- Volume 144, Issue 5, 1998

Amino acid sequences of two of the three bacteriocins from Leuconostoc mesenteroides TA33a were determined and their sequence-structure relationships investigated. Leucocin B-TA33a consists of 31 amino acid residues, with a molecular mass of 3466 Da. Leucocin B-TA33a does not belong to the pediocin family of bacteriocins, but shares 62% homology with mesenterocin 52B. A partial sequence of 36 amino acids of leucocin C-TA33a (4598 Da) was determined. Leucocin C-TA33a belongs to the class II bacteriocins having the consensus YGNGV motif. The third bacteriocin, leucocin A-TA33a, is identical to leucocin A-UAL 187. Circular dichroism spectra of the leucocins in aqueous solution and micellar SDS indicated that they undergo a structural transition when in a membrane-mimicking environment. Theoretical predictions from circular dichroism data suggest that leucocins A-, B- and C-TA33a adopt a β-structure (48%) in membrane-mimicking environments. Sequence alignments and secondary structure predictions for the N-terminus of leucocins A- and C-TA33a predicted that Cys-9 and Cys-14 are connected by a disulfide bridge and form two β-strands.

The zygomycete fungus Rhizomucor pusillus (previously called Mucor pusillus) secretes an aspartic proteinase containing two asparagine-linked, high-mannose type oligosaccharide chains at Asn79 and Asn188. For structural elucidation of the carbohydrate moieties, the protein was divided into two portions, an N-terminal portion containing Asn79 and a C-terminal portion containing Asn188, by a specific autocatalytic cleavage under alkaline conditions. Each of the asparagine-linked oligosaccharides was then released by peptide-N-glycosidase F digestion and pyridylaminated with a fluorescent reagent, 2-aminopyridine, at the reducing end. High-performance liquid chromatography analyses showed that the structure of the asparagine-linked oligosaccharide chain attached to residue Asn79 was Man5 GlcNAc2, and that bound to residue Asn188 was Man5 GlcNAc2 and Man5GlcNAc2. These observations suggest that the processing of mannose residues in asparagine-linked oligosaccharides in the Golgi apparatus of Rhizomucor resembles that in mammalian cells.

Biofilms comprising a pure and a mixed culture of sulphate-reducing bacteria (SRB) were grown in continuous culture. When exposed to 20 or 200 μM Cd, both cultures accumulated Cd but the mixed culture accumulated more and continued to accumulate Cd during the experiment, whereas accumulation by the pure cultures ceased after 4-6 d. Unlike the pure culture, the mixed culture also accumulated both protein and carbohydrate throughout the experiment proportionally to Cd which showed that accumulation required the production of biofilm material. Electron microscopy showed the presence of polysaccharide and particulates in both pure and mixed cultures, irrespective of the presence of Cd. However, energy-dispersive X-ray analysis (EDXA) showed that accumulation of Cd in the form of CdS occurred in biofilms exposed to Cd while back-scattered electron imaging of sections indicated that the accumulation of Cd was localized in a superficial layer of the biofilm. The mechanism of uptake, therefore, appeared to be entrapment and/or precipitation of CdS at the biofilm surface. The relatively low Cd uptake by the pure culture biofilm was attributed to its less efficient growth and polysaccharide production. These results indicate that mixed SRB cultures are more effective than pure cultures for metal removal and underlines significant differences between the biology of pure and mixed cultures.

The fission yeast Schizosaccharomyces pombe shows bipolar growth and is a convenient model for studying cell polarity and polar growth. This paper shows that the related Schiz. japonicus var. japonicus can switch to unipolar growth and can exist in both yeast and mycelial phases. On solid media, the yeast phase is unstable and prone to switch to the mycelial form, which shows unipolar growth by tip elongation. The hyphae can colonize the body of the substrate (true mycelium) or just its surface (pseudo-mycelium). The yeast-to-mycelium transition and the growth of the mycelium are regulated by a nutritional gradient and are associated with extensive vacuolation. The mycelium can convert into arthroconidia or return to the yeast phase in response to environmental changes. These environmentally controlled morphological transitions make Schiz. japonicus var. japonicus an attractive model for the investigation of cell polarity and morphogenesis.

