factors. Strain 334A fimbriae have therefore been designated as CS7 fimbriae. Surveillance with CS7 anti-serum of ETEC with no known attachment factors showed two other strains of serotype O15:H11, one strain of serotype O103:H49 and 20 strains of serotypes O114:H49 and H− to possess CS7 fimbriae.

Candida albicans ATCC 26555 switched at high frequency (10-1 to 10-3) between several phenotypes identified by colony morphology on a defined mineral amino-acid-containing agar medium supplemented with arginine and zinc (LAZ medium). When cells taken from colonies exhibiting distinct morphologies were plated directly onto LAZ agar, spontaneous conversion to all the variant phenotypes occurred at combined frequencies of 21 × 101 to 95 × 10-3 However, when cells taken from the different colonial phenotypes were plated directly onto an undefined medium (yeast extract/peptone/dextrose; YPD medium), or first incubated in liquid YPD medium and then cloned on YPD agar, all colonies observed exhibited the same phenotype (smooth-white). When cells from the smooth-white colonies were plated as clones on LAZ agar, the original switch phenotype reappeared. These results suggest that environmental conditions such as the growth medium (and possibly the temperature) influence switching by suppressing phenotype expression, but have no effect on genotype. The variant colony morphologies also appeared to be associated with differences in the relative proportions of yeast and mycelial cells. Zymolyase digests of wall preparations obtained from cells belonging to different colonial phenotypes were analysed by SDS-PAGE. After blotting to nitrocellulose paper, the mannoproteins were stained with Concanavalin A, with a polyclonal antiserum enriched in antibodies against mycelium-specific wall components, and with a monoclonal antibody raised against a high-molecular-mass mannoprotein band (260 kDa) specific to the walls of mycelial cells. The results suggest that phenotypic switching might be associated with changes in the degree of glycosylation in high-molecular-mass mannoproteins, or in the way these mannoproteins are bound to other cell wall components.