- Volume 134, Issue 6, 1988

The NAD-dependent glutamate dehydrogenase from Phycomyces spores was purified more than 300-fold. Estimation of M r by gel filtration gave a value of 98000 whereas after SDS-PAGE one major band of M r 54000 was found, suggesting that the enzyme is a dimer. The enzyme was virtually dependent on the presence of AMP for activity and showed half-maximal activation at 9·5 and 43 μm-AMP in the direction of amination and deamination respectively. ADP was nearly as effective at 20-fold higher concentrations. Other nucleotide monophosphates were ineffective and nucleoside triphosphates were slightly inhibitory. Hyperbolic kinetics were found for all substrates yielding K m values of about 10 mm for ammonium, 1 mm for 2-oxoglutarate and 0·1 mm for NADH in the direction of amination, and 10 mm for glutamate and 0·7 mm for NAD in the direction of deamination.

In contrast to a previous report, strains of Klebsiella pneumoniae were found to take up and phosphorylate the disaccharide sucrose via the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS). In addition to the two soluble and general components enzymeI and HPr of the PTS, a sucrose-specific enzymeIIScr (gene scrA), together with the enzymeIII, coded for by the gene crr, were needed for the vectorial phosphorylation of sucrose to generate intracellular sucrose 6-phosphate. This sugar phosphate is hydrolysed by a hydrolase (invertase, gene scrB) to generate glucose 6-phosphate and free fructose. The latter is converted to fructose 6-phosphate by an ATP-dependent fructokinase (gene scrK), an enzyme which is part of the sucrose and not of the fructose catabolic pathway. Analysis of different mutants of K. pneumoniae strain 1033, and of Escherichia coli K12 derivatives carrying R′scr plasmids isolated from K. pneumoniae, showed that the genes scrA, B, and K, together with a gene scrR for a repressor, form a genetic unit located on the chromosome of K. pneumoniae. These genes and the corresponding sucrose metabolic pathway are very similar to a previously described scr system encoded on plasmid pUR400 and found in other enteric bacteria.

The alkali-insoluble glucan was isolated from regenerating spheroplasts and intact cells of Candida albicans. Sequential enzymic hydrolysis of this fraction by Zymolyase 100T and purified chitinase and subsequent gel filtration produced a fraction which was enriched in glycosaminoglycans. This fraction was analysed by partial acid hydrolysis, TLC and GLC-MS. The GLC-MS peaks identified included 2,3,4,6-tetra-O-methylglucitol acetate and 2,3,4-tri-O-methylglucitol acetate of β-1,6-glucan and the 3,6-di-O-methyl-2-N-methylglucosaminitol acetate of chitin. In addition, 3-O-methyl-2-N-methylglucosaminitol acetate was identified, which indicated a branch point in chitin. These data provide evidence for a covalent linkage between chitin and β-(1,6)-glucan through a glycosidic linkage at position 6 of N-acetylglucosamine and position 1 of the glucose in the glucan.

Several strains of Acetobacter xylinum were screened for in vivo cellulose and acetan production, and for in vitro synthesis of a prenyl-diphosphate-hexasaccharide, using UDP-Glc, UDP-GlcA and GDP-Man as sugar donors. The lipid-bound saccharide was synthesized only by acetan-producing strains. Previous work has shown that the in vitro-synthesized lipid-linked saccharides have the same structure as the acetan repeating unit. The present results strongly suggest a precursor-product relationship. The strains that produced acetan lost their ability to do so by ageing of the culture.

Growth of two independently isolated strains of methicillin-resistant Staphylococcus aureus (MRSA) in increasing concentrations of methicillin (step-selection) resulted in increased resistance in these strains. When chromosomal DNA from the step-selected variants was probed using DNA sequences previously demonstrated to be associated with methicillin resistance in MRSA strains, amplification of the homologous chromosomal sequence was identified. Growth of these step-selected strains in the absence of methicillin resulted in loss of the amplified sequence, while the original sequence remained. There are differences between the two strains in the stability of maintenance of amplified sections. Prolonged storage of the variants on a high concentration of methicillin resulted in loss of amplified sections without concomitant loss of methicillin resistance. Thus amplification may be only one of at least two molecular mechanisms available to S. aureus to increase methicillin resistance in response to step-selection. Probing of cells of the highly resistant sub-population of a heterogeneously resistant MRSA strain showed that duplication of this mec-associated DNA is not involved in the mechanism of heteroresistance.

