- Volume 141, Issue 10, 1995

Summary: Novel recombinant plasmids derived from the Kluyveromyces lactis killer plasmid k2 have been constructed to study plasmid biology and gene function. In vivo recombination between native resident k2 and suitable disruption vectors, employing the KlTRP1 gene fused to a plasmid promoter as selection marker, yielded ORF2 and ORF6 deletion plasmids at high frequencies. As judged from Southern hybridization and plasmid restriction mapping analyses, these novel hybrids, termed rk2/2 and rk2/6, respectively, carry deletions in their putative DNA (ORF2) and RNA (ORF6) polymerase structural genes with central regions replaced by the input marker DNA. Long-term selection for TRP1 over 350 generations of growth did not favour maintenance of hybrids over wild-type k2. Thus, neither rk2/2 nor rk2/6 was fully functional and able to displace parental k2, indicating that both target genes are essential for plasmid integrity or maintenance. Recombinant plasmids were reduced in copy number relative to k2 with rk2/2 more drastically affected than rk2/6 implying a direct involvement of the ORF2 product in plasmid replication and an indirect maintenance function for the ORF6 gene product.

Summary: A canCHS1A gene encoding the chitin synthase of Candida albicans was cloned. DNA sequencing and comparison with another canCHS1 gene described elsewhere indicated that the canCHS1A gene encoded a polypeptide with 775 amino acid residues, a protein with one less amino acid than that encoded by the canCHS1 gene. A six-base alteration was observed between the two genes, suggesting that the canCHS1A gene is a variant gene of the canCHS1. The pH profile/activity relationship of canChs1A in permeabilized cells was identical to that of canChs1. The canChs1A enzyme was competitively inhibited by polyoxin D (5.2 μM) and nikkomycin Z (12 μM). When the cloned gene was expressed in a Saccharomyces cerevisiae chs2 mutant that exhibited aberrant morphology, the normal structure was restored. We conclude that the function of the canCHS1A gene is similar to that of sacCHS2 in S. cerevisiae.

Summary: Cytosolic fractions of mycelial extracts from Aspergillus nidulans, A. flavus, and three different isolates of A. fumigatus, grown to stationary phase in Czapek-Dox-AOAC medium, were tested by immunoblotting for the presence of antigens reactive to 80 serum samples from aspergilloma patients. Fifty control serum samples were used to determine the specificity of the reactions. In the A. fumigatus cytosolic fraction a group of four main antigenic bands (p90, p60, p40 and p37) was consistently recognized (in total or partial form) by 90% of the serum samples from the aspergilloma patients. This group of antigens was designated as the ‘cytosolic fraction complex’ (CFC). As confirmed by two-dimensional electrophoresis followed by immunoblotting with aspergilloma serum samples, each of the four antigenic bands is formed of several isoforms of acidic glycopeptides with slightly different pls. All the isoforms are at least N-glycosylated, as demonstrated by endoglycosidase H removal of a considerable amount of sugar residues. The relationship of these antigens with certain other A. fumigatus antigens previously reported in the literature, and their potential use in the immunodiagnosis of aspergilloma, are discussed.

Summary: The resurgence of tuberculosis, the emergence of multiple drug resistant tuberculosis, and the increasing prevalence of mycobacterial disease in AIDS patients have increased the importance of defining new mycobacterial antigens that can be utilized in the development of improved diagnostic reagents and more effective vaccines. In this report, a highly immunogenic Mycobacterium kansasii protein (MK35) and the gene encoding this antigen were characterized. MK35 gene probes reacted with genomic DNA from M. avium, M. bovis BCG, M. intracellulare and M. tuberculosis but not with DNA isolated from nine other mycobacterial species. Nucleotide sequence analysis showed that the MK35 gene encodes a 26 kDa protein which contains a consensus bacterial lipoprotein processing sequence. In addition, detergent-phase separation studies strongly suggested that MK35 is a lipoprotein. Skin test assays demonstrated that MK35 elicited a strong response in guinea pigs sensitized with M. kansasii but did not react in M. tuberculosis-sensitized guinea pigs. These results further suggest that mycobacterial lipoproteins are immunogenic antigens that should be considered in the development of new mycobacterial vaccines and diagnostic reagents.

