- Volume 132, Issue 12, 1986

Summary: Sporothrix schenckii cells were grown on a medium containing yeast extract, neopeptone and glucose at 20°C to obtain a mixture of mycelia and conidia, and at 35°C to obtain yeast-like cells. The organism was maintained in the mycelial form, and its transformation to yeast at the higher temperature proceeded via conidia and ‘intermediate cells’ that then gave rise to yeast by a blastic mechanism. Cell-free extracts were analysed by PAGE at pH 8.0 and acid phosphatases (EC 3.1.3.2) were revealed by a sensitive detection reagent at pH 5.0. Mycelial, conidial and yeast extracts all had some acid phosphatase activity (M-I, C-I and Y-I) at the origin, although the proportion was highest for the yeast extracts. All of the bands that penetrated the gels had different electrophoretic mobilities. Mycelial and conidial extracts each had one other isoenzyme (M-II and C-II), while the yeast extracts had a total of five electrophoretically distinct acid phosphatases. Isoenzyme Y-II was further resolved into five closely related bands (Y-IIa to Y-Ile), the relative intensities of which varied with the phosphate nutrition of the yeast cells and the history of the extracts. The acid phosphatase isoenzymes were inhibited to various extents by sodium fluoride, L(+)-tartrate and phosphate, and showed interactions with citrate as opposed to acetate as the background buffer at pH 5.0.

Summary: The mechanism of inactivation of hexokinase PII of Saccharomyces cerevisiae by D-xylose was characterized. Inactivation was dependent on the presence of MgATP and was irreversible. Inactivation involved phosphorylation of the protein. Observation of the carbon catabolite repression of selected enzymes showed that invertase and maltase synthesis were not repressed when hexokinase PII was phosphorylated.

Summary: During pole formation in Bacillus subtilis the inner and outer surfaces of the nascent pole are enlarged by almost exactly the same extent. This means that the stress is almost uniformly distributed throughout the polar wall. This differs from the situation in the cylindrical side wall, where most of the stress is exerted in the outer portions of the intact wall. Because the stress is shared more uniformly, the maximum strain in any part of the polar wall is reduced, compared with the maximum strain within the side wall. The lowered stress may account, in part, for the resistance of the polar wall to hydrolysis by autolytic enzymes under certain conditions. The shape of the newly completed pole is significantly different from the spherical shape that the hydrostatic pressure would tend to produce. It does, however, achieve the shape that maximizes the polar volume under the restrictions arising due to expansion along the circumference not being possible near the junction of cylindrical and polar wall.

Summary: Mutations in three Escherichia coli K12 genes, tonB, exbB and the newly discovered semA, reduce sensitivity to the low Mr polypeptide antibiotic microcin E492. The products of the tonB and exbB genes were previously shown to be involved in the uptake of siderophore-complexed iron and in the action of a number of colicins. Strains mutated at or close to semA (collectively referred to as sem mutations) remained fully sensitive to these colicins, and grew as well as wild-type strains under conditions of iron starvation. Expression of a number of sem-lacZ operon fusions was not affected by iron limitation, and sem mutations did not affect the production of iron-regulated outer membrane proteins which are known or thought to be involved in iron uptake. Hfr conjugation and P1 phage transduction experiments indicated that semA is located close to pabB at 40 min on the E. coli K12 chromosome. This places semA close to the mng locus, wherein mutations result in decreased manganese sensitivity. However, strains carrying the semA mutation exhibited increased manganese sensitivity.

Summary: The structures of the colicin Ib plasmid (ColIb), the delta transfer factor and a plasmid determining kanamycin resistance and colicin Ib production called KColIb, were compared. Radiolabelled mini-ColIb plasmids and isolated DNA fragments of ColIb were used as probes for nitrocellulose blots of digests of the other two large plasmids. The structure of delta was consistent with it having one large deletion of about 10 MDa in the SB fragment and two insertions of approximately 6 MDa and 12 MDa in the SB and SA fragments of the ColIb plasmid. It was hypothesized that KColIb had six small insertions in SA, SB, SE and near the junction of the SB and SD fragments. However, ColIb, KColIb and delta were homologous for at least 70% of their lengths. The highly conserved regions in the three plasmids were the regions that corresponded to fragments SA, SC and SD of ColIb. In addition, delta and KColIb differed from ColIb at similar sites. The possible evolution of these plasmids is discussed.