Gas vesicle formation in halophilic archaea is encoded by a DNA region (the vac region) containing 14 different genes: gvpACNO and gvpDEFGHIJKLM. In Halobacterium salinarum PHH1 (which expresses the p-vac region from plasmid pHH1), gas vesicles are spindle shaped, whereas predominantly cylindrical gas vesicles are synthesized by the chromosomal c-vac region of H. salinarum PHH4 and the single chromosomal mc-vac region of Haloferax mediterranei. Homologous complementation of gvp gene clusters derived from the chromosomal c-vac region led to cylindrical gas vesicles in transformants and proved that the activity of the c-gvpA promoter depended on a gene product from the c-gvpE-M DNA region. Heterologous complementation experiments with transcription units of different vac regions demonstrated that the formation of chimeric gas vesicles was possible. Comparison of micrographs of wild-type and chimeric gas vesicles indicated that the shape was not exclusively determined by GvpA, the major structural protein of the gas vesicle wall. More likely, a dynamic equilibrium of several gvp gene products was responsible for determination of the shape. Transmission electron microscopy of frozen hydrated, wild-type gas vesicles showed moiré patterns due to the superposition of the front and back parts of the ribbed gas vesicle envelope. Comparison of projections of model helices with the moiré pattern seen on the cylindrical part of the gas vesicles provided evidence that the ribs formed a helix of low pitch and not a stack of hoops.

A gene encoding the major antigenic protein of phytoplasma associated with sweet potato witches’ broom (SPWB) was cloned and analysed by screening the genomic library of SPWB phytoplasma with monoclonal antibodies for SPWB phytoplasma. The entire predicted structural gene encoded an antigenic protein composed of 172 amino acids with a computed molecular mass of 19.15 kDa and a pl value of 9.78. The -10 region of the promoter and the terminator region of the gene were identified and found to be similar to those of prokaryotes. The hydropathy profile of the deduced amino acid sequence consisted of two distinct regions, a strongly hydrophobic N-terminus and a highly hydrophilic C-terminus. This major antigenic protein was also present in phytoplasma associated with peanut witches’ broom (PNWB) and the two showed homology based on the results of Western blot analysis. Southern hybridization. Northern hybridization, primer extension analysis and PCR. The homologous genes of the antigenic protein of SPWB phytoplasma arid PNWB phytoplasma were not found in other phytoplasmas tested.

Bacillus thuringiensis subspecies kurstaki HD73, toxic for lepidopteran larvae, contains two large self-transmissible plasmids of approximately 75 kb, pHT73 and pAW63. The conjugative plasmid pHT73 has been studied extensively and has been shown to harbour the toxin gene cry1Ac, the transposon Tn4430 and several insertion sequences. In this study it was demonstrated that the minor plasmid pAW63 is also self-transmissible and about 10-30 times more efficient in mobilizing plasmid pBC16. To facilitate direct selection for pAW63 transfer, the plasmid was tagged with the tetracycline resistance transposon Tn5401 and in intraspecies matings it was found that after 2 h, all recipients had acquired a copy of the plasmid. Mating experiments demonstrated that pAW63 could be transferred to Bacillus thuringiensis subsp. israelensis. Bacillus cereus, Bacillus licheniformis, Bacillus subtilis and Bacillus sphaericus, and that the conjugative functions were expressed in these hosts. Hybridization studies showed that the replicons of pAW63 and pHT73 were distinct from one another. Sequences homologous to transposon Tn4430 and several insertion sequences were, however, shown to reside on both plasmids.

The telomers of several linear plasmids of Rhodococcus opacus (formerly Nocardia opaca) were studied. The plasmids pHG201, pHG204 and pHG205 carry proteins bound to their ends, as shown by gel retardation experiments. A sequence hybridizing with the terminal sequence of pHG207, a recombinant linear plasmid consisting of the left part of pHG204 and the right part of pHG205, which was analysed in a previous study by the authors, could be detected in all linear plasmids of the wild-type R. opacus strains MR11 and MR22. However, only pHG204 and pHG206 carry terminal inverted repeats (TIRs) like pHG207. Cloning and sequencing of the terminal fragment of pHG204 revealed a nearly perfect TIR of 1016 bp. In contrast, the termini of pHG201 and pHG205 share little homology. Sequence analysis of the two end fragments of pHG201 revealed a similarity of only 65% within the terminal 34/32 bp and a perfect TIR of only 3 bp. The results support the assumption that long TIRs are not absolutely necessary for replication and maintenance of linear plasmids.

