- Volume 147, Issue 10, 2001

The active efflux of toxic compounds by (multi)drug transporters is one of the mechanisms that bacteria have developed to resist cytotoxic drugs. The authors describe the role of the lactococcal secondary multidrug transporter LmrP in the resistance to a broad range of clinically important antibiotics. Cells expressing LmrP display an increased resistance to the lincosamide, streptogramin, tetracycline and 14- and 15-membered macrolide antibiotics. The streptogramin antibiotic quinupristin, present in the fourth-generation antibiotic RP 59500, can inhibit LmrP-mediated Hoechst 33342 transport, but is not transported by LmrP, indicating that quinupristin acts as a modulator of LmrP activity. LmrP-expressing Lactococcus lactis cells in which a proton-motive force is generated accumulate significantly less tetracycline than control cells without LmrP expression. In contrast, LmrP-expressing and control cells accumulate equal amounts of tetracycline in the absence of metabolic energy. These findings demonstrate that the increased antibiotic resistance in LmrP-expressing cells is a result of the active extrusion of antibiotics from the cell.

Listeriolysin O (LLO) is a major virulence factor secreted by the pathogenic Listeria monocytogenes and acts as pore-forming cytolysin. Based on sequence similarities between LLO and perfringolysin (PFO), the cytolysin from Clostridium perfringens of known crystallographic structure, two truncated LLO proteins were produced: LLO-d123, comprising the first three predicted domains, and LLO-d4, the last C-terminal domain. The two proteins were efficiently secreted into the culture supernatant of L. monocytogenes and were able to bind to cell membranes. Strikingly, when expressed simultaneously, the two secreted domains LLO-d123 and LLO-d4 reassembled into a haemolytically active form. Two in-frame linker insertions were generated in the hinge region between the d123 and d4 domains. In both cases, the insertion created a major cleavage site for proteolytic degradation and abolished cytolytic activity, which might suggest that the region connecting d123 and d4 participates in the interaction between the two portions of the monomer.

Salmonella enterica serovar Enteritidis is a leading cause of food poisoning in the USA and Europe. Although Salmonella serovars share many fimbrial operons, a few fimbriae are limited to specific Samonella serovars. SEF14 fimbriae are restricted to group D Salmonella and the genes encoding this virulence factor were acquired relatively recently. Genomic, genetic and gene expression studies have been integrated to investigate the ancestry, regulation and expression of the sef genes. Genomic comparisons of the Salmonella serovars sequenced revealed that the sef operon is inserted in leuX in Salmonella Enteritidis, Salmonella Paratyphi and Salmonella Typhi, and revealed the presence of a previously unidentified 25 kb pathogenicity island in Salmonella Typhimurium at this location. Salmonella Enteritidis contains a region of homology between the Salmonella virulence plasmid and the chromosome downstream of the sef operon. The sef operon itself consists of four co-transcribed genes, sefABCD, and adjacent to sefD there is an AraC-like transcriptional activator that is required for expression of the sef genes. Expression of the sef genes was optimal during growth in late exponential phase and was repressed during stationary phase. The regulation was coordinated by the RpoS sigma factor.

Clostridium difficile is a nosocomial pathogen that causes a range of chronic intestinal diseases, usually as a result of antimicrobial therapy. Macrolide-lincosamide-streptogramin B (MLS) resistance in C. difficile is encoded by the Erm B resistance determinant, which is thought to be located on a conjugative transposon, Tn5398. The 9630 bp Tn5398 element has been cloned and completely sequenced and its insertion site determined. Analysis of the resultant data reveals that Tn5398 is not a classical conjugative transposon but appears to be a mobilizable non-conjugative element. It does not carry any transposase or site-specific recombinase genes, nor any genes likely to be involved in conjugation. Furthermore, using PCR analysis it has been shown that isolates of C. difficile obtained from different geographical locations exhibit heterogeneity in the genetic arrangement of both Tn5398 and their Erm B determinants. These results indicate that genetic exchange and recombination between these determinants occurs in the clinical and natural environment.

