- Volume 137, Issue 4, 1991

Summary: Spleen cells from mice immunized with a Bordetella pertussis N-lauroyl sarcosine membrane extract (SME) were used to generate hybridoma cells lines producing monoclonal antibodies (mAbs). Seven mAbs were shown to be specific to B. pertussis lipo-oligosaccharide (LOS) by immunoblotting of SME or purified LOS following SDS-PAGE. All mAbs reacted with the B. pertussis Tohama I strain of the LOS AB phenotype, and did not react with the atypical variant strain 134 of the LOS B phenotype. The immune reactivity of the mAbs was retained after treatment of SME with proteinase K and was lost after sodium periodate treatment. No cross-reactivity was observed with the mAbs when tested against B. parapertussis and other Gram-negative bacteria. However, all mAbs reacted with B. bronchiseptica. Binding assays with live B. pertussis cells demonstrated that mAbs strongly reacted with cell surface exposed antigenic determinants. High bacterial cell lytic capability was observed for five of these mAbs. Concentrations between 0·22 and 2·2 μg mAb ml–1 (0·1 and 1 μg per 450 μl assay) purified by protein A were required to kill at least 50% of the bacteria. Competition immunoassays with biotinylated antibodies showed that the bacteriolytic and non-bacteriolytic mAbs were directed to different epitopes of the B. pertussis LOS A.

Summary: The chloramphenicol acetyltransferase gene (cat) of a 3·9 kb chloramphenicol resistance (CmR) plasmid from Staphylococcus intermedius, designated pSCS1, was cloned into an Escherichia coli plasmid vector. Sequence analysis revealed a high degree of base similarity with the cat gene of the S. aureus CmR plasmid pC221 but there were several differences in the regulatory region. A lesser degree of similarity was observed between the cat gene of the S. intermedius plasmid and the cat gene of the S. aureus plasmid pC194.

Natural abundance 13C nuclear magnetic resonance spectroscopy identified the disaccharide trehalose as the major organic osmolyte synthesized by Escherichia coli grown in continuous culture under nitrogen limitation in the presence of 0·5 m-NaCl. Trehalose accumulation was dependent on both the growth phase of the culture and the osmolality of the growth medium, but independent of the solute used to increase the osmolality as long as the solute was non-penetrant. The penetrant solute glycerol did not induce trehalose synthesis indicating that the loss of cell turgor rather than increasing medium osmolality per se was the mechanism stimulating trehalose synthesis. Under conditions of either carbon or nitrogen limitation osmoadaptation was distinctly biphasic. The initial response consisted of a rapid (within 30 min) accumulation of K+ and a concurrent synthesis of the amino acid glutamate; trehalose synthesis occurred during the second slower phase of osmoadaption. Chloramphenicol severely inhibited trehalose accumulation indicating that the enzyme(s) involved in trehalose synthesis were inducible.

Agaricus bisporus was cultivated axenically on wheat straw compost. Analysis of this culture medium during growth and fruiting showed that the lignin fraction of straw was degraded preferentially during the vegetative growth phase, whereas cellulose was degraded after the emergence of the fruit bodies. A novel technique was developed, whereby natural or synthetic radiolabelled lignin was mixed intimately with axenic compost and the rate of mineralization to CO2 throughout the life-cycle of A. bisporus was monitored continuously without culture disturbance. Mineralization rates were maximal during the vegetative growth phase and the onset of fruiting brought a decrease of this activity. A mutant strain of A. bisporus, which was unable to develop fruiting bodies, was shown to mineralize radiolabelled lignin continuously.

Summary: Mutants unable to utilize acetate as sole source of carbon and energy were isolated from ethyl-methanesulphonate-mutagenized cultures of a prototrophic Bacillus stearothermophilus strain. Three groups of mutants had identifiable defects. Thirteen of the mutants lacked one or both enzymes of the inducible anaplerotic glyoxylate shunt, and three mutants were auxotrophic for isoleucine and valine when grown on acetate, in a manner analogous to acetohydroxyacid synthase I mutants of enteric bacteria. One mutant was defective specifically in acetate uptake, providing direct evidence for an acetate transport system in B. stearothermophilus. The acetate uptake system was uncoupler-sensitive and had a relatively high K m (in the range 300–350 μm). Escherichia coli was also shown to possess a saturable acetate uptake system, but its K m was much lower (15 μm).

Summary: The permissive temperature for sporulation of Bacillus megaterium 27 (up to 42 °C) was found to be 4–5 °C lower than that for growth. The non-permissive temperature suppressed the initial phases of sporulation characterized by the synthesis of an extracellular proteinase but the cells retained the ability to sporulate for several hours. Neither growth at supraoptimal temperatures nor heat shock applied at the end of the growth phase increased the permissive sporulation temperature. The organism synthesized at least ten heat-shock proteins, the dominant one being HSP 69. These proteins were also found in cells after 3 h of incubation at 43·5 °C but their presence did not ensure the ability to sporulate at this temperature. The rise of temperature provoked an imbalance between synthesis and degradation of cellular proteins, whose role in suppression of sporulation is discussed.