Summary: Cells of the oral bacterium Streptococcus oralis CN3410 produce lateral tufts of cell-surface fibrils of two lengths. Treatment of cells with trypsin resulted in loss of the tufts and release of longer fibrils intact. SDS-PAGE analysis of trypsin extracts containing fibrils revealed two groups of high molecular mass polypeptides which were denoted group A (molecular mass 227-246 kDa) and group B (molecular mass 175-208 kDa). Antibodies were raised to these two groups of trypsin-extracted polypeptides (TEPs) and to purified fibrils, and the reactivities of the three different antisera were found to be similar both on nitrocellulose blots of cell-surface polypeptides and in ELISA with whole cells. Similar patterns of TEPs were obtained from cells of a spontaneously derived mutant strain, KP34V, which lacked the short fibril components of tufts. Cells of strain KP34V had similar cell-surface hydrophobicity to strain CN3410 cells, and adhered to the same extent to parotid salivary pellicle or human buccal epithelial cells (BECs) as the wild-type cells. Trypsin treatment of strain CN3410 cells abolished their surface hydrophobicity and ability to adhere to BECs, but did not affect streptococcal cell binding to experimental salivary pellicle. Antibodies to TEPs or fibrils had no effect on cell adhesion to BECs or salivary pellicle. The results imply that the short fibril components of tufts are not involved in the cell adhesion properties tested. It is suggested that the TEPs are components of long fibrils, but they are not determinants of streptococcal cell adhesion to pellicle or to epithelial cells.

Summary: Sequencing 280 bp of the internal transcribed spacer (ITS) between the 16S and 23S rRNA genes in a collection of 46 clinical isolates of the Mycobacterium avium-intracellulare complex (MAI complex) identified nine different sequences, grouping these isolates in nine ‘ITS sequevars’. This analysis extends the subdivision within the MAI complex to 18 ITS sequevars and also improves discrimination from other mycobacterial species. Evaluation of the sequevar grouping among different clinical sources revealed strong association of the M. avium sequevar Mav-B with AIDS and with lymphadenitis in children (18 out of 20 and 3 out of 3 respectively). Isolates from elderly patients with pulmonary disease and not suspected of being HIV infected belonged predominantly to M. intracellulare ITS sequevars and sequevars not assigned to either M. avium or M. intracellulare. On the other hand, animal isolates were of both the Mav-A and Mav-B sequevars. We conclude that ITS sequevar typing is an accurate way of identifying distinct MAI complex strains. The observed differences between clinical sources suggest that ITS sequevars reflect possibly important, biologically and clinically relevant polymorphisms between MAI complex organisms.

Summary: Strains of Pseudomonas aeruginosa isolated from patients with cystic fibrosis (CF) never spread systemically. This may be due to serum sensitivity since these strains are very sensitive to complement-mediated bactericidal activity. A serum-resistant mutant, P. aeruginosa TUM3 HSR, was obtained from serum-sensitive strain TUM3 from a CF patient in order to clarify the mechanism of failure of systemic spread. LPS profiles on silver-stained gels and immunological analysis revealed that a long O-polysaccharide side chain was overproduced on the LPS molecules of TUM3 HSR as compared with the LPS of TUM3. The clearance rate from the bloodstream in mice was compared in the two strains. The number of TUM3 bacteria in 1 ml of blood, 10 min after injection into the tail vein, significantly decreased from 1.7 × 108 to 3.7 × 105 c.f.u. ml-1. In contrast, TUM3 HSR was not eliminated during the same period (decrease from 1.9 × 108 to 3.4 × 107c.f.u. ml-1). Interestingly, these isogenic strains were not killed by 40% murine serum, probably reflecting immaturity of the complement-mediated killing system in mice. These results pointed to a correlation between LPS structure and blood clearance rate in mice. This was confirmed by examining blood clearance kinetics using the smooth-LPS strain Salmonella typhimurium LT2 and LPS-deficient mutants derived from it. S. typhimurium LT2 resisted blood clearance while the LPS-deficient mutants were cleared rapidly. None of the S. typhimurium strains were killed by murine serum. The number of P. aeruginosa TUM3 and S. typhimurium LPS-deficient mutants trapped in the liver following injection into the peripheral circulation was greater than that of their counterparts. These results indicate that the long O-polysaccharide side chain of LPS may play a crucial role in evading phagocytosis by the reticuloendothelial system (RES), and therefore, may control the establishment of systemic infection by Gram-negative bacteria. The interaction between complement C3 on bacteria and C3 receptors on macrophages may also be involved in the trapping mechanism by the RES. It was expected that the level of C3 bound on the cell surface would be higher in P. aeruginosa TUM3 or the LPS-deficient mutants derived from S. typhimurium LT2. However, our flow-cytometric results demonstrated that the level of C3 was almost identical in isogenic strains.