The fluorescent amplified fragment length polymorphism (AFLP) fingerprinting method was tested for its ability to identify and subtype the most important Campylobacter species found in veterinary infections. Sixty-nine reference strains and 19 clinical isolates of Campylobacter jejuni subsp. jejuni, Campylobacter jejuni subsp. doylei, Campylobacter upsaliensis, Campylobacter coli, Campylobacter lari, Campylobacter fetus subsp. fetus, C. fetus subsp. venerealis, Campylobacter hyointestinalis subsp. hyointestinalis, C. hyointestinalis subsp. lawsonii, Campylobacter mucosalis, Campylobacter helveticus and Campylobacter sputorum were subjected to analysis. The topology of the dendrogram obtained by numerical analysis of the AFLP profiles did not reflect the phylogenetic relationships as derived from 16S rDNA sequence comparison. However, except for C. lari, AFLP analysis grouped the strains that belonged to the same genomic species into distinct clusters. C. lari strains were separated into two distinct AFLP groups, which corresponded with nalidixic-acid-sensitive and -resistant variants of C. lari. These results correlated with data from whole-cell protein profiling. Within C. jejuni, C. hyointestinalis and C. fetus, strains could be identified at the subspecies level. AFLP analysis also allowed the subtyping of most species at the strain level. It is concluded that AFLP analysis is a valuable tool for concurrent identification of campylobacters at the species, subspecies and strain levels. In addition, the data confirm and extend previous reports showing that C. lari is a heterogeneous species that may comprise multiple taxa.

A 2019 bp DNA fragment containing the replicon of pSW800 from Pantoea stewartii SW2 was cloned and characterized. This replicon contains two genes – repA and repB, which encode a 36·5 kDa replication initiation protein (RepA) and a peptide of 18 aa, respectively. These two genes overlap by 8 bases with repB situated upstream. The replicon also transcribes an antisense RNA (RNAI) that inhibits the expression of repA and repB. The ribosome-binding sequence (RBS) of repA is likely to be hidden in a stem–loop structure, inhibiting the translation of repA. Furthermore, translation of repB is likely to disrupt the stem–loop structure, which is one of the criteria allowing the translation of repA to begin. A mutagenesis study revealed that a sequence (5′-GCACGGG-3′) located 111 nt upstream from repA is crucial; mutation of this sequence prevented the translation of repA. Additionally, this region and the stem–loop structure containing the RBS of repA may form an RNA pseudoknot. Results in this study demonstrate that a mechanism similar to that regulating plasmid replication in the IncB, IncIα and IncL/M groups also regulates pSW800 replication.

Two overlapping promoters, osmC p1 and osmC p2, direct the transcription of the osmC gene of Escherichia coli. The proximal promoter, osmC p2, is induced upon entry into stationary phase under the control of Eσs, the RNA polymerase that uses the σs (RpoS) sigma factor. Transcription from the distal promoter, osmC p1, is independent of σs. Previous analysis demonstrated that the osmolarity of the growth medium modulates expression of both promoters. The use of an E. coli genomic library showed that the cloned nhaR gene was able to stimulate transcription of an osmC–lac reporter fusion. NhaR is a positive regulator of the LysR family, previously identified as an activator of nhaA, a gene encoding a Na+/H+ antiporter involved in adaptation to Na+ and alkaline pH in E. coli and other enteric bacteria. NhaR was shown to activate only the expression of osmC p1 and to be necessary for the induction of this promoter by LiCl, NaCl and sucrose. Therefore, activation by NhaR is responsible for the osmotic induction of osmC p1. In contrast to its action on nhaA, NhaR activation of osmC p1 is independent of H-NS. Activation of osmC p1 by NhaR requires a site located just upstream of the atypical −35 region of the promoter.