Streptomycetes differ from other prokaryotic organisms in their mycelial life cycle and in possessing a large, linear, GC-rich chromosome. To deduce structural features of the Streptomyces origin of chromosomal replication, the oriC sequences of three Streptomyces species (S. antibioticus, S. chrysomallus and S. lividans) were compared. In Streptomyces, the oriC region contains 19 DnaA boxes whose location, orientation and spacing are conserved. The consensus sequence of the DnaA box identified within Streptomyces oriC is (T/C)(T/C)(G/A/C)TCCACA (preferred bases underlined). The interactions of DnaA with DNA fragments containing single, two or three DnaA boxes were studied using surface plasmon resonance. The dissociation constant (K D) for specific binding of individual DnaA boxes varied between 12 and 78 nM. Streptomyces oriC does not contain the three AT-rich 13-mer direct repeats present in the 5′ part of the Escherichia coli oriC region. However, short AT-rich sequences are distributed among the DnaA boxes of Streptomyces oriC. Repeated attempts to unwind Streptomyces oriC have been unsuccessful. It remains to be elucidated whether DnaA interacts with putative accessory proteins which help in unwinding Streptomyces oriC.

To examine the ecology and evolution of microbial chitinases, especially the chitin-binding domain, one of the chitinase genes (chiA) from the marine bacterium Vibrio harveyi was analysed. The deduced amino acid sequence of ChiA is not very similar overall to other proteins, except for two regions, the putative catalytic and chitin-binding domains. Among all bacterial chitinases sequenced to date, there is no relationship between percentage similarity of catalytic domains and chitin-binding domains in pairwise comparisons, suggesting that these two domains have evolved separately. The chitin-binding domain appears to be evolutionarily conserved among many bacterial chitinases and is also somewhat similar to cellulose-binding domains found in microbial cellulases and xylanases. To investigate the role of the chitin-binding domain, clones producing versions of ChiA with or without this domain were examined. One version with the domain (ChiA1) bound to and hydrolysed chitin, whereas a truncated ChiA without the putative chitin-binding domain (ChiA2) did not bind to chitin but it could hydrolyse chitin, although not as well. ChiA1 diffused more slowly in agarose containing colloidal chitin than ChiA2, but diffusion of the Two proteins in agarose without colloidal chitin was similar. These results indicate that the chitin-binding domain helps determine the movement of chitinase along N-acetylglucosamine strands and within environments containing chitin.

Pseudomonas putida strain O2C2 is able to grow on toluene, m-xylene and p-xylene through benzoate and the corresponding methylbenzoates (toluates). The catabolic genes are encoded on a large TOL plasmid, pWW102, of >220 kb. The complete catabolic genes were cloned on four large overlapping restriction fragments covering a total of 28 kb of the plasmid, which was carefully mapped by restriction enzyme analysis. The presence of the xyl genes on the cloned DNA was confirmed by assay of representative enzymes of both operons. Virtually all the genes were located on the cloned DNA by hybridization of Southern blots with gene-specific probes from related pathways of other catabolic plasmids. Within the limitations of available restriction sites, the analysis showed that the genes are in two blocks. The major block carries the meta pathway operon xylXYZLTEGFJQKIH with the two regulatory genes xylSR immediately downstream. The upper pathway operon xylUWCMAB(N) is about 2-3 kb downstream of the regulatory genes and transcribed in the same direction as the meta pathway operon. Within each operon the gene order appears to be identical to that found in other TOL plasmids, but the relative location of the operons most closely resembles that found on plasmid pWW53, although there is no evidence of any xyl duplications on pWW102. The nucleotide sequence of the xylQ gene for the acetaldehyde dehydrogenase (acylating; ADA), together with the 3-end of the upstream xylJ (for 2-oxopent-4-enoate hydratase) and the 5-end of the downstream xylJ (for 4-hydroxy-2-oxovalerate aldolase), was determined. The xylQ gene was ligated into expression vector pTrc99a and high levels of XylQ protein were detected by enzyme assay and by SDS-PAGE. All three genes xylJQK showed a high degree of homology with genes encoding isofunctional proteins from other Pseudomonas meta pathways, the highest being with the naphthalene catabolic genes nahLOM from the plasmid of Pseudomonas sp. NCIB 9816. The implications of the sequence homologies to the evolution of these pathways are discussed.

