- Volume 137, Issue 6, 1991

The nematophagous fungus Arthrobotrys oligospora captures nematodes using adhesive polymers present on special hyphae (traps) which form a three-dimensional network. To understand further the adhesion mechanisms, A. oligospora surface polymers were visualized by transmisson electron microscopy and characterized by chemical methods. Both traps and hyphae were surrounded by a fibrillar layer of extracellular polymers which stained with ruthenium red. The polymer layer was resistant to most of the chemicals and enzymes tested. However, part of the layer was removed by sonication in a Tris-buffer or by extraction in a chaotropic salt solution (LiCI), and the structure of the polymers was modified by treatment with Pronase E. Chemical analysis showed that the crude extracts of surface polymers removed by sonication or LiCl solution contained neutral sugars, uronic acids and proteins. Gel chromatography of the extracts revealed that the major carbohydrate-containing polymer(s) had a molecular mass of at least 100 kDa, containing neutral sugars (75% by weight, including glucose, mannose and galactose), uronic acids (6 %) and proteins (19 %). There was more polymer in mycelium containing trap-bearing cells than in vegetative hyphae. SDS-PAGE of the extracted polymers showed that the trap-forming cells contained at least one protein, with a molecular mass of approx. 32 kDa, not present on vegetative hyphae. Examining the capture of nematodes by traps of A. oligospora in which the layer of surface polymers was modified, or removed by chemical or enzymic treatments, showed that both proteins and carbohydrate surface polymers were involved in the adhesion process.

Brefeldin A (BFA) inhibited in a dose-dependent manner secretion of the cell-surface enzyme acid phosphatase (APase) into the periplasm of Candida albicans and caused intracellular accumulation of enzyme protein. Cells grown in the presence of BFA became more dense, implying that cell-surface growth was also blocked by BFA treatment. The APase that was accumulated intracellularly migrated faster on SDS-PAGE, suggesting less N-linked glycosylation compared with the mature, periplasmic APase produced in the absence of BFA. Pulse-chase experiments and gel-filtration of oligosaccharides released by Endo H treatment suggested that the coreglycosylated precursor form of APase accumulated in the presence of BFA. These results strongly suggested that endoplasmic reticulum (ER)-to-Golgi transport in C. albicans was inhibited by BFA. Aberrant membrane structures were observed in BFA-treated cells. Within 1 h of BFA removal these structures were replaced with rough ER membranes, suggesting that the accumulated membranes were derived from the ER.

Summary: The purification, location, properties and regulation of glutamine synthetase (GS) from the facultative methylotroph Hyphomicrobium X were investigated. The enzyme was purified to homogeneity by differential centrifugation, Blue Sepharose CL-6B chromatography and Sephadex G-25 gel filtration. The specific activity of the purified enzyme was 44.7 μmol min-1 (mg protein)-1. GS was cytoplasmic in location with no direct association with DNA, membranes or the enzyme glutamine: 2-oxoglutarate aminotransferase (GOGAT). The molecular mass of the native enzyme was 638 kDa; it was composed of 12 subunits of molecular mass 53 kDa. Double reciprocal plots showed that the enzyme followed Michaelis-Menten kinetics. The apparent K m values for hydroxamate and glutamine in the γ-glutamyl transferase assay were 7.0 and 5.0 mM respectively; those of ammonia, glutamate and ATP in the biosynthetic assay were 0.032, 31.1 and 0.37 mM respectively. GS activity was controlled by covalent modification, the presence of specific divalent cations and feedback inhibition by several end-products of glutamine metabolism.

Bacterial conversion of 4-chlorobiphenyl (4-CB) usually proceeds through a pathway involving an initial oxidation of the unsubstituted ring in the 2,3 position followed by a 1,2 meta-cleavage. The meta-cleavage product (MCP) is converted through a single hydrolysis step into chlorobenzoic acid. However, several other acidic metabolites that were not expected as part of this pathway have already been described. In this paper, we used strains of Pseudomonas putida carrying cloned genes from Pseudomonas testosteroni B-356 that are involved in polychlorinated biphenyl (PCB) degradation to demonstrate that several acidic metabolites found in the culture media of various bacteria grown in the presence of 4-CB result from alternative novel bioconversion pathways of MCP. The degradation products of MCP through these pathways were identified as analogues with saturated or shorter side chains or as 4′-chlorophenyl-2-picolinic acid; pathways leading to their formation are proposed.

