- Volume 147, Issue 8, 2001

Simple sequence repeats, due to their high variability, are widely used for molecular epidemiology of pathogenic micro-organisms. However, their usefulness is restricted by their high instability and low information content. Here, a locus, CKTNR, in the fungal pathogen Candida krusei is described which displays considerable sequence, as well as length, heterogeneity. Alleles of this locus, which contains a degenerate trinucleotide repeat, appear to be stable. The CKTNR polymorphism could serve as the basis for a molecular typing system of C. krusei. Furthermore, analysis of the CKTNR allele distribution suggested that C. krusei reproduces mainly clonally.

Glucose-repressible alcohol dehydrogenase II, encoded by the ADH2 gene of the yeast Saccharomyces cerevisiae, is transcriptionally controlled by the activator Adr1, binding UAS1 of the control region. However, even in an adr1 null mutant, a substantial level of gene derepression can be detected, arguing for the existence of a further mechanism of activation. Here it is shown that the previously identified UAS2 contains a distantly related variant of the carbon source-responsive element (CSRE) initially found upstream of gluconeogenic genes. In a mutant defective for the CSRE-binding factor Cat8, derepression of an ADH2-lacZ fusion was reduced to about 12% of the wild-type level. Gene expression in a cat8 adr1 double mutant decreased almost to the basal level of the glucose-repressed promoter. CSREADH2 present in a single copy turned out to be a weak UAS element, while a significant synergism of gene activation was found in the presence of at least two copies. Its importance for regulated gene activation was confirmed by site-directed mutagenesis of the CSRE in the natural ADH2 control region. Direct binding of Cat8 to CSREADH2 could be shown by electrophoretic retardation of the corresponding protein/DNA complex in the presence of a specific antibody. In contrast to what was shown previously for CSRE sequence variants, no significant influence of the isofunctional activator Sip4 on CSREADH2 was detected. In conclusion, these results show a derepression of ADH2 by synergistically acting regulators Adr1 (interacting with UAS1) and Cat8, binding to UAS2 (=CSREADH2).

The site-specific recombination system of temperate lactococcal bacteriophage TP901-1 is unusual in several respects. First, the integrase belongs to the family of extended resolvases rather than to the λ integrase family and second, in the presence of this integrase, a 56 bp attP fragment is sufficient for efficient recombination with the chromosomal attB site in the host Lactococcus lactis subsp. cremoris MG1363. In the present work, this attB site was analysed and a 43 bp attB region was found to be the smallest fragment able to participate fully in recombination. In vitro studies showed that the TP901-1 integrase binds this 43 bp attB fragment, the 56 bp attP and a larger attP fragment with equal affinity. Mutational analysis of the 5 bp common core region (TCAAT) showed that the TC dinucleotide is essential for recombination, but not for binding of the integrase, whereas none of the last three bases are important for recombination. When a number of attL sites, obtained by recombination between an attB site containing a mutation in this TC dinucleotide and a wild-type attP site, were sequenced, a mix of sites with the wild-type or the mutated sequence was obtained. These results are consistent with the hypothesis that the TC dinucleotide constitutes the TP901-1 overlap region. A 2 bp overlap region has been observed in recombination reactions catalysed by all other members of the resolvase/invertase family tested so far. By selecting for attB sites with a decreased ability to participate in recombination, two bases located outside the core region of attB were shown to be involved in the in vitro binding of the TP901-1 integrase.

Full exploitation of the information available in bacterial genome sequences requires the availability of facile tools for rapid genetic manipulation. One bacterium for which new genetic tools are needed is the methylotroph Methylobacterium extorquens AM1. IncQ and small IncP vectors were shown to be unsuitable for use in this bacterium, but a spontaneous mutant of a small IncP plasmid was isolated that functioned efficiently in M. extorquens AM1. This plasmid was sequenced and used as a base for developing improved broad-host-range cloning vectors. These vectors were found to replicate in a wide variety of bacterial species and have the following advantages: (1) high copy number in Escherichia coli; (2) small size (7·2 and 8·0 kb); (3) complete sequences; (4) variety of unique restriction sites; (5) blue–white screening via lacZα; (6) conjugative mobilization between bacterial species; and (7) readily adaptable into species-specific promoter-probe and expression vectors. Two low-background promoter-probe vectors were constructed based on these cloning vectors with either lacZ or xylE as reporter genes; these were shown to report gene expression effectively in M. extorquens AM1. Specific expression vectors were developed for use in M. extorquens AM1, which were shown to express foreign genes at significant levels, and a simple strategy is outlined to develop specific expression vectors for other bacteria. The strong mxaF promoter was used for expression, since E. coli lac-derived promoters were expressed at very low levels. This suite of genetic tools will enable a more sophisticated analysis of the physiology of M. extorquens AM1, and these vectors should also be valuable tools in the study of a variety of bacterial species.