Summary: DNA-DNA hybridization studies support the conclusion that coding sequences of the fertility inhibition gene, finO, are present on the F plasmid and map downstream of the transfer region, entirely within 99.19-2.0 F. In addition, the results indicate that the IS3a element at 100/0-1.26 F is inserted within the F finO coding region. This insertion may have inactivated the finO gene on the F plasmid.

Summary: Some general characteristics of five phages (Mm1, ϕM2, ϕM3, Mm4 and Mm5) infecting Micromonospora are presented. All were naked, showing an icosahedral head and long non-contractile tail; they differed in their size and the presence of specific structures at the end of the tail. The phages were temperate, and four immunity groups were delimited (Mm4 and Mm5 were in the same group). Mm4 and Mm5 produced plaques on 13 of 20 strains of Micromonospora tested, whereas the remaining three phages could infect only Micromonospora sp. IMET 8002. No phage was able to infect any of four Streptomyces strains tested. Phages Mm4 and Mm5 both exhibited a buoyant density of 1.513 ± 0.002 g ml−1 in CsCl density gradients, and showed a similar pattern of structural polypeptides. All five phages had a single molecule of double-stranded DNA; their sizes (kb) were 50.6 (Mm1), 18.9 (ϕM2), 65.5 (ϕM3), 44.0 (Mm4) and 44.4 (Mm5), as determined after digestion with 13 restriction enzymes. The restriction patterns of Mm4 and Mm5 showed the presence of common size fragments.

Summary: A mutant of Hansenula polymorpha defective in β-isopropylmalate dehydrogenase was isolated. It was transformed with YEp13, a shuttle vector containing the Saccharomyces cerevisiae LEU2 gene, either using the protoplasting method or by treatment with LiCl. Leu+ transformants were recovered at a frequency of 30-60 μg DNA)−1. The transformants were shown to contain an autonomously replicating plasmid by stability tests, hybridization experiments and by the recovery of YEp13 from transformed cells via transformation of Escherichia coli. YEp13 linearized by BamHI digestion was also effective in transformation; the linearized plasmid was shown to recircularize and replicate autonomously in the transformants.

Summary: Piliated Neisseria gonorrhoeae forming small, transparent colonies (P+O−) on clear typing agar have been grown in prolonged continuous culture to ascertain how different growth environments might affect gonococcal physiology and the expression of virulence determinants. Virulence of the penicillin-sensitive P9-2 and the penicillin-resistant KW1 strains was assessed by their ability to survive in polypropylene chambers implanted into the flanks of guinea pigs. Initial continuous culture experiments in the defined medium of Manchee et al. (FEMS Microbiology Letters 7, 115-118, 1980) indicated that growth was actually cystine-limited, rather than the anticipated glucose-limited. Surprisingly, cysteine was not completely metabolized and ammonium salts remained in excess. The molar growth yield on glucose (Y Glc) was 65 g dry wt mol−1 and 45% of the glucose carbon metabolized was converted to biomass. Gonococci, whilst retaining the P+0− phenotype for over 100 generations of growth, did not survive in the subcutaneous chambers when inoculated at a variety of doses. When the cystine and glucose concentrations were increased and decreased respectively, growth became glucose-limited, the Y Glc increased to 108 g mol−1 for strain KW1 and 75% of the metabolized glucose carbon was converted to biomass. After 17 generations of growth, however, only 2% of the gonococci retained the P+O− phenotype and P−O− bacteria predominated. Nevertheless, these bacteria were virulent in the chamber model, as was strain P9-2, which also retained only 2% of the P+O− phenotype during glucose-limited continuous culture. By contrast, the P+O− phenotype was retained during prolonged cystine- or oxygen-limited growth but only the latter was virulent. SDS-PAGE of membrane extracts confirmed that opaque colonies (O+) selected from the glucose-limited cultures contained a heat-modifiable protein (protein II) whereas transparent colony types lacked such proteins. The initial phenotype of virulent gonococci recovered from the subcutaneous chambers was P+O− but opaque variants dominated after several days. A 40 kDa outer-membrane protein was apparently induced during oxygen-limited continuous culture whereas a 44 kDa protein was absent during cystine-limited growth.

