- Volume 139, Issue 7, 1993

Summary: Luteolin is a flavonoid produced by plants which is required for induction of nod genes in Rhizobium meliloti. R. meliloti absorbed luteolin at higher rate than all other bacteria tested, including R. leguminosarum. The flavonoids naringenin and quercetin, which do not induce the expression of nodulation genes of R. meliloti, were absorbed at a lower rate by this species, suggesting a certain degree of species specificity of flavonoid absorption. Luteolin accumulated preferentially in the outer membrane, but a small amount was always found in the inner membrane. Luteolin strongly inhibited NADH oxidase, an enzyme of the respiratory chain, raising the possibility that the site of luteolin absorption in the outer membrane allows the protection of the respiratory chain located in the inner membrane from an excess of flavonoids. The incorporation of luteolin was found to be affected in some exo or nod mutants of R. meliloti. The exoB mutant, which does not produce exopolysaccharides, accumulated lower amounts of luteolin in the outer membrane than the exo + parent. Among the nod mutants affected in nodulation genes, those not expressing any of the three nodD genes accumulated luteolin at a significantly lower level in both the outer and the inner membrane. A strain overexpressing the nod genes, particularly the nodD genes, absorbed luteolin at a higher level in both membranes. These results indicate that absorption of luteolin by R. meliloti involves several gene products, including the NodD protein.

Summary: The sequence of the small subunit nuclear ribosomal DNA (18S rDNA) was determined for seven selected species of the teliospore-forming yeasts and Filobasidiaceae in the basidiomycetous yeasts. A phylogenetic tree, including published reference sequences, was inferred from 1623 sites which could be unambiguously aligned. The molecular phylogeny, using a chytridiomycete as an outgroup, divided the eight basidiomycetous yeasts into two groups which correlated well with both septal ultrastructure (simple pore or dolipore) and cellular xylose (present or absent). The first group included the teliospore-forming yeasts Rhodosporidium toruloides and Leucosporidium scottii, and Erythrobasidium hasegawianum, which is currently a member of the Filobasidiaceae. The second group was formed by the filobasidiaceous yeasts, comprising a Cystofilobasidium capitatum/Leucosporidium lari-marini/Mrakia frigida branch, and a Filobasidium floriforme/Filobasidiella neoformans branch. Our molecular data support the principal chemotaxonomic and ultrastructural evidence, which indicates a very close affinity between C. capitatum and L. lari-marini.

Summary: The metabolism of copper in the yeast Saccharomyces cerevisiae has been studied with respect to the distribution and stability to exchange of newly arrived 64Cu. Cells pre-incubated with 10 m-Cu2+ accumulated 64Cu into two pools distinguishable by cellular locale and lability to exchange with extracellular cold copper. One pool was non-exchangeable and was localized to protoplasts. Size-exclusion chromatography of a soluble cell (protoplast) extract showed that this 64Cu was associated with up to four species. Two were identified as copper metallothionein and Cu,Zn superoxide dismutase based on comparisons of chromatograms derived from strains in which the genes for these two proteins had been deleted. A third species was identified as copper-glutathione based on chromatographic and biochemical assays. A second pool was exchangeable and was localized to the cell wall. In contrast to its rapid copper-stimulated exchange (t1/2 % 1 min), this pool exhibited only slow efflux (10% 64Cu loss per 60 min). Zn2+ did not stimulate the loss of 64Cu from this pool indicating that it was selective for copper. This pool was released into the supernatant upon protoplast formation and was found in the cell wall debris obtained when cells were mechanically disrupted. This 64Cu eluted in the void volume (peak Pv) of the column used to size-fractionate copper-binding species. The metal in Pv was exchangeable in vivo and in vitro. However, the corresponding chromatographic fraction obtained from copper-naive cells when labelled in vitro could bind less than 20 % of the 64Cu bound to it in vivo indicating that the deposition of copper in this pool was primarily cell-dependent. In fact, this deposition was shown to be dependent on the cellular reduction of medium sulphate or sulphite to the level of sulphide, or on the addition of sulphide to the 64Cu uptake buffer. 64Cu in the non-exchangeable protoplast pool was not mobilized by cellular sulphide generation, indicating that cellular sulphide generation did not causally lead to the partitioning of 64Cu to the cell wall pool. The data indicate that the appearance of copper sulphide(s) on the cell wall in S. cerevisiae is gratuitous and does not represent a sulphide-based mechanism of copper resistance in this yeast.