Chromosomal gene replacement in cyanobacteria often relies upon the availability of drug resistance markers, and thus multiple replacements have been restricted. Here, a versatile gene replacement system without this restriction is reported in a unicellular cyanobacterium, Synechococcus sp. PCC 7942. The system is based upon the dominance of a streptomycin-sensitive rps12 gene encoding a ribosomal S12 protein over a streptomycin-resistant rps12-R43 allele with a Lys-43→→→Arg substitution. To demonstrate the utility of this method, a cassette consisting of the wild-type rps12 gene and a kan gene conferring kanamycin resistance was integrated into the rps12-R43 mutant at the psbAI locus encoding photosystem II D1 protein, resulting in streptomycin-sensitive merodiploids. Despite spontaneous gene conversion in these merodiploids to produce streptomycin-resistant progeny at frequencies ranging from 1×10−5 to 5×10−5, homologous recombination could be induced by transforming the merodiploids with template plasmids carrying psbAI 5′ and 3′ non-coding sequences flanking the D1 coding sequence, which was then replaced by either the gfp ORF for a green fluorescent protein or a precise deletion. Depending on the replication ability of the template plasmids, at most 3–16% of streptomycin-resistant progeny of the merodiploids after transformation were homogenote recombinants with concomitant loss of the kan gene, even in these polyploid cyanobacteria. The rps12-mediated gene replacement thus makes it possible to construct mutants free from drug resistance markers and opens a way to create cyanobacterial strains bearing an unlimited number of gene replacements.

The pdx-4 mutation in Streptomyces venezuelae ISP5230 confers a growth requirement for pyridoxal (pdx) and is a marker for the genetically mapped cluster of genes associated with chloramphenicol biosynthesis. A gene regulating salvage synthesis of vitamin B6 cofactors in S. venezuelae was cloned by transforming a pdx-4 mutant host with the plasmid vector pDQ101 carrying a library of wild-type genomic DNA fragments, and by selecting for complementation of the host’s pdx requirement. However, the corresponding replicative plasmid could not be isolated. Southern hybridizations and transduction analysis indicated that the complementing plasmid had integrated into the chromosome; after excision by a second crossover, the plasmid failed to propagate. To avoid loss of the recombinant vector, a pdx-dependent Streptomyces lividans mutant, KAA1, with a phenotype matching that of S. venezuelae pdx-4, was isolated for use as the cloning host. Introduction of pIJ702 carrying an S. venezuelae genomic library into S. lividans KAA1, and selection of prototrophic transformants, led to the isolation of a stable recombinant vector containing a 2·5 kb S. venezuelae DNA fragment that complemented requirements for pdx in both S. venezuelae and S. lividans mutants. Sequence analysis of the cloned DNA located an intact ORF with a deduced amino acid sequence that, in its central and C-terminal regions resembled type-I aminotransferases. The N-terminal region of the cloned DNA fragment aligned closely with distinctive helix–turn–helix motifs found near the N termini of GntR family transcriptional regulators. The overall deduced amino acid sequence of the cloned DNA showed 73% end-to-end identity to a putative GntR-type regulator cloned in cosmid 6D7 from the Streptomyces coelicolor A3(2) genome. This location is close to that of pdxA, the first pdx marker in S. coelicolor A3(2) identified and mapped genetically in Sir David Hopwood’s laboratory. The S. venezuelae gene and S. coelicolor pdxA are postulated to be homologues regulating vitamin B6 coenzyme synthesis from pdx.