Both GcvA and Lrp are required for normal regulation of the gcv operon. Moving the GcvA-binding sites 3 and 2 and the Lrp-binding region either closer to, or further away from, the gcv promoter by approximately one helical turn of DNA resulted in a less than twofold decrease in glycine-mediated activation or inosine-mediated repression of a gcvT::lacZ fusion. Moving these sites approximately two helical turns of DNA away from the gcv promoter resulted in a further loss of both activation and repression; moving these sites approximately three helical turns of DNA from the gcv promoter resulted in an essentially complete loss of both glycine-mediated activation and inosinemediated repression. However, when these sites were moved by approximately 1.5 and 2.5 helical turns of DNA away from the gcv promoter, there was a complete loss of both glycine-mediated activation and inosine-mediated repression of the gcvT::lacZ fusion. The flexibility in the absolute distance of the GcvA- and Lrp-binding sites relative to the gcv promoter, but strict orientation dependence of these sites is consistent with a possible protein-protein interaction of either GcvA, Lrp, or both of these proteins with RNA polymerase. Because of the location of these target sites relative to the gcv promoter, it is also likely that DNA looping is required for this mechanism of regulation.

The ppc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) of an extremely thermophilic bacterium, Rhodothermus obamensis, was directly sequenced by the thermal asymmetric interlaced (TAIL) PCR method. An ORF for a 937 amino acid polypeptide was found in the gene. The ppc gene had a high G+C content (66.2 mol%) and the third position of the codon exhibited strong preference for G or C usage (85.0 mol%). The calculated molecular mass was 107 848 Da, which was consistent with the molecular mass of the enzyme as determined by SDS-PAGE (100 kDa). The amino acid sequence of R. obamensis PEPC was closely related to that of PEPC from another thermophile, a Thermus sp., and from a mesophile. Corynebacterium glutamicum, exhibiting 45.3% or 37.7% identity and 61.5% or 56.5% similarity, respectively. By Southern analysis, the ppc gene was found to be present in a single copy in the genomic DNA of this organism. The cloned gene was expressed in Escherichia coli using a pET expression vector system and a thermostable recombinant PEPC was obtained. Comparison of the deduced amino acid sequences of the thermophilic and mesophilic PEPCs revealed distinct or common preferences for specific amino acid composition and substitutions in the two thermophilic enzymes.

The aspartate carbamoyltransferase (ATCase) genes of psychrophilic Vibrio strain 2693 were cloned by complementation in Escherichia coli and the enzyme was partly characterized. The genes constitute a pyrBl operon homologous to the cognate structure in E. coli where pyrB and pyrl respectively encode the catalytic and the regulatory chains of ATCase. The strong sequence similarities noted between Vibrio and E. coli ATCases include extensive conservation of residues involved in interactions between subunits, suggesting that the two enzymes have very similar tertiary and quaternary structures. Vibrio ATCase is, however, not activated by ATP and not synergistically inhibited by CTP and UTP. It is also much more thermolabile than E. coli ATCase. With respect to Pyrococcus abyssi and E. coli ATCases, Vibrio ATCase presents marked differences in composition which could be related to its psychrophilic character. The results of these structural and functional comparisons indicate that Vibrio 2693 ATCase is a suitable model for biochemical studies on structure-function relationships in a ‘cold’ allosteric enzyme. The operon is expressed from a promoter which is immediately followed by a pyrimidine-rich leader ORF terminating within a putative transcription attenuator. These genetic and enzymic data strengthen the evolutionary relationship already noted between Vibrionaceae and Enterobacteriaceae.

