H-NS and Lrp serve as positive modulators of traJ expression from the Escherichia coli plasmid pRK100.

Conjugative transfer of F-like plasmids is a tightly regulated process. The TraJ protein is the main positive activator of the tra operon which encodes products required for conjugative transfer of F-like plasmids. Nucleotide sequence analysis revealed potential Lrp and H-NS binding sites in the traJ regulatory region. Expression of a traJ-lacZ fusion in hns and lrp mutant strains showed that both are positive modulators of traJ expression. Competitive RT-PCR demonstrated that H-NS and Lrp exert their effect at the transcriptional level. Electrophoretic mobility-shift assays showed that H-NS and Lrp proteins bind to the traJ promoter. Conjugative transfer of pRK100 was decreased in hns but not in lrp mutant strains. Together, the results indicate H-NS and Lrp function as activators of traJ transcription.

Binary toxin producing Clostridium difficile strains.

Clostridium difficile produces three toxins, TcdA, TcdB and CDT. TcdA and TcdB are single-stranded molecules acting as glucosyltransferases specific for small GTPases. CDT is an actin specific ADP-ribosylating binary toxin characteristically composed of two independent components, enzymatic CDTa (48 kDa) and binding CDTb (99 kDa). The cdtA and cdtB genes were sequenced in two CDT-positive strains of C. difficile (CD 196 and 8864) and at least two CDT-negative strains with truncated form of binary toxin genes are known (VPI 10463 and C. difficile genome strain 630). The prevalence of binary toxin producing strains is estimated to be from 1.6% to 5.5%, although a much higher proportion has been reported in some studies. The role of the binary toxin as an additional virulence factor is discussed.

The bacteriophages of ruminal prevotellas.

Rumen bacteriophage-lyzed bacterial strains of the genus Prevotella were isolated and preliminarily characterized. The strain TCl-1 the species P. bryantii was the only prevotella strain successfully infected with filter sterilized rumen fluid from a black-and-white Holstein cow. Two types of plaques were observed, both rather small and turbid. Preliminary electron microscopy observation showed that several morphologically different bacteriophages were present in these plaques. The plaque eluates were further used for the infection of other prevotella strains. The plaques produced by the bacteriophages were observed with two strains, i.e. P. bryantii B(1)4 and P. brevis GA33. The bacteriophages from both strains were examined by transmission electron microscopy and several morphologically different bacteriophages were observed, among others also a large virion with an icosahedral head with the diameter of approximately 120 nm. The bacteriophage was identified in plaques of bacterial cells of the strain GA33 and has an approximately 800 nm long helical tail, which places it among the largest ruminal bacteriophages described to date. Other bacteriophages from the same indicator strain as well as from P. bryantii B(1)4 strain were smaller and tail structures were not observed in all of them.

The role of the bacitracin ABC transporter in bacitracin resistance and collateral detergent sensitivity.

The bacitracin resistance of Bacillus licheniformis, a producer of bacitracin, is mediated by the ABC transporter Bcr. Bacillus subtilis cells carrying bcr genes on high-copy number plasmids developed collateral detergent sensitivity, as did human cells with overexpressed multidrug resistance P-glycoprotein. Resistance against bacitracin and sensitivity of resistant cells to detergents were shown to be inseparable phenomena associated with the membrane part of Bcr transporter, namely protein BcrC. A fused protein, consisting of ATP-binding protein BcrA and membrane component BcrC was constructed. It resembled a half molecule of P-glycoprotein and was capable of providing a significant degree of antibiotic resistance and detergent sensitivity.

A nonsense mutation abrogates production of a functional enterotoxin A in Clostridium difficile toxinotype VIII strains of serogroups F and X.

Clostridium difficile strains of toxinotype VIII from serogroups F and X are described as toxin B-positive, toxin A-negative (TcdB+ A-), although they harbour almost the entire tcdA gene. To identify the reason for the lack of TcdA detection, we analyzed catalytic and ligand domains of TcdA-1470 of the type strain of serogroup F, strain 1470. Using recombinant fragments, the C-terminal immunodominant ligand domain TcdA3-1470, spanning amino acid residues 1694-2711 (corresponding to VPI 10463 sequence), was detected in Western blots. Similar experiments using the recombinant N-terminal catalytic fragment TcdAc1-2-1470 (amino acid positions 1-544) failed. In addition, this fragment showed no glucosylation activity. We determined the size and the position of alterations in the ligand domain tcdA3-1470 by DNA sequencing. Within the N-terminal fragment tcdAc1-2-1470, a nonsense mutation was identified introducing a stop codon at amino acid position 47. Identical mutations were found in the two serogroup X strains 17663 and 10355. The mutation might explain the lack of TcdA production observed in strains of serotypes F and X.

Virulence determinants of uropathogenic Escherichia coli in fecal strains from intestinal infections and healthy individuals.