Summary: The cell association of copper in the yeast Saccharomyces cerevisiae can involve both binding to the cell wall and the accumulation of copper within the cell. The former process requires the concurrent generation of H2S by the cell via the reduction of sulphate. The contributions of each of these processes to the uptake of 64Cu by wild type and met3-containing (ATP sulphurylase-deficient) strains have been kinetically dissected. The Michaelis constant for uptake (4 m) is independent of the type of cell association which is occurring, suggesting, although not requiring, that both processes are associated with a common kinetic intermediate. The time dependence of the cell-association of 64Cu also suggests the presence of this intermediate pool of bound copper. The Vmax for uptake includes a constant contribution from accumulation of 64Cu within the plasmalemma [0.1 nmol min-1 (mg protein)-1] plus that fraction of the 64Cu within the intermediate pool which diffuses away and is trapped on the cell wall as a metal sulphide. This latter contribution to V max can be two- to three-times greater than the intracellular uptake depending on the amount and type of sulphur supplementation provided in the 64Cu2+ uptake buffer. Both processes are energy-dependent although the sulphide-dependent periplasmic accumulation is somewhat more sensitive to metabolic inhibition. This can be attributed to the ATP required for the activation of sulphate prior to its reduction to the level of sulphite and then sulphide. Periplasmic 64Cu accumulation is strongly inhibited by Zn2+ and Ni2+. This inhibition is due to competition for cell-generated sulphide; in the presence of 64Zn2+, the decrease in 64Cu bound is quantitatively related to the amount of 65Zn which becomes cell-associated. In contrast, intracellular 64Cu uptake is not inhibited by these two metals (at 50 M) showing that the copper translocation pathway is metal-specific. These observations suggest a model for the way newly arrived copper is handled at the cell membrane and is partitioned for intracellular uptake.

Summary: β-Glucosidase activity was detected in the plasma-membrane fraction of plasmodia of Physavum polycephalum. The enzyme activity was also found in the extracellular (medium) fraction after maximal growth had been attained, though the activity in this fraction was negligible during the exponential growth phase. The specific activities in both fractions were increased appreciably when the plasmodia were cultured in a glucose-free medium. The β-glucosidase was purified from the culture fluid by gel-filtration, ion-exchange and hydrophobic chromatographies. The purified enzyme appeared homogeneous on polyacrylamide-gel electrophoresis, and the molecular mass was estimated to be about 65 kDa. The enzyme showed highest activity at pH 4·5 and at 55 °C. The β-Glucosidase from the membrane fraction showed very similar properties to that from the culture medium, suggesting that the extracellular enzyme was derived from the membrane. The enzyme was active on laminarin and lichenan as well as on p-nitrophenyl-β-D-glucoside; it was strongly inhibited by D-glucono-l,5-lactone, N-bromosuccinimide and Hg2+.

Summary: The starvation-survival of Escherichia coli in seawater was assessed by plate and epifluorescence counts, 3H-label decrease, cellular DNA concentrations, and metabolic activities. These assays were performed on two types of populations, adapted and non-adapted to seawater. The number of viable cells in the adapted population remained constant throughout starvation-survival in sterile seawater. In contrast, a significant decrease in the ability of the non-adapted E. coli to form colonies on plates following starvation-survival in sterile seawater was observed. However, this drop in viable counts was not mirrored by the epifluorescence counts and 3H-label, which did not show major changes for either population during the experiments, indicating maintenance of the number of cells. In addition, a significant increase in and subsequent maintenance of DNA content and thymidine incorporation was observed for both populations during starvation-survival in sterile seawater. The changes in cell-attached exoproteolytic activity and electron transport system activity showed that adapted and non-adapted E. coli cells maintain their metabolic potential. Cell-free exoproteolytic activity was drastically reduced in both populations. Adapted cells showed higher electron transport system activity and thymidine incorporation than non-adapted cells at the onset of starvation-survival. The effect of previous adaptation on E. coli starvation-survival, as assessed by plate counts and 3H-label decrease, w as also observed in raw seawater. It seems from these data that the biological potential of E. coli cells suspended in sterile seawater has not been switched off or impaired seriously.