Amplification of sequences from Streptomyces venezuelae ISP5230 genomic DNA using PCR with primers based on conserved prokaryotic pabB sequences gave two main products. One matched pabAB, a locus previously identified in S. venezuelae. The second closely resembled the conserved pabB sequence consensus and hybridized with a 3·8 kb NcoI fragment of S. venezuelae ISP5230 genomic DNA. Cloning and sequence analysis of the 3·8 kb fragment detected three ORFs, and their deduced amino acid sequences were used in BLAST searches of the GenBank database. The ORF1 product was similar to PabB in other bacteria and to the PabB domain encoded by S. venezuelae pabAB. The ORF2 product resembled PabA of other bacteria. ORF3 was incomplete; its deduced partial amino acid sequence placed it in the MocR group of GntR-type transcriptional regulators. Introducing vectors containing the 3·8 kb NcoI fragment of S. venezuelae DNA into pabA and pabB mutants of Escherichia coli, or into the Streptomyces lividans pab mutant JG10, enhanced sulfanilamide resistance in the host strains. The increased resistance was attributed to expression of the pair of discrete translationally coupled p-aminobenzoic acid biosynthesis genes (designated pabB/pabA) cloned in the 3·8 kb fragment. These represent a second set of genes encoding 4-amino-4-deoxychorismate synthase in S. venezuelae ISP5230. In contrast to the fused pabAB set previously isolated from this species, they do not participate in chloramphenicol biosynthesis, but like pabAB they can be disrupted without affecting growth on minimal medium. The gene disruption results suggest that S. venezuelae may have a third set of genes encoding PABA synthase.

Mupirocin (pseudomonic acid) is a polyketide antibiotic, targeting isoleucyl-tRNA synthase, and produced by Pseudomonas fluorescens NCIMB 10586. It is used clinically as a topical treatment for staphylococcal infections, particularly in contexts where there is a problem with methicillin-resistant Staphylococcus aureus (MRSA). In studying the mupirocin biosynthetic cluster the authors identified two putative regulatory genes, mupR and mupI, whose predicted amino acid sequences showed significant identity to proteins involved in quorum-sensing-dependent regulatory systems such as LasR/LuxR (transcriptional activators) and LasI/LuxI (synthases for N-acylhomoserine lactones – AHLs – that activate LasR/LuxR). Inactivation by deletion mutations using a suicide vector strategy confirmed the requirement for both genes in mupirocin biosynthesis. Cross-feeding experiments between bacterial strains as well as solvent extraction showed that, as predicted, wild-type P. fluorescens NCIMB 10586 produces a diffusible substance that overcomes the defect of a mupI mutant. Use of biosensor strains showed that the MupI product can activate the Pseudomonas aeruginosa lasRlasI system and that P. aeruginosa produces one or more compounds that can replace the MupI product. Insertion of a xylE reporter gene into mupA, the first ORF of the mupirocin biosynthetic operon, showed that together mupR/mupI control expression of the operon in such a way that the cluster is switched on late in exponential phase and in stationary phase.

Ralstonia eutropha JMP134(pJP4) is able to grow on minimal media containing the pollutants 3-chlorobenzoate (3-CB) or 2,4-dichlorophenoxyacetate (2,4-D). tfd genes from the 88 kb plasmid pJP4 encode enzymes involved in the degradation of these compounds. During growth of strain JMP134 in liquid medium containing 3-CB, a derivative strain harbouring a ∼∼95 kb plasmid was isolated. This derivative, designated JMP134(pJP4-F3), had an improved ability to grow on 3-CB, but had lost the ability to grow on 2,4-D. Sequence analysis of pJP4-F3 indicated that the plasmid had undergone a deletion of ∼∼16 kb, which included the tfdA–tfdS intergenic region, spanning the tfdA gene to a previously unreported IS1071 element. The loss of the tfdA gene explains the failure of the derivative to grow on 2,4-D. A ∼∼23 kb duplication of the region spanning tfdR-tfdD II C II E II F II-tfdB II-tfdK-ISJP4-tfdT-tfdC I D I E I F I-tfdB I, giving rise to a 51-kb-long inverted repeat, was also observed. The increase in gene copy number for the tfdCD(DC)EF gene cluster may provide an explanation for the derivative strain’s improved growth on 3-CB. These observations are additional examples of the metabolic plasticity of R. eutropha JMP134, one of the more versatile pollutant-degrading bacteria.