Summary: The activities of the enzymes involved in ammonium assimilation and glutamate biosynthesis were determined in wild-type and NADP-glutamate dehydrogenase (GDH) null mutant strains of Kluyveromyces lactis. The specific NADP-GDH activity from K. lactis was fivefold lower than that found in Saccharomyces cerevisiae. The glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were similar to those reported in S. cerevisiae. The NADP-GDH null mutant was obtained by transforming the uraA strain MD2/1 with a linearized integrative yeast vector harbouring a 390 bp fragment of the NADP-GDH structural gene. This mutant grew as well as the parent strain on ammonium, but showed GS and GOGAT activities higher that those found in the wild-type strain, implying that the GS-GOGAT pathway could play a leading role in glutamate biosynthesis in K. lactis. Southern blotting analysis of K. lactis chromosomes separated by contour-clamped homogeneous electric field electrophoresis, indicated that the NADP-GDH structural gene is localized on chromosome VI.

Summary: The intra- and extracellular concentrations of a-amylase in Aspergillus oryzae have been measured during batch culture of a wild-type strain and two recombinant strains. The mean intracellular level for the two recombinant strains was about four to five times the level of the wild-type strain. The recombinant strains also had a higher a-amylase productivity, whereas the residence time of the intracellular a-amylase pool was approximately the same for the three strains. At high glucose concentrations there was a low constitutive synthesis of a-amylase, whereas at low glucose concentrations derepression resulted in an increased production rate. Shifts from a glucose- to a maltose-limited chemostat showed that maltose induces both the production and secretion of a-amylase. Finally, from immunoblots, both a glycosylated and an unglycosylated a-amylase have been detected.

Summary: The effect of medium hypo-osmolality on cell volume and intracellular amino acid composition was studied in the protozoan parasite Giardia intestinalis. When G. intestinalis was exposed to hypotonic medium, it initially swelled by 40% of its original volume and then decreased its volume, thus demonstrating a regulatory volume decrease process (RVD). These processes were accompanied by a rapid release of intracellular neutral amino acids, especially alanine, but not by amino acids with net charges such as glutamate and ornithine. The net alanine efflux was Na+ and Cl- independent, and sensitive to medium osmolality. Alanine efflux was sigmoidal with respect to medium osmolality, with an approximately linear relationship over a range of 250 mOsm kg-1. Alanine efflux was also sensitive to temperature, and an Arrhenius plot gave a Q 10 of 3.6 and an activation energy of 25 kcal mol-1 (105 kJ mol-1), suggesting that a carrier-type transport protein, or uniport, was involved in the net alanine efflux under hypotonic conditions. This volume-activated (VA) alanine efflux was not inhibited by ionophores or chloride channel blockers. Of the potential inhibitors tested, only p-hydroxymercuribenzoate inhibited net alanine efflux. This thiol reagent also inhibited giardial RVD, suggesting that alanine efflux plays a significant role in this process. The VA (alanine) uniport was able to transport 2-aminoisobutyric acid (AIB), a structural analogue of alanine which is frequently used for the characterization of eukaryotic alanine transport, but which is not transported by Giardia under isotonic conditions. On the basis of AIB uptake under hypotonic conditions and lack of transactivation of AIB efflux from AIB-loaded cells by external 10 mM alanine or glycine under isotonic conditions, it is evident that the VA (alanine) uniport is different from the previously reported (alanine) antiport.

Summary: Cells of the yeast Saccharomyces cerevisiae sporulated in nutrient-rich medium containing l-lysine. Sporulation was specific to the presence of L-lysine and was initiated when the cellular content of this basic amino acid reached approximately 0·2-0·5 mmol (g cells)−1, at early stationary phase. The formation of asci was most efficient at pH 7·0 and 50-100 mM L-lysine; in these optimum conditions, the sporulation frequency reached about 60% after 5 d incubation. The L-lysine-dependent sporulation system in nutrient-rich conditions was distinct from the currently used potassium-acetate-dependent system in nutrient-deficient conditions. Analysis of the L-lysine-dependent system indicated that, prior to entrance into meiosis and/or sporulation processes, the yeast cells change in shape, their pool sizes for L-cysteine and glutathione alter, and they synthesize a protein with a molecular mass of 15 kDa. A low level of cAMP was not required for the entrance into meiosis and/or sporulation.