Summary: The 2.55 kb Staphylococcus aureus plasmid, pRJ5, confers constitutive resistance to macrolide–lincosamide–streptogramin B (MLS) antibiotics. pRJ5 is nearly identical to the inducible MLS resistance plasmid pT48, and has homology with the S. aureus plasmids pE194 and pSN2. The HindIII-C and/or Hind-B fragments were required for stable maintenance of the plasmid and probably carry palA. Plasmids pRJ5 and pT48 were shown to belong to the same incompatibility group, Inc12 (L). DNA sequencing showed that pRJ5 contains a 28 bp direct tandem duplication in the leader/attenuator region of ermC. This is likely to change the secondary structure of the methylase mRNA, allowing constitutive expression of ermC. The type of mutation found on plasmid pRJ5 is different from those observed in similar 2.5 kb constitutive MLS-resistance plasmids isolated from other Gram-positive bacteria, including staphylococci.

Summary: A Butyrivibrio fibrisolvens glnA gene encoding glutamine synthetase (GS) was cloned on a recombinant plasmid pGS4 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. The nucleotide sequence of a 2423 bp DNA segment containing the GS-coding region of B. fibrisolvens was determined and the complete amino acid sequence (701 residues) was deduced. Comparisons of the derived B. fibrisolvens GS protein sequence with the amino acid sequences of GS from other bacteria indicate that it is the second reported example of a type III GS, originally identified in the obligate anaerobe Bacteroides fragilis. The presence of GS in B. fibrisolvens cells and the regulation of the cloned GS in E. coli cells was demonstrated by Western blot analysis.

Summary: Lactose metabolism is an important industrial trait in dairy lactococci. In Lactococcus lactis, lactose is taken up via the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS) and is subsequently metabolized via the glycolytic and tagatose 6-phosphate pathways. Genes for the lactose-specific PEP-PTS proteins, phospho-β-galactosidase and tagatose 6-phosphate pathway enzymes are encoded by a single 8 kb operon, lac ABCDFEGX, and there is a divergently transcribed lacR repressor gene. Transcriptional fusions of both the lac operon promoter and the lacR promoter to the luxAB genes of Vibrio fischeri were used to investigate the regulation of expression of both promoters. In vivo bioluminescence assays demonstrated that lacR negatively regulates the lac operon and also autoregulates itself. Induction of transcription occurred for both promoters during growth on lactose: sevenfold for lacR and fivefold for the lac operon. The lacR promoter was demonstrated to be a particularly strong promoter, being approximately four times more efficient than the lac operon promoter. Both promoters provide good potential for the inducible expression of foreign proteins in Lactococcus.

Summary: The physicochemical characteristics of the lactococcin A immunity protein, as deduced from its gene sequence, were used to devise a procedure for its purification. The protein was purified from cell extracts by cation-exchange and reverse-phase chromatography. As judged from the amino acid composition and amino acid sequencing, the immunity protein is not post-translationally processed by cleavage at its N- or C-terminus. Consequently, the absorption coefficient at 280 nm, the isoelectric point, and the molecular mass of the immunity protein may be calculated to be, respectively, 8·2 × 103 M−1 cm−1, 10·2 and 11163 Da from the amino acid sequence predicted from the nucleotide sequence. The immunity protein is a major cell protein component – one cell may contain (to an order of magnitude) 105 molecules – and it is in part associated with the cell membrane, as judged by immunoblot analysis of membrane vesicle-associated proteins. Exposing lactococcin-A-sensitive cells to an excess of the immunity protein did not affect the lactococcin-A-induced killing of the cells, indicating that the immunity protein does not protect cells by simply binding to lactococcin A, nor to externally exposed domains on the cell surface. Exposing immune-positive cells to antiserum against the immune protein did not sensitize the cells to lactococcin A, suggesting that the immunity protein in fact does not act extracellularly.