The adhC1 gene from Acinetobacter baumannii 8399, which encodes a glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), was identified and cloned after mapping the insertion site of Tn3-HoHo1 in a recombinant cosmid isolated from a gene library. Sequence analysis showed that this gene encodes a protein exhibiting significant similarity to alcohol dehydrogenases in bacterial, yeast, plant and animal cells. The expression of the adhC1 gene was confirmed by the detection of GSH-FDH enzyme activity in A. baumannii and Escherichia coli cells that expressed the cloned gene. However, the construction and analysis of an A. baumannii 8399 adhC1::Tn3-HoHo1 isogenic derivative revealed the presence of adhC2, a second copy of the gene encoding GSH-FDH activity. Enzyme assays and immunoblot analysis showed that adhC2 encodes a 46·5 kDa protein that is produced in similar amounts under iron-rich and iron-limited conditions. In contrast, the expression of adhC1, which encodes a 45 kDa protein with GSH-FDH activity, is induced under iron limitation and repressed when the cells are cultured in the presence of free inorganic iron. The differential expression of adhC1 is controlled at the transcriptional level and mediated through the Fur iron-repressor protein, which has potential binding sites within the promoter region of this adhC copy. The expression of both adhC copies is significantly enhanced by the presence of sub-inhibitory concentrations of formaldehyde in the culture media. Examination of different A. baumannii isolates indicates that they can be divided into two groups based on the type of GSH-FDH they produce. One group contains only the constitutively expressed 46·5 kDa protein, whilst the other produces this GSH-FDH type in addition to the iron-regulated isoenzyme. Further analysis showed that the presence and expression of the two adhC genes does not confer resistance to exogenous formaldehyde, nor does it enable it to utilize methylated compounds as a sole carbon source when cultured under iron-rich as well as iron-deficient conditions.

Regions of the Streptomyces venezuelae ISP5230 chromosome flanking pabAB, an amino-deoxychorismate synthase gene needed for chloramphenicol (Cm) production, were examined for involvement in biosynthesis of the antibiotic. Three of four ORFs in the sequence downstream of pabAB resembled genes involved in the shikimate pathway. BLASTX searches of GenBank showed that the deduced amino acid sequences of ORF3 and ORF4 were similar to proteins encoded by monofunctional genes for chorismate mutase and prephenate dehydrogenase, respectively, while the sequence of the ORF5 product resembled deoxy-arabino-heptulosonate-7-phosphate (DAHP) synthase, the enzyme that initiates the shikimate pathway. A relationship to Cm biosynthesis was indicated by sequence similarities between the ORF6 product and membrane proteins associated with Cm export. BLASTX searches of GenBank for matches with the translated sequence of ORF1 in chromosomal DNA immediately upstream of pabAB did not detect products relevant to Cm biosynthesis. However, the presence of Cm biosynthesis genes in a 7·5 kb segment of the chromosome beyond ORF1 was inferred when conjugal transfer of the DNA into a blocked S. venezuelae mutant restored Cm production. Deletions in the 7·5 kb segment of the wild-type chromosome eliminated Cm production, confirming the presence of Cm biosynthesis genes in this region. Sequencing and analysis located five ORFs, one of which (ORF8) was deduced from BLAST searches of GenBank, and from characteristic motifs detected in alignments of its deduced amino acid sequence, to be a monomodular nonribosomal peptide synthetase. GenBank searches did not identify ORF7, but matched the translated sequences of ORFs 9, 10 and 11 with short-chain ketoreductases, the ATP-binding cassettes of ABC transporters, and coenzyme A ligases, respectively. As has been shown for ORF2, disrupting ORF3, ORF7, ORF8 or ORF9 blocked Cm production.

Chryseobacterium meningosepticum is an aerobic Gram-negative rod widely distributed in natural environments. Unlike many bacteria, it produces a phosphate-irrepressible periplasmic alkaline phosphatase (AP). This work describes cloning of the gene encoding that enzyme from C. meningosepticum CCUG 4310 (NCTC 10585), and preliminary characterization of its product. The gene, named pafA, encodes a protein (PafA) of 546 amino acids with a calculated molecular mass of the mature peptide of 58682 Da. PafA exhibits high sequence identity with the PhoV AP of Synechococcus PCC 7942 (49·9% identity) and with the Cda Ca2+-dependent ATPase of Myroides odoratus (51·9% identity), while being more distantly related to the PhoD AP of Zymomonas mobilis (22·1% identity) and to the PhoA AP of Escherichia coli (14·0% identity). PafA was partially purified; it exhibits optimal activity at pH 8·5 and is active towards a broad spectrum of substrates including both phosphomonoesters and ATP, with preferential activity for the latter compound. The present findings allow definition of a new family of APs including 60 kDa, periplasmic enzymes whose expression is not influenced by freely available Pi in the medium. Moreover, PafA can be considered an evolutionary intermediate between Ca2+-ATPase of M. odoratus and the APs PhoV of Synechococcus PCC 7942 and PhoD of Z. mobilis.