Summary: Trichodesmium is a marine filamentous cyanobacterium with the exceptional ability to fix atmospheric nitrogen during the day without differentiating the specialized oxygen-protective cells known as heterocysts. The localization of the Fe-protein of nitrogenase (dinitrogenase reductase) was examined in cross-sectioned colonies of three species: Trichodesmium thiebautii, T. tenue and T. erythraeum, using immunogold/transmission electron microscopy and immunofluorescence/light microscopy. The enzyme was confined to a limited subset of cells (on average 14%) in the sections, randomly distributed within the colonies in all three species. The frequency of nitrogenase-containing cells varied on a diurnal basis, being highest and comparatively constant during the day, the period of active nitrogen fixation. The percentage of nitrogenase-labelled cells decreased in the evening and approached zero just before dawn. After sunrise a period of rapid synthesis of nitrogenase followed. Although the frequency of nitrogenase-containing cells was constant during the day, the relative concentration of nitrogenase within the labelled cells clearly increased until noon and then successively decreased in the afternoon/night. The data imply that in three species of the non-heterocystous cyanobacterium Trichodesmium there are a limited number of cells, or possibly whole trichomes (filaments), which are specialized for nitrogen fixation. Most likely the same set of cells is active throughout the day, with the variation in activity being due to a variation in the quantity of nitrogenase within each cell. Comparisons between nitrogenase-containing Trichodesmium cells and heterocysts are discussed.

Summary: Washed cells of Agrobacterium radiobacter prepared from a fructose-limited continuous culture (D 0.045 h-1) transported D-[U-14C]fructose in a linear manner for up to 4 min at a rate several-fold higher than the rate of fructose utilization by the growing culture. D-[U-14C]Fructose transport exhibited a high affinity for fructose (K T < 1 μM) and was inhibited to varying extents by osmotic shock, by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and by unlabelled sugars (D-fructose/D-mannose > D-ribose > D-sorbose > D-glucose/D-galactose/D-xylose; no inhibition by D-arabinose). Prolonged growth of A. radiobacter in fructose-limited continuous culture led to the selection of a novel strain (AR100) which overproduced a fructose-binding protein (FBP) and showed an increased rate of fructose transport. FBP was purified from osmotic-shock fluid using anion-exchange fast protein liquid chromatography (FPLC). The monomeric protein (Mr 34200 by SDS-PAGE and 37700 by gel-filtration FPLC) bound D-[U-14C]-fructose stoichiometrically (1.17 nmol nmol FBP-1) and with high affinity (KD 0.49 μM) as shown by equilibrium dialysis. Binding of D-[U-14C]fructose by FBP was variably inhibited by unlabelled sugars (D-fructose/D-mannose > D-ribose > D-sorbose; no inhibition by D-glucose, D-galactose or D-arabinose). The N-terminal amino acid sequence of FBP (ADTSVCLI-) was similar to that of several sugar-binding proteins from other species of bacteria. Fructose transport and FBP were variably induced in batch cultures of A. radiobacter by growth on different carbon sources (D-fructose > D-ribose/D-mannose > D-glucose; no induction by succinate). An immunologically similar protein to FBP was produced by Agrobacterium tumefaciens and various species of Rhizobium following growth on fructose. It is concluded that fructose is transported into A. radiobacter and related organisms via a periplasmic fructose/mannose-binding-protein-dependent active-transport system, in contrast to the phosphotransferase system used by many other species of bacteria.