Summary: The genes encoding an oxygen-labile stereospecific L-tartrate dehydratase (L-Ttd, EC 4.2.1.32) have been identified as the orfZ1 and orfZ2 genes located upstream of the rpsU-dnaG-rpoD operon at 67 min in the Escherichia coli linkage map. They were previously cloned and sequenced by M. Nesin and others (Gene 51, 149-161, 1987) and have now been independently cloned, partially resequenced, and designated as an operon (ttdAB) containing two translationally coupled genes. The enzyme behaves as a tetramer (Mr 105000) containing two pairs of non-identical subunits, TtdA (Mr 32589) and TtdB (Mr 22641), which otherwise resembles the homodimerie iron-sulphur-containing Class I fumarases of E. coli and Bacillus stearothermophilus. The amino acid sequences of the TtdA-TtdB subunits are colinearly related to a single fumarase subunit, indicating a common evolutionary ancestry. E. coli can use L-, D- and meso-tsrtrates as aerobic growth substrates and as reducible substrates for supporting anaerobic growth on glycerol. L-Ttd was induced during anaerobic growth on glycerol plus L- and meso -tartrates, and a stereospecific D-tartrate dehydratase was induced by all three stereoisomers under comparable conditions. No meso-tartrate dehydratase was detected, nor were any dehydratases detected after aerobic growth on tartrate minimal media suggesting that different catabolic routes operate under aerobic conditions.

Summary: Protoplast fusion was used to hybridize strains 99-6 (h-cR) and 7-141 (h-cGL) in the Birmingham collection of Aspergillus nidulans. A sparsely conidiating, brown-pigmented diploid strain (MA7) was isolated. Inocula from MA7 were haploidized on medium containing Benlate and a haploid progeny sample collected. Heterokaryon compatibility testing of selected progeny strains assayed each linkage group in turn and established that h-cR and h-cGL were hetero-allelic for heterokaryon incompatibility (het) loci located on linkage groups III and V. Two selected MA7 progeny strains were sexually crossed to an h-cGL strain. Compatibility classification of the progenies of these crosses indicated that three het genes were located on linkage group III and two on linkage group V. Combination of these results with previous work using h-c groups A, B and Q in comparison with h-cGL indicates that a minimum number of eight and a maximum number of 18 het loci, spread over five linkage groups, are necessary to explain the compatibility relationships among these five h-c groups.

Summary: Twenty-two monoclonal antibodies (mAbs) specific for smooth lipopolysaccharide (S-LPS) were generated by fusion of spleen cells from mice infected with the rough Brucella melitensis strain B115 with the NSO myeloma. According to reactivity in enzyme-linked immunosorbent assay (ELISA) with O-polysaccharide (O-PS) and absence of reactivity with rough lipopolysaccharide (R-LPS), it was postulated that these mAbs recognized epitopes present on the O-PS. Most of the mAbs reacted equally well in ELISA and immunoblotting with S-LPS types of Brucella A and M dominant strains and were designated as specific for common (C) epitopes. Three mAbs were highly specific for M dominant S-LPS. All these mAbs, in contrast to a mAb specific for the A epitope, showed little or no cross-reactivity with Yersinia enterocolitica O:9 S-LPS. S-LPS of B. melitensis B115 was extracted and analysed by immunoblotting and ELISA with mAbs specific for A, M and C epitopes. Reactivity of the mAbs with this S-LPS was compared to reactivity with S-LPS of A and M dominant smooth Brucella strains. The results suggest that S-LPS of B. melitensis B115 bears mainly C epitopes and a few M epitopes. The very weak reactivity of this S-LPS with the mAb specific for the A epitope and the fact that the mAbs specific for C and M epitopes showed little or no cross-reactivity with Y. enterocolitica O:9 S-LPS suggest that O-PS from this rough strain could be used to distinguish Y. enterocolitica O:9 infection from Brucella infection. The potential value of the rough B. melitensis strain B115 as a vaccine strain is also discussed.

Summary: A monoclonal antibody (mAb) was raised against extracellular polysaccharides from Mucor racemosus after intrasplenic immunization of mice. An indirect ELISA and a dot-blot assay were developed with this mAb. The IgG antibody was found to be very specific for all mould species tested belonging to the order of Mucorales, except species belonging to the genus Mortierella sensu stricto. No cross-reactions were observed with other moulds or yeasts. The immunoreactivity of the polysaccharides of these moulds with this mAb is based on carbohydrate epitopes, in which fucose residues probably play an important role. The mAb may be suited for specific detection of species of the genera Mucor, Rhizopus, Rhizomucor, Thamnidium, Absidia, Syncephalastrum and species belonging to the Mortierella isabellina group in food, and possibly for diagnosis of mucormycosis in humans.