The phosphate-deficiency response in Bacillus subtilis is regulated by PhoP PhoR, a pair of two-component regulatory proteins. PhoR is a histidine kina and PhoP is a response regulator. Genetic evidence indicates that the Pho-regulon genes, which are induced or repressed under phosphate starvation conditions, are regulated by PhoP and PhoR at the transcriptional level. It has previously been shown that PhoP binds to four Pho-regulon promoters in both unphosphorylated and phosphorylated forms. This study demonstrates that another Pho-regulon gene promoter, the tuaA promoter preceding the operon which is responsible for cell wall teichuronic acid synthesis, is also transcriptionally regulated and is bound by PhoP. The binding affinity for phosphorylated PhoP was about 10-fold higher than that for unphosphorylated PhoP. Both unphosphorylated and phosphorylated PhoP bound upstream of the -20 region in the tuaA promoter. By aligning the Phop binding sites within the Pho-regulon promoters, a consensus core PhoP-binding region composed of four TT(A/T)ACA direct repeats, each separated by 5.2 non-conserved nucleotides was identified. PhoP, phosphorylated or unphosphorylated, binds to such a sequence in all Pho-regulon promoters studied. Phosphorylated PhoP binds to the core binding region with high affinity and to additional regions surrounding this region with similar or lower affinity.

Individual rrn operons and their flanking regions have been analysed in a study of the molecular basis of ribotype variation in the seventh pandemic clone of Vibrio cholerae. The genome of an early isolate of the seventh pandemic clone had nine rrn operons of which two were in tandem with other rrn operons. The site for Bgll, the most discriminatory enzyme used for ribotyping, was found to be present in the 16S sequence of three of the operons of the earliest isolate. This site was observed to be gained or lost in specific operons in many later isolates, presumably by recombination, and this gave most of the ribotype variation. Additional rrn recombination events were uncovered by analysis of the 16S-23S intergenic spacers associated with each operon. Spacers of 431, 509, 607 and 711 bp were found. A total of at least eight rrn recombination events were detected. Three rrn loci were primarily involved in this recombination, with four new forms generated from that in the early strains for operon B and two new forms each for operons C and G. In addition there was variation due to deletion of tandem operons. The frequency of recombination between rrn operons was very high as there were nine new ribotypes found among 47 isolates sampled over the 33 year period of study. This means that any variation could undergo precise reversion by the same recombination event within the time frame covered by the study. Recombination between rrn operons may be a factor in ribotype variation in all systems. The recombination observed is thought to be that which results in concerted evolution and the data give an indication of the rate involved.

Oral streptococci such as Streptococcus gordonii bind the abundant salivary enzyme x-amylase. This interaction may be important in dental plaque formation and metabolism, thus contributing to the initiation and progression of dental caries and periodontal disease, the two most common plaque-mediated diseases. The conjugative transposon Tn916 was used to insertionally inactivate gene(s) essential to the expression of amylase-binding components of S. gordonii Challis, and a mutant deficient in amylase-binding (Challis Tn1) was identified. While wild-type strains of S. gordonii released both 20 kDa and 82 kDa amylase-binding proteins into culture supernatants, Challis Tn1 expressed the 82 kDa but not the 20 kDa protein. The 20 kDa amylase-binding protein was isolated from culture supernatants of S. gordonii Challis by hydroxyapatite chromatography. A partially purified, functionally active 20 kDa protein was sequenced from blots, and the N-terminal sequence obtained was found to be DEP (A) TDAAT(R)NND. A novel strategy, based on the single-specific-primer polymerase chain reaction technique, enabled the gene inactivated by Tn916 to be cloned. Analysis of the resultant nucleotide sequence revealed an open reading frame of 585 bp, designated amylase-binding protein A (abpA), encoding a protein of 20 kDa (AbpA), immediately downstream from the insertion site of Tn916. This protein possessed a potential signal peptide followed by a region having identity with the N-terminal sequence of the 20 kDa amylase-binding protein. These results demonstrate the role of the 20 kDa protein in the binding of amylase to S. gordonii. Knowledge of the nature of amylase-binding proteins may provide a better understanding of the role of these proteins in the colonization of S. gordonii in the oral cavity.

The course of nucleoid movement during and upon release from protein synthesis inhibition by chloramphenicol in filaments of Escherichia coli pbpB(Ts) was analysed. Cells were grown at 42 °C in glucose minimal medium for two mass doublings and were treated with chloramphenicol to generate fusion (coalescence) of the nucleoids. Upon release from protein synthesis inhibition, the large distance between the border of the fused nucleoids and the cell poles immediately decreased, before full recovery of the rates of mass growth and length increase at 30 °C. This indicates that nucleoids can reoccupy the DNA-free cell ends independently of cell elongation. During filamentation at 42 °C, the pbpB cells established initial constrictions at midcell and at one-quarter and three-quarter positions. Nevertheless, divisions only started 75 min after chloramphenicol removal at 30 °C, when most nucleoids had moved back into the vacated cell ends. No ‘guillotine-like’ constrictions at the site of the nucleoids occurred.This suggests that segregating nucleoids postpone division recovery at previously established sites. The results are discussed in the light of a working model for transcription/translation-mediated chromosome segregation and nucleoid occlusion of cell division.