Mutants of Saccharomyces cerevisiae characterized by osmotic fragility showed a marked fibrillar structure on the inner wall surface when studied by two electron microscopic techniques, i.e. freeze-etching of whole native cells and metal shadowing of isolated cell walls. The walls of the mutant cells were more permeable to macromolecules than were those of the wild-type parental strain. The synthesis and assembly of (1→3)-β-d-glucan wall microfibrils studied in protoplasts of mutant cells were not impaired. It is suggested that the osmotic fragility of the mutant cells is related to the deficiency of the wall structure as a consequence of the srb1 mutation affecting biogenesis of the amorphous (glucan) component.

Summary: The relationship between the vegetative cell cycle and sexual competence in the green alga Chlamydomonas eugametos was studied using synchronized cultures. The gametic stage was found to be a regular part of the cell cycle; it extended from the beginning of the cell cycle to about 2 h prior to the commitment point for cell division. All variations in the length of this period caused by external growth conditions were accompanied by corresponding changes in the period of mating competence. The period varied depending on the fluence rate, temperature, and nitrate or CO2 concentration. We conclude that the length of the period in the cell cycle in which cells are mating competent is determined by the length of the precommitment period and that environmental factors do not influence mating competence directly, but rather affect the growth rate of the cells.

We have constructed four deletion derivatives of the cloned dnaK gene. Plasmid pDD1, in which the last 10 amino acids of the DnaK protein have been replaced by three different amino acids derived from the pBR322 vector, was as effective as plasmid pKP31, from which it was derived, in restoring the ability of a dnaK null mutant, Escherichia coli BB1553, to plate λ phage and to grow at high temperatures. The other three mutations, involving much larger deletions of the dnaK gene, did not restore the ability to plate λ phage or the ability to grow at high temperatures. Plasmid pKUC2, which contains the whole dnaK gene and its promoters, was capable of restoring the ability of E. coli BB1553 to plate λ phage but, surprisingly, it did not restore the ability to grow at high temperatures, even though it was shown that the DnaK protein was efficiently expressed in these cultures. By transposon mutagenesis and sub-cloning, we have shown the presence of a second gene in plasmid pKP31 which is required for high-temperature growth of E. coli BB1553. This gene, which we call htgA, is presumably also defective in the dnaK null mutant E. coli BB1553. We have also demonstrated that the inability of E. coli K756 to grow above 43.5 °C is complemented by sub-clones which contain the htgA gene, but not by plasmid pKUC2.

Summary: Rhodococcus rhodochrous ATCC 4273 (Nocardia corallina) has a restriction-modification system with the same recognition sequence, methylation site and cleavage site as the SalI restriction-modification system. Both the restriction endonuclease and the DNA-methyltransferase (DNA-MTase) have been partially purified and characterized. The nuclease has requirements for activity similar to SalI, and a native M r of about 46000. The DNA-MTase is a protein with an M r of about 67000. No DNA homology was detected between the cloned SalI restriction-modification genes of Streptomyces albus and R. rhodochrous chromosomal DNA.

Summary: ϕvML3 is a virulent prolate-headed bacteriophage that attacks a genetically well-studied strain family including Lactococcus lactis subsp. lactis strains 712, C2 and ML3. A restriction map was constructed for ϕvML3 using a wide variety of restriction endonucleases. The DNA was highly refractory to in vitro restriction; this is a common feature of lytic lactococcal phages. Genome size was estimated as 23 kb, which is similar to the sizes of other phages of the same morphological group. The presence of heat-dissociable DNA fragments was noted in gel electrophoresis of restriction enzyme digests. Cohesive ends were confirmed by pretreating the DNA with T4 ligase, which rendered the composite fragment insensitive to heat. The phage ϕvML3 genome thus consists of linear and non-permuted double-stranded DNA with complementary cohesive ends. The lysin gene from this phage acts as a specific lysis agent against the lactic streptococci. This gene has been cloned and sequenced previously. Further specific subcloning of EcoRV fragments of ϕvML3 DNA has located the lysin gene in the central region of the genome, orientated from right to left. This location was confirmed by hybridization of a lysin gene probe to ϕvML3 DNA. The lysin gene probe showed homology to a number of other prolate-headed phages including P001. The lysin gene of P001 was shown to be located in the central region of its genome. However, the isometric phage P107 did not hybridize, in spite of encoding its own lysin gene.