Summary: Studies indicated that prior growth of Staphylococcus aureus 196E on glycerol or maltose led to cells with repressed ability to produce staphylococcal enterotoxin A (SEA). A PTS- mutant (196E-MA) lacking the phosphoenolpyruvate phosphotransferase system (PTS), derived from strain 196E, showed considerably less repression of SEA synthesis when cells were grown in glycerol or maltose. Since SEA synthesis is not repressed in the PTS− mutant, repression of toxin synthesis by glycerol, maltose or glucose in S. aureus 196E appears to be related to the presence of a functional PTS irrespective of whether the carbohydrate requires the PTS for cell entry. With lactose as an inducer, glucose, glycerol, maltose or 2-deoxyglucose repressed the synthesis of β-galactosidase in S. aureus 196E. It is postulated that these compounds repress enzyme synthesis by an inducer exclusion mechanism involving phosphorylated sugar intermediates. However, inducer exclusion probably does not explain the mechanism of repression of SEA synthesis by carbohydrates.

Summary: This study reports the presence of sialic acid in Actinomyces viscosus strains T14V and T14AV. Mild acid hydrolysis of whole organisms released a compound which reacted positively in the periodate-thiobarbituric acid, direct Ehrlich's and resorcinol assays, and which co-chromatographed on paper with authentic N-acetylneuraminic acid. Strain T14V contained 10-fold greater concentrations of sialic acid than did strain T14AV. Sialic acid content was dependent upon the stage of growth of the culture, reaching a maximum in early stationary phase. Epifluorescence microscopy of fluorescein isothiocyanate (FITC)-conjugated Limulus polyphemus agglutinin (LPA), a lectin specific for sialic acid, revealed a uniform distribution of bound lectin on the surfaces of strains T14V and T14AV. Additional evidence for surface localization was obtained by demonstration of whole-cell agglutination of both strains with LPA. All LPA interactions with A. viscosus were inhibited by the presence of 0.1 M-N-acetylneuraminic acid. Neuraminidases from Clostridium perfringens, Arthrobacter ureafaciens and Vibrio cholerae did not release detectable amounts of sialic acid, but the extracellular enzyme from A. viscosus cleaved amounts equivalent to those obtained by acid hydrolysis. Other laboratory strains (W1053, M100, W859, 5-5S, RC45, ATCC 19246, and 'binder') as well as recent clinical isolates of A. viscosus were agglutinated by LPA and released sialic acid upon mild acid hydrolysis. Surface-available sialic acid has been implicated in the inhibition of alternative complement pathway activation and subsequent opsonophagocytosis. Thus the occurrence of surface sialic acid in A. viscosus may represent a mechanism of pathogenesis for this oral bacterium.

Summary: The effect of the glucose analogue 5-thio-D-glucose (5TG) on the yeast Saccharomyces cerevisiae was studied. Derepression of mitochondrial respiratory chain cytochromes, alcohol dehydrogenase (isoenzyme II), NADH dehydrogenase and maltase was inhibited by 0.5-2 mM-5TG. Growth rate was only slightly affected. Ethanol was efficiently produced with 2 mM-5TG in medium initially containing 0.25% glucose. Mutants resistant to the growth inhibitory effects of 5TG on glycerol medium showed resistance to the catabolite repressing effects of glucose. Other mutants, known to be catabolite repression resistant, showed resistance to 5TG. The analogue seems to inhibit derepression of glucose repressible enzymes with greater potency than glucose itself.

Summary: The effect of glutamine synthesis on sucrose metabolism in Neurospora crassa was studied. Different inhibitors of glutamine synthetase were used to inhibit glutamine synthesis in mutants having a low ammonium assimilation capacity. Sucrose utilization was impaired, as indicated by a lower concentration and synthesis of intermediates of the tricarboxylic acid cycle and reduced release of CO2. We propose that a coordinated regulation of carbon and nitrogen utilization is achieved through sensing of the carbon and nitrogen flows through glutamine synthesis, mediated by changes in the intracellular content of ATP, which is reduced as a consequence of glutamine synthesis.