Summary: We have studied the regulation of the extracellular chymoelastase protease (PrI) of the fungal entomopathogen Metarhizium anisopliae. This enzyme is involved in the penetration of insect cuticle by Metarhizium and other entomopathogenic fungi. Changes in mRNA sequences during starvation-induced synthesis of chymoelastase were investigated by comparing poly(A+)RNAs and their complementary DNA in rapidly growing or nutrient-deprived cultures of Metarhizium. Hybrid-selected translation revealed that three novel polypeptides (41, 40·2 and 29·8 kDa) were produced rapidly (<1 h) during nutrient deprivation; the most intensely translated species (41 kDa) was identified as the primary translation product of Pr1 by examination with anti-Pr1-IgG. Measurement of starvation-specific RNA levels by dot-blot hybridization with [32P]cDNA showed that transcripts were not present in spores but were produced when the formation against a hard surface of infection structures was induced by depletion of endogenous nutrient reserves. Starvation-specific mRNA species failed to appear when new RNA synthesis was blocked with actinomycin D, indicating that control is at the level of transcription. Immunoblot analysis with anti-Pr1-IgG showed that Pr1 protein increased in concert with the appearance of Pr1 transcripts and active enzyme. Two very short lived precursors of Pr1, the primary translation product (41 kDa) and an intermediate (40·8 kDa) in addition to mature Pr1 (30 kDa) were detected by immunoprecipitation of [35S]methionine-labelled cell extracts with anti-Pr1-IgG. Pulse-labelling experiments demonstrated that a time of about 7 min was required for [35S]methionine to be processed into extracellular Pr1. These results suggest that regulation of Pr1 gene expression occurs during starvation which, coupled with fast processing, allows very rapid secretion of Pr1. Isolates of four other entomopathogenic fungi (Verticillium lecanii, Beauveria bassiana, Tolypocladium niveum and Paecilomyces farinosus) produced Pr1-type enzymes during nutrient deprivation, suggesting our results may have widespread application in understanding entomopathogenicity.

Summary: In the psychrophilic bacterium Vibrio sp. strain ANT-300, the temperature-related characteristics of protein synthesis in cells grown at 0 °C differed from those of cells grown at 13 °C. Cells grown at 0 °C and 13 °C transported amino acids at the same rates, dependent on the temperature at which rates were measured. The rates of protein synthesis in extracts of cells grown at 0 °C and at 13 °C differed, as a result of the changes in the properties of the soluble fraction involved in protein synthesis. Concurrently, levels of more than 24 polypeptides in the soluble fraction changed considerably. These results suggest that the difference in temperature dependence of protein synthesis in cells grown at various temperatures may be brought about by specific changes in the levels of a small number of polypeptides (less than 15% of the total number of proteins detected by silver-staining) in response to a change in temperature.

Summary: The glycopeptide antibiotic teicoplanin belongs to the same group as vancomycin and ristocetin and is a valuable tool for studying the autolytic system of sensitive Gram-positive bacteria. Teicoplanin, at a concentration of 1 μg ml–1, caused rapid lysis of exponential phase cells of Streptococcus faecalis. Bacillus spp. were most sensitive to the antibiotic; effective lysis occurred at 0·1 μg teicoplanin ml–1. The bacteriolytic effect depended on the antibiotic concentration, the growth phase and growth rate of the target organism. Antibiotic added to overnight cultures did not cause lysis. Mg2+ (50 mm) was unable to prevent lysis. Mutants with decreased autolytic activity were more resistant to teicoplanin and lysed more slowly than the wild-type. Growth of bacteria in slightly acidic medium protected the cells against the lytic effect of teicoplanin typically observed at pH 7 or 8. This pH-dependent antibiotic tolerance was demonstrated with both bacilli and streptococci. Bacterial lysis was prevented by the presence of Ac-l-Lys(Ac)-d-Ala-d-Ala and normal growth was observed when this peptide was added simultaneously with teicoplanin. Bacteria pretreated with teicoplanin, washed and transferred to fresh medium or buffers behaved as if the antibiotic was still present; in neutral or slightly alkaline conditions strong lysis occurred, whereas in acidic buffer only bacteriostasis was observed. In contrast to vancomycin, teicoplanin induced some lysis of bacteria in hypertonic media, presumably by affecting the integrity of the cell membrane.

Summary: A study was made of the effects of adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP) and choline on the morphology and growth of a wild-type strain (A 3/5) and a highly branched, “colonial” mutant strain (C106) of Fusarium graminearum. Addition of up to 50 mm-cAMP or cGMP to the medium had no effect on the specific growth rate of strain A 3/5. For strain A 3/5, but not for strain C106, exogenous cAMP caused significant decreases in both mean hyphal extension rate (E) and hyphal growth unit length (G), i.e. cAMP caused mycelia of strain A 3/5 to branch profusely. By contrast, for both strains, cGMP caused significant increases in both E and G, i.e. exogenous cGMP caused mycelia to branch more sparsely. The effects of exogenous cGMP and choline in increasing E and G were synergistic, but the effects of cGMP and choline counteracted the effect of cAMP. The mutant phenotype of strain C106 was not correlated with altered levels of endogenous cAMP or cGMP.

Summary: Activities of glycolytic enzymes were determined in elutriation fractionated cultures of Saccharomyces cerevisiae grown on different carbon sources. Almost pure fractions of single cells at the G1 stage of cell division were obtained for some of the growth conditions tested, whereas other stages were enriched in particular fractions. Specific activities of glucose-6-phosphate dehydrogenase and alcohol dehydrogenase were found to be constant during the cell cycle, as reported by van Doorn et al. (1988a) , Journal of Bacteriology 170, 4808–4815, and (1988b), Journal of General Microbiology 134, 785–790. In contrast to the earlier reports, the activities of hexokinase, phosphofructokinase, pyruvate kinase and trehalase were also constant in different stages of the cell cycle. For hexokinase and phosphofructokinase it was shown that the apparent specific activity in a cell-free extract strongly diminished when extracts contained less than 0·5-1 mg protein ml−1. In the experiments of van Doorn et al. (1988a) the protein content of the outer fractions was up to 20 times lower than that of the central fractions, suggesting an alternative explanation for the observed changes in enzyme activities during the cell cycle. Therefore, we want to rectify the observations presented by van Doorn et al. (1988a) , and conclude that the activities of the glycolytic enzymes do not vary greatly during the cell cycle of S. cerevisiae.