Summary: The pheA gene encoding the bifunctional P-protein (chorismate mutase: prephenate dehydratase) was cloned from Pseudomonas stutzeri and sequenced. This is the first gene of phenylalanine biosynthesis to be cloned and sequenced from Pseudomonas. The pheA gene was expressed in Escherichia coli, allowing complementation of an E. coli pheA auxotroph. The enzymic and physical properties of the P-protein from a recombinant E. coli auxotroph expressing the pheA gene were identical to those of the native enzyme from P. stutzeri. The nucleotide sequence of the P. stutzeri pheA gene was 1095 base pairs in length, predicting a 365-residue protein product with an M r of 40844. Codon usage in the P. stutzeri pheA gene was similar to that of Pseudomonas aeruginosa but unusual in that cytosine and guanine were used at nearly equal frequencies in the third codon position. The deduced P-protein product showed sequence homology with peptide sequences of the E. coli P-protein, the N-terminal portion of the E. coli T-protein (chorismate mutase: prephenate dehydrogenase), and the monofunctional prephenate dehydratases of Bacillus subtilis and Corynebacterium glutamicum. A narrow range of values (26-35%) for amino acid matches revealed by pairwise alignments of monofunctional and bifunctional proteins possessing activity for prephenate dehydratase suggests that extensive divergence has occurred between even the nearest phylogenetic lineages.

Summary: We have compared methicillin-resistant (Mcr) Staphylococcus aureus isolates from Australia, the UK and the USA with regard to chromosomal inserts of the macrolides-lincosamides-streptogramin B (MLS)-resistance transposon Tn554. The American isolates were known to have a distinctive Tn554 insert, designated insert 6, which was closely associated epidemiologically with the methicillin-resistance phenotype. Southern blots of DNA from Australian and London, UK Mcr isolates were hybridized with a range of probes related to Tn554. The isolates had similar or identical Tn554 inserts, and we consider them to be a single group, designated ‘Australondon'. Australondon isolates were compared in detail with a deletion mutant, ANS62, that had lost the methicillin-resistance determinant mec, plus other resistance determinants resident in the mec region of the chromosome, and with an American Mcr isolate containing Tn554 insert 6. The Australondon isolates had three Tn554 inserts. Sequence analysis with the polymerase chain reaction showed that all of these inserts differed from classical Tn554 in that the 3'-terminal residues of the transposons were reverse complements of the usual GATGTA. One of the Australondon inserts, designated 6B, closely resembled Tn554 insert 6 in the sequence of its left flanking chromosomal DNA. This insert was found to abut the deletion from the mec region which results in strain ANS62. We infer that Tn554 insert 6B is part of the mec region of the chromosome in Australondon isolates, supporting the idea that insert 6 of the American isolates is also part of this chromosomal region.

Summary: Restriction fragment length polymorphisms (RFLPs) of rRNA genes were evaluated as a tool for intra-and inter-species differentiation of Peptostreptococcus isolates. RFLPs from a collection of 20 clinical isolates and five ATCC strains representing five Peptostreptococcus spp. (P. anaerobius, P. asaccharolyticus, P. magnus, P. micros and P. prevotii) were obtained by hybridization of Southern blots of HindIII- or EcoRI-digested genomic DNA with three probes: probe A, a 0.98 kb HindIII fragment with a partial 16S rRNA gene sequence from P. anaerobius ATCC 27337; probe B, cloned Escherichia coli rrnB operon in plasmid pKK3535; and probe C, E. coli 16S and 23S rRNA. The hybridization patterns varied, but all yielded RFLPs useful for both intra- and inter-species differentiation. RFLPs of P. asaccharolyticus clinical isolates were closely related to each other and differed significantly from those of the ATCC type strains. The profiles of P. prevotii differed from those of the other four species studied, and based on the HindIII- and EcoRI-generated RFLPs, the strains in this species are more heterogeneous than the other four species studied.