Expression of the phenol degradation pathway in Pseudomonas putida strain PaW85 requires coordinated transcription of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and phenol monooxygenase, respectively, and the chromosomally encoded catechol degradation catBCA operon. Transcriptional activation from the pheBA and catBCA promoters is regulated by CatR and the catechol degradation pathway intermediate cis,cis-muconate. Here it is shown that physiological control mechanisms are superimposed on this regulatory system. Transcriptional activation from the pheBA and catBCA promoters is growth-phase-regulated in P. putida cells grown on rich medium (LB medium). CatR-mediated transcription from these promoters is silenced on rich medium until the transition from exponential to stationary phase. A slight positive effect (threefold) of stationary-phase-specific sigma factor σS on transcription from the pheBA promoter was observed. Expression of the catBCA promoter was not influenced by the activity of this sigma factor. In contrast to rich growth medium, transcription from the pheBA and catBCA promoters in minimal medium containing a mixture of glucose and sodium benzoate was rapidly induced in exponential culture. It was shown that the presence of amino acids in the culture medium causes exponential silencing of the pheBA and catBCA promoters. The possibility that a hypothetical repressor protein could be involved in physiological control of transcription from the pheBA and catBCA promoters is discussed.

A key intermediate for biodegradation of various distinct aromatic growth substrates in Comamonas testosteroni is protocatechuate (Pca), which is metabolized by the 4,5-extradiol (meta) ring fission pathway. A locus harbouring genes from C. testosteroni BR6020 was cloned, dubbed pmd, which encodes the enzymes that degrade Pca. The identity of pmdAB, encoding respectively the α- and β-subunit of the Pca ring-cleavage enzyme, was confirmed by N-terminal sequencing and molecular mass determination of both subunits from the separated enzyme. Disruption of pmdA resulted in a strain unable to grow on Pca and a variety of aromatic substrates funnelled through this compound (m- and p-hydroxybenzoate, p-sulfobenzoate, phthalate, isophthalate, terephthalate, vanillate, isovanillate and veratrate). Growth on benzoate and o-aminobenzoate (anthranilate) was not affected in this strain, indicating that these substrates are metabolized via a different lower pathway. Tentative functions for the products of other pmd genes were assigned based on sequence identity and/or similarity to proteins from other proteobacteria involved in uptake or metabolism of aromatic compounds. This study provides evidence for a single lower pathway in C. testosteroni for metabolism of Pca, which is generated by different upper pathways acting on a variety of aromatic substrates.

Although gene clusters for the degradation of biphenyls and polychlorobiphenyls have been extensively characterized, comparatively little is known about the regulation of their expression. In the present work, different aspects of transcription of the bph locus of the potent polychlorobiphenyl degrader Burkholderia sp. strain LB400 were investigated. An RNA blot analysis of the entire gene cluster revealed that the transcription of all genes encoding biphenyl catabolic enzymes responded similarly to the presence of biphenyl, succinate or a mixture of the two. One region of the locus, encompassing ORF0, was separately transcribed and differently regulated. A single start position was mapped for this monocistronic transcript. Synthesis of the adjacent RNA, encoding subunits of biphenyl dioxygenase, was strongly biphenyl-inducible. In this case, four major 5′-ends were mapped between 25 and 70 bp upstream of the start codon of gene bphA1. Sequence elements between approximately positions 710 and 1080 upstream were required in cis for full functioning of the respective promoter(s) (P bphA1 ). ORF0− mutants of strain LB400 retained the ability to grow on biphenyl, but showed decreased concentrations of bphA1A2 RNA and decreased lacZ expression in strains harbouring a reporter system with a bphA1–lacZ transcriptional fusion. This effect was compensated by the introduction of an intact ORF0 in trans, indicating that the ORF0 gene product mediates activation of P bphA1 .

‘Pseudomonas azelaica’ HBP1 degrades 2-hydroxybiphenyl (2-HBP) and 2,2′-diHBP by employing a meta-cleavage pathway encoded by the hbpCAD genes. The regulatory gene hbpR, located directly upstream of the hbpCAD genes and oriented in the opposite direction, encodes a transcription activator protein belonging to the so-called XylR/DmpR subclass within the NtrC family. HbpR activates transcription from two separate σ54-dependent promoters upstream of the hbpC and the hbpD genes, in the presence of the pathway substrates 2-HBP and 2,2′-diHBP. The DNA region upstream of the hbpC gene displays an unusual organization, containing two adjacent 0·3 kb regions that share 71% sequence identity. The DNA region most proximal to the hbpC promoter harbours one pair of putative upstream activating sequences (UASs C-1/C-2) and a small cryptic ORF that shows homology to hbpR itself. The second, more distal, region contains a second pair of putative UASs (UASs C-3/4) and the 5′-part of the hbpR gene. Transcriptional fusions in Escherichia coli between different deletions of the hbpR–hbpC intergenic region and the genes for bacterial luciferase revealed that most if not all of the transcriptional output from the hbpC promoter is mediated from the proximal UASs C-1/C-2. However, when the UASs C-1/C-2 were deleted and UASs C-3/C-4 were placed in an appropriate position with respect to the promoter region, the hbpC promoter was still inducible with 2-HBP, albeit at a lower level. Transcription studies in E. coli and ‘P. azelaica’ revealed that the divergently oriented hbpR gene is expressed constitutively from a σ70-dependent promoter situated within the cryptic ORF. The presence of UAS pair C-3/C-4 mediated a slightly higher promoter activity for transcription of hbpR.