A 4 kb fragment of chromosomal DNA was cloned from a clinical strain of methicillin-resistant Staphylococcus aureus. It comprises part of a section of the chromosome that was lost when the strain was cured of resistance to methicillin and to other antimicrobial agents. The fragment mediates an increased level of methicillin resistance when inserted into a shuttle vector and transformed back into the sensitive strain generated when the original DNA was deleted.

Several methods of introducing mobilizable cloning vectors by conjugation into the photosynthetic bacterium Rhodobacter sphaeroides have been examined. The efficiency of transfer was sufficiently high to enable a bank of Rb. sphaeroides genes in Escherichia coli to complement non-photosynthetic mutants of Rb. sphaeroides, thus providing a generally applicable method of isolating Rb. sphaeroides genes. With a mutant incapable of synthesizing reaction centres and light-harvesting LH1 complexes as a recipient, the transfer of puf genes encoding reaction centre and light-harvesting LH1 polypeptides was examined in some detail. The spectroscopic and electrophoretic properties of this mutant and the newly photosynthetic transconjugant strain were consistent with the efficient transfer and expression of puf genes.

The puf operon of the photosynthetic bacterium Rhodobacter sphaeroides encodes reaction centre and B875 (LH1) antenna polypeptides of the photosynthetic apparatus. This region of the genome was used to establish the applicability of random transposon Tn5 mutagenesis in this bacterium. Four Tn5 insertions have been mapped and one of the mutants characterized using a variety of techniques in order to establish that the fluorescence properties and polypeptide composition were consistent with the absence of reaction centre polypeptides. Following the ‘rescue’ of the transposon along with flanking regions of the puf operon, re-introduction of this construction into wild-type Rb. sphaeroides yielded the original mutation. This demonstrates that following homologous recombination, localized mutagenesis can direct Tn5 into a predetermined region of the Rb. sphaeroides chromosome. Accordingly, puf genes borne on plasmid pSRC2 were mutagenized with Tn5 in Escherichia coli, and the sites of insertion mapped physically. pSRC2 derivatives containing Tn5 were transferred to wild-type Rb. sphaeroides. puf genes have been mapped by correlating the photosynthetic properties of resulting strains with the sites of Tn5 insertion into pSRC2.

Four mutants of the photosynthetic bacterium Rhodobacter sphaeroides were isolated which were incapable of photosynthetic growth due to inability to synthesize bacteriochlorophyll. A Rb. sphaeroides gene bank was constructed in the mobilizable vector pSUP202 and was transferred into these mutants using the helper plasmid pRK2073. Three clones that produced photosynthetic transconjugants from one or more of the bch mutants were isolated and characterized. These clones were used as probes to estimate levels of specific transcripts in cells undergoing a 100-fold increase in bacteriochlorophyll content. The maximum level of transcripts was observed at an early stage of photosynthetic membrane synthesis when only 7% of the eventual level of pigment had been synthesized.

Stable ampicillin-resistant transformants of Synechococcus PCC 6301 were isolated using as donor fragments chromosomal DNA cloned into ColEl-derived vectors. Using dark-incubated recipient cells, transformation was achieved reproducibly at frequencies of approximately 10−5 per cell and 3 × 102 per μg of donor DNA. These frequencies were 102- to 104-fold lower than those reported previously by other workers for the closely related strain PCC 7942 but only 5- to 10-fold lower than found by us in parallel experiments with both strains. Different donor fragments of PCC 6301 DNA gave different characteristic transformation frequencies which were greatly reduced by short internal deletions. The results are most simply explained by a model of integration of circular plasmid DNA into the recipient chromosome by a single crossover event.

R68.45 and other similar broad-host-range (IncP) plasmids carrying a tandem repeat of the 2·1 kb insertion element IS21 mobilize the chromosome of many different Gram-negative bacteria. To analyse the structure of R68.45-chromosome cointegrates, whose involvement in the mobilization process had been postulated previously, we selected for the stable integration of R68.45-like plasmids into the Pseudomonas aeruginosa chromosome. Two plasmids were chosen: pME28, a transfer-deficient, mobilizable RP1 derivative with an inactive replication control (trfA) gene, and pME487, an R68.45 derivative with a trfA(ts) mutation causing temperature-sensitive replication. Chromosomally integrated pME28 and pME487 were found to be flanked by single IS21 elements. This structure is in agreement with a ‘cut-and-paste’ mode of R68.45 transposition. pME28 and pME487 showed a low specificity of insertion but rarely < 0.1%) induced auxotrophic mutations. Hfr (high-frequency-of-recombination) donors of P. aeruginosa could be obtained by chromosomal integration of pME487 or pME28; in the latter case, the transfer functions lacking from pME28 had to be provided in trans on an autonomous plasmid. Hfr donors gave higher conjugational linkage and transferred longer stretches of the P. aeruginosa chromosome than did R68.45 donors. This suggests that the integration of R68.45 into the donor chromosome is short-lived in P. aeruginosa.