Summary: The genetic determinant for abortive infection of bacteriophage (Abi) from the lactococcal plasmid pCI829 was cloned on a 6.2 kb StuI fragment in Escherichia coli using the shuttle vector pSA3. In Lactococcus lactis subsp. lactis MG1363Sm the resulting recombinant plasmid pCI816 conferred complete insensitivity to the small isometric-headed phage 712 and a reduced plaque size in the case of the prolate-headed phage c2. The determinant was further localized by subcloning and nuclease Ba131 deletion analysis; approximately 2.0 kb of DNA was essential for the expression of the Abi+ phenotype. Nucleotide sequence analysis of this region revealed a putative open reading frame of 1887 base pairs preceded by a putative promotor sequence and ribosome-binding site which exhibited similarity to consensus E. coli and Bacillus subtilis transcription/translation signals. Hybridization experiments indicated that this region was not homologous to the abi determinant from the phenotypically similar lactococcal plasmid pCI750.

Summary: Iron-uptake mutants of Neisseria gonorrhoeae strain 340 were obtained following treatment with streptonigrin, and one such mutant (Fud14) was characterized. N. gonorrhoeae strain Fud14 was unable to grow with human transferrin or haemoglobin as the sole source of iron, but grew normally with heat-inactivated normal human serum or haemin. Internalization of 55Fe from transferrin by strain Fud14 was only 25% of the parent level. Strain Fud14 (≤1 × 108 c.f.u.) did not grow in subcutaneous chambers implanted in mice, whereas the parent strain was infective at an ID50 of 4.3 × 101 c.f.u. Supplementation of chambers with either normal human serum or haemin resulted in the establishment of strain Fud14 in vivo for at least 240 h post-inoculation. Electroporation of Fud14 with wild-type DNA and selection for growth on medium containing human transferrin resulted in a recombinant (Fud15) that was capable of utilizing haemoglobin, and was virulent in mice. These results suggest that a gonococcal strain defective in the ability to utilize in vivo iron sources is not capable of survival in vivo.

Summary: A novel β-lactamase enzyme produced by a strain of Pseudomonas paucimobilis is described. The enzyme differs from other recorded β-lactamases from Gram-negative aerobic bacteria. It was constitutive, and had the characteristics of a penicillinase. One single band of β-lactamase activity at pI 4.6 was seen on iso-electric focusing. The enzyme had a molecular mass of 30 kDa. The β-lactamase was strongly inhibited by tazobactam, sulbactam and clavulanic acid but not by the thiol residue inhibitors p-chloromercuribenzoate and p-chloromercuriphenylsulphonic acid, or by metallo-enzyme inhibitors. Plasmid DNA was not demonstrable, suggesting that the enzyme was chromosomally encoded.

Summary: Bacteroides fragilis strains were classified as virulent or avirulent on the basis of their clearance from the subcutaneous tissues of mice. To determine the factors which may contribute to the virulence of B. fragilis strains, we studied encapsulation, hydrophobicity, growth rate, serum sensitivity, agglutination with erythrocytes of different origin, and neuraminidase production. The strains of the virulent group displayed a higher growth rate in broth and a lower sensitivity to the bactericidal activity of serum than the strains of the avirulent group. They also agglutinated different types of erythrocytes more strongly than did the avirulent strains. No significant differences were found between the two groups of strains as regards encapsulation, hydrophobicity and neuraminidase activity.