Several dipeptides, containing the N 3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) moiety linked to protein and non-protein amino acids, exhibited a strong growth-inhibitory and bactericidal effect against Bacillus subtilis. FMDP-dipeptides were efficiently transported into bacterial cells by a di-tripeptide permease and subsequently cleaved by intracellular Mn2+/Co2+-dependent peptidases. Cleavage rates [0.1-5.6 μmol min-1 (mg protein)-1] were about two orders of magnitude lower than transport rates [40-200 μmol min-1 (mg dry wt)-1]. The released FMDP inactivated glucosamine-6-phosphate (GlcN-6-P) isomerase, an enzyme catalysing the first committed step in a biosynthetic pathway leading to amino sugar-nucleotide precursors of bacterial peptidoglycan. Inhibition of GlcN-6-P isomerase precluded peptidoglycan biosynthesis and resulted in a strong bacteriolytic effect. Results of the studies on consequences of GlcN-6-P isomerase inhibition upon the action of FMDP-dipeptides provided evidence demonstrating that the lack of endogenous GlcN-6-P could be a reason for the triggering of bacterial autolysis. Peptides containing the inhibitors of GlcN-6-P isomerase are one of the very few antimicrobial agents known that exhibit both bactericidal and fungicidal effects.

The gene encoding a 102 kDa autolysin has been cloned from an expression library of Listeria monocytogenes EGD genomic DNA, using a direct screening protocol. The encoded protein has two domains, an N-terminal enzymic domain showing a high level of homology to the amidase domain of the major autolysin (atl) of Staphylococcus aureus, and a C-terminal, putative cell-wall-binding domain containing four imperfect direct repeats. In order to examine the role of the enzyme, the autolysin-encoding gene was insertionally inactivated by site-specific integration of a temperature sensitive plasmid. The enzyme accounts for 66% of the total lytic enzyme activity when L. monocytogenes walls are used as substrate and several of the major autolytic bands are missing on renaturing gels when compared to the wild-type. The enzyme does not appear to be directly involved in cell separation but has a role in motility. Characterization of the recombinant enzyme expressed in Escherichia coli has revealed it to be an amidase and to be able to hydrolyse a range of peptidoglycan substrates.

Pseudomonas aeruginosa PAO1 grew in defined synthetic medium with any of a broad variety of single sulfur sources, including sulfate, cysteine, thiocyanate, alkanesulfonates, organosulfate esters and methionine, but not with aromatic sulfonates, thiophenols or organothiocyanates or isothiocyanates. During growth with any of these compounds except sulfate, cysteine or thiocyanate, a set of 10 sulfate starvation-induced (SSI) proteins was strongly up-regulated, as observed by two-dimensional protein electrophoresis of total cell extracts. A comparable level of up-regulation was found for the hydrolytic enzyme arylsulfatase, which has previously been used as a marker enzyme for the sulfate starvation response. One of the SSI proteins was identified by N-terminal sequencing as a high-affinity periplasmic sulfate-binding protein, and another was related to thiol-specific antioxidants, but the N-terminal sequences of the other SSI proteins revealed no similarity to N-termini of proteins of known function, and they probably represent uncharacterized enzymes involved in sulfur scavenging when preferred sulfur sources are absent. To study the role that cysteine biosynthetic intermediates play in the synthesis of these proteins in vivo, we isolated mini-Tn5 transposon mutants of P. aeruginosa with insertions in the cysN and cysl genes, which encode subunits of ATP-sulfurylase and sulfite reductase, respectively. These two genes were cloned and sequenced. cysl showed high similarity to the cognate gene in Escherichia coli, whereas cysN encoded a 69.3 kDa protein with two domains corresponding to the E. coli CysN and CysC proteins. Sulfate no longer repressed synthesis of the SSI proteins in cysN mutants, but repression was restored by sulfite; in the cysl mutant, sulfate, sulfite and sulfide all led to repression of SSI protein synthesis. This suggests that there are at least two independent corepressors of the sulfate starvation response in this species.