The replication genes (rep) of the virulence plasmid pYVe439-80 of Yersinia enterocolitica were localized and characterized by restriction endonuclease analysis. Comparison with pIBl, a virulence plasmid of Y. pseudotuberculosis, indicates that while the plasmids carry homologous rep genes their location with respect to the highly conserved ‘calcium region’ is different. This replication function is thermosensitive. Mini-derivatives of pYVe439-80 appear to be rather unstable. The region of pYVe439-80 containing homology to the incD determinant of F was shown to contain a plasmid-stabilization system (par). The region encoding par was characterized by restriction endonuclease analysis. pIBl contained an homologous par region but located differently. The pYV plasmids thus underwent rearrangements during their divergent evolution. While the positions of rep and par in the two plasmids are inverted with respect to the surrounding loci, our determination of the orientation of each locus rules out the hypothesis of a simple inversion of a quadrant of pYV. The gene encoding YOP5, a 26 kDa protein encoded by pIBl, was cloned on a mobilizable vector and introduced in Y. enterocolitica W22708 containing pYVe227 (indistinguishable from pYVe439-80), mutated in the homologous gene. The recombinant Y. enterocolitica secreted YOP5. Hence, the transcriptional activation and secretion systems of pYVe227 act on a yop gene from pIBl and on its product, indicating that these systems are interchangeable.

Two outer-membrane (OM) proteins of Yersinia enterocolitica YOMP-C and YOMP-F appear to function as porins. Mutants that were YOMP-C− and YOMP-F− exhibited changes in cephaloridine and [3H]glucose uptake and increased resistance to β-lactam antibiotics (especially cephalosporins) and tetracycline. Alterations in OM permeability may contribute to antibiotic resistance in Yersinia.

Five temperate phages were isolated from strain 4042B of Bacillus thuringiensis subsp. aizawai. The phages, which were heteroimmune, could also be distinguished by their host ranges, plaque and particle morphologies, serological specificities, and locations of restriction endonuclease cleavage sites on their chromosomes. Besides maintaining a stable lysogenic relationship with the 4042B host strain, each phage formed a stable lysogen with Bacillus cereus.

Benomyl treatment (at 100 μg ml−1) of Candida albicans 1001, and other strains derived from it, determined the appearance of morphological mutants similar to those derived from UV irradiation treatment. A permanent alteration in the morphogenesis of these mutant strains determined their inability to grow by budding, to form oval yeast cells or blastospores (Y−phenotype) and their growth as long filamentous forms, mostly with the appearance of pseudomycelium, giving rise to rough colonies (R phenotype). In order to carry out a genetic complementation analysis, we isolated morphological mutants that carried other genetic markers (nutritional, conditional lethal) adequate for crosses by means of protoplast fusion. Wild-type hybrids of regular mononuclear oval yeast cells and smooth colonies were obtained by crossing pairs of complementing mutants, whereas hybrids from crosses of non-complementing mutants still retained their morphological alterations. Our results define two complementation groups, which represent two genes relevant for dimorphism, whose alteration interferes with the correct transition from blastospores to mycelium.

A 2·8 kb PstI fragment of Bacillus subtilis 168W DNA has been cloned into Escherichia coli HB101 and B. subtilis AG5 using pAC3 as a shuttle plasmid. The new plasmid (pBRG1), of 10·2 kb, complemented flaD mutations which show reduced production of autolysin(s), filamentation and non-motility (deficiency of flagella). Deletion experiments showed that the suppressive gene is located between the HindIII and XbaI sites (1·0 kb apart) in pBRG1. The integration of a plasmid having chloramphenicol resistance closely linked to the flaD gene into the B. subtilis AC703 chromosome and its genetic analysis indicated that the cloned fragment contained the flaD gene itself. A high-copy-number plasmid carrying the cloned gene did not lead to an increase in autolysin production above the wild-type level, but it changed the colony morphology from smooth to rough. Among several autolysin-deficient mutations, lyt-151 was suppressed only by the high-copy-number plasmid carrying the cloned gene.