To help understand the subcellular machinery responsible for cell wall formation in a fungus, we determined the abundance and subcellular distribution of chitin synthetase (chitin synthase, EC 2.4.1.16) and chitosomes in the asexual life cycle of Mucor rouxii. Cell-free extracts of ungerminated sporangiospores, hyphae/mycelium in exponential and stationary phase, and yeast cells were fractionated by isopycnic centrifugation in sucrose density gradients. The total amount of chitin synthetase per cell increased exponentially during aerobic germination of spores. In all developmental stages, the profile of chitin synthetase activity encompassed a broad range of sucrose density (d = 1·12–1·22) with two distinct zones: a low-density chitosome zone (d = approx. 1·12–1·16) and a highdensity, mixed-membrane zone (d = approx. 1·16–1·22). Chitosomes were a major reservoir of chitin synthetase in all stages of the life cycle, including ungerminated spores. Two kinds of chitin synthetase profiles were recognized and correlated with the growth state. In nongrowing cells (ungerminated sporangiospores and stationary-phase mycelium), the profile was skewed toward lower densities with a sharp chitosome peak at d = 1·12–1·13. In actively growing cultures (aerobic mycelium or anaerobic yeast cells), the entire profile of chitin synthetase was displaced toward higher densities; the average buoyant density of chitosomes was higher (d = 1·14–1·16), and more chitin synthetase was associated with denser (d = 1·16–1·23) membrane fractions. In all life cycle stages, chitosomal chitin synthetase was almost completely zymogenic. In contrast to the enzyme from spores or from growing cells, samples of chitosomal chitin synthetase from stationary-phase mycelium were unstable and contained a high proportion of larger vesicles in addition to the typical microvesicles. The presence of chitosomes in ungerminated spores indicates that these cells are poised to begin synthesizing somatic (= vegetative) cell walls at the onset of germination. The increased buoyant density of chitosomes in actively growing cultures suggests that the composition of these microvesicles changes significantly as they mobilize chitin synthetase to the cell surface.

Summary: Pseudomonas putida mt-2, harbouring the TOL plasmid PWW0, was grown continuously on benzoate in a phauxostat at a non-limited rate. The gradual decrease in the population carrying the complete TOL plasmid was caused predominantly by a growth-rate advantage of spontaneous mutants carrying a partially deleted plasmid (TOL− cells). The growth-rate difference (v) was quantified both by measuring the increase in the dilution rate (from 0.68 to 0.79 h−1; v = 0.11 h−1) and by mathematical analysis of the ingrowth of TOL− cells (v = 0.12 h−1). The latter procedure also established that the segregation rate was of the order of magnitude 10−5 h−1. Similar values for the growth-rate advantage and the segregation rate were found when both benzoate and succinate were present in non-limiting concentrations. It is suggested that the growth-rate disadvantage of the wild-type strain is caused by inhibitory effects of an intermediate in the degradation of benzoate via the plasmid-encoded meta-pathway.

Summary: Pseudomonas putida mt-2, harbouring the TOL plasmid pWW0, was grown in chemostat culture under succinate-, sulphate-, ammonium- or phosphate-limitation at different dilution rates. The fraction of mutant cells lacking the plasmid-encoded enzymes for the degradation of toluene and xylene (TOL– cells), was determined. Genetic analysis revealed that all TOL– cells isolated harboured partially deleted plasmids, lacking the TOL catabolic genes. The growth-rate advantage of the TOL– cells was quantified from the kinetics of their increase as a fraction of the total population. At a dilution rate of 0·1 h–1 no growth-rate advantage of TOL– cells was found when phosphate or ammonium were limiting. Under sulphate-limitation, ingrowth of TOL– cells was evident but did not follow a straightforward pattern. Under succinate-limitation the growth-rate advantage was the highest, particularly at low dilution rates (about 50% at D = 0·05 h–1) In phauxostat culture, at the maximal growth rate, the growth-rate advantage of TOL– cells was less than 1%. The specific activity in TOL+ cells of the plasmid-encoded enzyme catechol 2,3-dioxygenase was relatively high at a low growth rate.

A number of strains of halophilic archaeobacteria of the genera Halobacterium and Haloferax were able to grow anaerobically using fumarate as electron acceptor. The species showing the best anaerobic growth with fumarate were Haloferax volcanii and Haloferax denitrificans. The two Haloarcula species tested did not show anaerobic growth enhancement with fumarate. During anaerobic growth of Haloferax volcanii in the presence of fumarate, succinate accumulated in the medium with a stoichiometry of only 0-16-0-23 mmol succinate per mmol fumarate consumed; this can be explained by the use of succinate for assimilatory purposes. The ability to reduce fumarate to succinate did not correlate with the ability to grow anaerobically using nitrate, dimethylsulphoxide or trimethylamine N-oxide as terminal electron acceptors. Anaerobic respiration with fumarate as electron acceptor supplies the halophilic archaeobacteria with an additional mode of energy generation in the absence of molecular oxygen.