Summary: The fatty acid composition of the cellular lipids of Rhodococcus rhodochrous NCIMB 13064 grown on various long-chain haloalkanes has been investigated and the influence of halogen substituents, carbon chain length and the position of halogen substitution in the growth substrate explored. Of the total fatty acids present in cells grown on 1-chloro-, 1-bromo- and 1-iodohexadecane, 75, 90 and 81%, respectively, were substituted in the w position by the corresponding halogen but only 1% of the fatty acids present after growth on 1-fluorotetradecane were fluorinated in this position. The extent of the halofatty acid incorporation with different halogen substituents in the growth substrate appears to reflect the degree to which oxygenase attack is restricted to the non-halogenated end of the haloalkane. Studies of the fatty acid composition of cells after growth on a series of 1-chloroalkanes containing an even number of carbon atoms between C10 and C18 indicated chlorofatty acid incorporation from C12 to C18 substrates at levels ranging from 21% with C12 to 75% with C16. The chlorofatty acids formed by initial oxidation of the chloroalkane were chain-lengthened or chain-shortened by from two to eight carbon atoms, with accompanying desaturation in some instances. Substantial quantities of a methyl-branched C19:0 chlorofatty acid were also present with several chloroalkane substrates. When the fatty acid composition of cells after growth on 1-bromoalkanes containing an odd number of carbon atoms between C11 and C17 was examined, the incorporation of bromofatty acids was observed with C13, C15 and C17 substrates; a maximum of 76% was recorded for the C15 bromoalkane. As with even chain-length chloroalkanes, both chain-lengthening and -shortening occurred predominantly via two-carbon units so that most bromoacids present possessed an odd number of carbon atoms. When 1-bromododecane or 2-bromododecane were substrates, overall incorporations of bromofatty acids into the lipid fraction were very similar, demonstrating that the position of halogen substitution in the haloalkane was not critical in determining the extent of incorporation of the haloacids into cellular lipids. The results of the study indicate a mechanism by which degradation products of chlorinated paraffins could enter the biological food chain.

Summary: Pyruvate carboxylase (PC) was purified to homogeneity from an overexpressing strain of the purple photosynthetic bacterium Rhodobacter capsulatus using a rapid dye-ligand affinity chromatography procedure, in which dye-bound enzyme was specifically eluted with a low concentration of acetyl-CoA, an allosteric activator of the enzyme. The enzyme purified by this method was obtained in 75% yield with a specific activity of 40 U (mg protein)-1. In contrast, affinity chromatography on a monomeric avidin column, commonly used in the purification of biotin-containing carboxylases, resulted in a yield of < 40%, with a specific activity of 10 U (mg protein)-1 The enzyme purified by the dye-linked procedure had a subunit molecular mass of 140000 Da and was absolutely dependent on acetyl-CoA for activity. Acetyl-CoA was also effective in protecting the enzyme from thermal denaturation. The enzyme was inhibited by 2-oxoglutarate and, to a lesser extent, L-aspartate, with sigmoidal kinetics with respect to acetyl-CoA concentration. The amino acid composition, pH optimum and kinetic constants for pyruvate, ATP and bicarbonate were determined. An N-terminal sequence of 26 residues was obtained, which was homologous to the N-terminal regions of several eukaryotic PCs, propionyl-CoA carboxylases and acetyl-CoA carboxylase.

Summary: Strain N106, a newly isolated soil actinomycete classified in the genus Nocardioides on the basis of its chemotaxonomy, produced an extracellular chitosanase and was highly active in chitosan degradation. A gene library of Nocardioides sp. N106 was constructed in the shuttle vector pFD666 and recombinant plasmids carrying the chitosanase gene (csnN106) were identified using the 5′-terminal portion of the chitosanase gene from Streptomyces sp. N174 as a hybridization probe. One plasmid, pCSN106-2, was used to transform Streptomyces lividans TK24. The chitosanase produced by S. lividans(pCSN106-2) is a protein of 29.5 kDa, with a pl 8.1, and hydrolyses chitosan by an endo-mechanism giving a mixture of dimers and trimers as end-products. N-terminal sequencing revealed that the mature chitosanase is a mixture of two enzyme forms differing by one N-terminal amino acid. The csnN106 gene is 79.5% homologous to the csn gene from Streptomyces sp. N174. At the amino acid level, both chitosanases are homologous at 74.4% and hydrophobic cluster analysis revealed a strict conservation of structural features. This chitosanase is the third known member of family 46 of glycosyl hydrolases.

Summary: HPLC techniques have been applied to study amino acid uptake and release by Trichomonas vaginalis under a variety of conditions. Studies on the growth of T. vaginalis in complex media and the survival of the parasite in simple media, with and without amino acids and/or maltose, have shown that the growth or survival of T. vaginalis is better in the presence of maltose than when it is absent, and that greater amounts of amino acids are consumed by T. vaginalis in the absence of maltose. The results are consistent with several amino acids, notably arginine, threonine, leucine and methionine, being used by T. vaginalis as energy substrates. T. vaginalis released alanine and glycine into the culture media, the excretion being greater in the presence of maltose. These studies have provided new data on the uptake and release of amino acids by T. vaginalis and pave the way for detailed analysis of key enzymes and the regulation of the pathways involved.