The nucleotide sequence of the aromatic amine utilization (aau) gene region from Alcaligenes faecalis contained nine genes (orf-1, aauBEDA, orf-2, orf-3, orf-4 and hemE) transcribed in the same direction. The aauB and aauA genes encode the periplasmic aromatic amine dehydrogenase (AADH) large and small subunit polypeptides, respectively, and were homologous to mauB and mauA, the genes for the large and small subunits of methylamine dehydrogenase (MADH). aauE and aauD are homologous to mauE and mauD and apparently carry out the same function of transport and folding of the small subunit polypeptide in the periplasm. No analogues of the mauF, mauG, mauL, mauM and mauN genes responsible for biosynthesis of tryptophan tryptophylquinone (the prosthetic group of amine dehydrogenases) were found in the aau cluster. orf-2 was predicted to encode a small periplasmic monohaem c-type cytochrome. No biological function can be assigned to polypeptides encoded by orf-1, orf-3 and orf-4 and mutations in these genes appeared to be lethal. Mutants generated by insertions into mauD were not able to use phenylethylamine, tyramine and tryptamine as a source of carbon and phenylethylamine, 3’-hydroxytyramine (dopamine) and tyramine as a source of nitrogen, indicating that AADH is the only enzyme involved in utilization of primary amines in A. faecalis. AADH genes are present in Alcaligenes xylosoxydans subsp. xylosoxydans, but not in other β- and γ-proteobacteria. Phylogenetic analysis of amine dehydrogenases (MADH and AADH) indicated that AADH and MADH evolutionarily diverged before separation of proteobacteria into existing subclasses.

From Ralstonia eutropha HF39 null-allele mutants were created by Tn5 mutagenesis and by homologous recombination which were impaired in growth on propionic acid and levulinic acid. From the molecular, physiological and enzymic analysis of these mutants it was concluded that in this bacterium propionic acid is metabolized via the methylcitric acid pathway. The genes encoding enzymes of this pathway are organized in a cluster in the order prpR, prpB, prpC, acnM, ORF5 and prpD, with prpR transcribed divergently from the other genes. (i) prpC encodes a 2-methylcitric acid synthase (42720 Da) as shown by the measurement of the respective enzyme activity, complementation of a prpC mutant of Salmonella enterica serovar Typhimurium and high sequence similarity. (ii) For the translational product of acnM the function of a 2-methyl-cis-aconitic acid hydratase (94726 Da) is proposed. This protein and also the ORF5 translational product are essential for growth on propionic acid, as revealed by the propionic-acid-negative phenotype of Tn5-insertion mutants, and are required for the conversion of 2-methylcitric acid into 2-methylisocitric acid as shown by the accumulation of the latter, which could be purified as its calcium salt from the supernatants of these mutants. In contrast, inactivation of prpD did not block the ability of the cell to use propionic acid as carbon and energy source, as shown by the propionic acid phenotype of a null-allele mutant. It is therefore unlikely that prpD from R. eutropha encodes a 2-methyl-cis-aconitic acid dehydratase as proposed recently for the homologous prpD gene from S. enterica. (iii) The translational product of prpB encodes 2-methylisocitric acid lyase (32314 Da) as revealed by measurement of the respective enzyme activity and by demonstrating accumulation of methylisocitric acid in the supernatant of a prpB null-allele mutant. (iv) The expression of prpC and probably also of the other enzymes is regulated and is induced during cultivation on propionic acid or levulinic acid. The putative translational product of prpR (70895 Da) exhibited high similarities to PrpR of Escherichia coli and S. enterica, and might represent a transcriptional activator of the sigma-54 family involved in the regulation of the other prp genes. Since the prp locus of R. eutropha was very different from those of E. coli and S. enterica, an extensive comparison of prp loci available from databases and literature was done, revealing two different classes of prp loci.