Growth of Saccharomyces cerevisiae on ammonia and glutamine decreases the expression of many nitrogen catabolic genes to low levels. To discriminate between ammonia- and glutamine-driven repression of GAP1, PUT4, GDH1 and GLN1, a gln1-37 mutant was used. This mutant is not able to convert ammonia into glutamine. Glutamine-limited continuous cultures were used to completely derepress the expression of GAP1, PUT4, GDH1 and GLN1. Following an ammonia pulse, the expression of GAP1, PUT4 and GDH1 decreased while the intracellular glutamine concentration remained constant, both in the cytoplasm and in the vacuole. Therefore, it was concluded that ammonia causes gene repression independent of the intracellular glutamine concentration. The expression of GLN1 was not decreased by an ammonia pulse but solely by a glutamine pulse. Analysis of the mRNA levels of ILV5 and HIS4 showed that the response of the two biosynthetic genes, GDH1 and GLN1, to ammonia and glutamine in the wild-type and gln1-37 was not due to changes in general transcription of biosynthetic genes. Ure2p has been shown to be an essential element for nitrogen-regulated gene expression. Deletion of URE2 in the gln1-37 background prevented repression of gene expression by ammonia, showing that the ammonia-induced repression is not caused by a general stress response but represents a specific signal for nitrogen catabolite regulation.

Broad-range 16S rDNA PCR (BR-PCR) applied to DNA from 32 clinical enterococcal isolates and 12 other enterococci from a clinical reference collection followed by species-specific hybridization analysis identified 25 strains of Enterococcus faecalis and 19 Enterococcus species. Randomly amplified polymorphic DNA (RAPD)analysis using upgma clustering on the same material revealed four different clusters at a similarity level of 49%. Based on partial 16S rDNA sequence analysis of variable regions V4 and V9, it was possible to divide the 19 type strains specifying the genus Enterococcus into 12 different 16S rDNA species groups. The type strain distribution then served as a template for the analysis of the other 44 strains which were assigned to four different species groups (a-d) based on their 16S rDNA motifs. There was good agreement with the RAPD clusters. Species group a was an individual species line containing 25 strains that were identified as E. faecalis. Group b also represented an individual species line of 12 strains identified as E. faecium. The remaining seven strains that formed species groups c and d could not be fully identified to species by this analysis. It was concluded that BR-PCR of 16S rDNA followed by partial sequence analysis of the PCR products is a reliable technique for the identification and classification of enterococci. Further division of unresolved species groups should be achievable if regions other than V4 and V9 of 16S rDNA are also analysed.

Fatty and mycolic acids and the pattern of glycolipids were studied in a collection of 34 strains of ‘Mycobacterium habana’ and in two strains of Mycobacterium simiae. Major glycolipids of these micro-organisms were assigned to the glycopeptidolipid (GPL) structural type, but both mycobacteria differed in the patterns obtained by TLC. The strains of ‘M. habana’ were separated into four groups (A-D), taking into account the presence or absence of several polar GPLs: group A contained GPL-I, GPL-II and GPL-III; group B contained GPL-I, GPL-II’, GPL-II and GPL-III; group C contained GPL-II’, GPL-II and GPL-III; group B did not contain any of these compounds. Fatty acids of both bacteria were similar, and ranged from 14 to 26 carbon atoms, hexadecanoic, octadecenoic and tuberculostearic acids being predominant. Mycolic acids were also similar by TLC and HPLC, and consisted of α-, α- and ketomycolates. Partial structural analysis by MS carried out in strains ‘M. habana’ TMC 5135 and M. simiae ATCC 25275T revealed that α- and ketomycolates ranged, in general, from 79 to 87 carbon atoms, and α-mycolates from 58 to 67 carbon atoms. The α- and ketomycolates belonged to several structural series, and minor variations were found between the two strains examined. The data obtained justified the synonymy between ‘M. habana’ and M. simiae but indicated, in turn, that the former can be distinguished on the basis of GPL analysis. Most strains of ‘M. habana’ can be defined by the presence of GPL-II and GPL-III, a finding that could be useful in the quality control of potential vaccine strains.