Twenty-five fecal Escherichia coli strains of serogroups O6 and O18 from patients with and without intestinal infections were analyzed for fimbrial adhesins pap, prs and sfa, hemolysin, cytonecrotic factor, colicins, capsules, antibiotic resistances, plasmid content and some plasmid encoded characteristics. A high percentage of strains expressing P fimbriae was found with an even higher percentage in strains isolated from intestinal infections. A correlation was found between colicinogenicity and P fimbriae production. None of the strains produced hemolysin while 28% had cytonecrotic factor type 1 sequences, demonstrating that cytonecrotic factor type 1 is not always associated with hemolysin production and indicating that the examined strains do not harbor a larger pathogenicity island. Plasmids and plasmid associated characteristics were more frequently associated with the O18 serogroup and chromosomal colicin V genes were found independently of plasmids.

Tn5431 arose by transposition of Tn3 into Tn1721.

To further characterize Tn5431, which is composed of Tn3 and Tn1721, DNA sequencing was carried out. It was demonstrated that Tn5431 arose by transposition of Tn3 into Tn1721. Multiple mutations in the Tn3 tnpA gene have occurred, rendering the Tn3 portion of Tn5431 transposition defective and ensuring the simultaneous transposition of the antibiotic resistance determinants on Tn3 and Tn1721.

Bacillus licheniformis bacitracin-resistance ABC transporter: relationship to mammalian multidrug resistance.

The nucleotide sequence of the Bacillus licheniformis bacitracin-resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP-binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydrophobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram-negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide-binding sites, BcrA protein and mammalian multidrug transporter or P-glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide-binding protein to determine the importance of various regions in transport.

Isolation and characterization of Tn917-generated bacitracin deficient mutants of Bacillus licheniformis.

Two Tn917-generated bacitracin deficient mutants of Bacillus licheniformis were isolated. Southern blot analysis of chromosomal DNA extracted from both insertional mutants showed that Tn917 inserted in the vicinity of the gene coding for the enzyme BA2 of the bacitracin synthetase enzyme complex. Measurements of bacitracin synthetase activity in cell-free extracts and positive hybridization signals in the vicinity of the BA2 gene indicate that in both bacitracin deficient mutants Tn917 could be inserted in the BA1 gene or in segments involved in regulation. Thus, it could be possible that the genes for bacitracin synthetase are clustered in the B. licheniformis genome.

Conjugal transfer of transposon Tn916 from Enterococcus faecalis to Bacillus licheniformis.

Transposon Tn916 was conjugally transferred from Enterococcus faecalis to Bacillus licheniformis bacterial strains ATCC 10716 and ATCC 9945A by filter and broth matings. The Tn916 transfer frequencies to B. licheniformis ranged from 10(-7) to 10(-5) selecting for tetracycline-resistant (Tcr) transconjugants depending on broth or filter mating. Movement of Tn916 was demonstrated when Tcr B. licheniformis transconjugants were mated with Bacillus subtilis strain W23. Tn916 insertion caused several auxotrophic and bacitracin deficient mutants. Southern blot analyses of HindIII chromosomal digests extracted from Tcr transconjugants showed that the transposon inserted at different sites in the B. licheniformis chromosome and the copy number of Tn916 varied. This approach should be useful for genetic studies of B. licheniformis.

Aerobactin uptake system, ColV production, and drug resistance encoded by a plasmid from an urinary tract infection Escherichia coli strain of human origin.

A study of Escherichia coli strains isolated from patients suffering from urinary tract infections in Ljubljana, Yugoslavia, revealed a plasmid encoding the aerobactin iron uptake system, ColV production, and drug resistance. The plasmid is conjugative and at least 85 kilobases in length.

Sporulation of a bacitracin-sensitive mutant of Bacillus licheniformis is self-inhibited by bacitracin.

A mutant of Bacillus licheniformis (BLU166) sensitive to its own antibiotic bacitracin was isolated and the mutation bcr-l was mapped close to the bacitracin synthetase genes. The sensitivity was shown to be specific for bacitracin. Two further bacitracin-sensitive strains were constructed, one (BLU171) with normal ability to synthesize bacitracin, and one (BLU170) a bacitracin non-producer. In addition to an increased sensitivity of growing cells to bacitracin, sporulation of the mutant strain BLU171 was self-inhibited by bacitracin. It is concluded that (1) there might exist at least two levels of resistance to bacitracin; (2) mutation bcr-1 affects a 'structural' component, which may protect the sensitive reaction of cell-wall biosynthesis; (3) sporulation is affected to a greater extent by bacitracin than vegetative growth; and (4) synthesis of bacitracin is independent of the presence of this resistance mechanism since the sensitive mutant produces similar amounts of the antibiotic to the wild-type strain.

Genetic mapping of the bacitracin synthetase gene(s) in Bacillus licheniformis.

The map position of a mutation in the bacitracin synthetase gene(s) in Bacillus licheniformis ATCC 10716 was determined by transduction with phage SP-15. Results indicate that it is linked to the lys and trp loci and is distinct from the known sporulation loci on the chromosome of Bacillus licheniformis. The defect(s) of the enzyme complex were analysed in terms of its ability to bind covalently 14C-labelled amino acid precursors of the bacitracin molecule.

Regulation of the major proline permease gene of Salmonella typhimurium.

The structural gene for the major proline permease is located in a tight cluster with genes coding for the proline degradative enzymes, proline oxidase and pyrroline-5-carboxylic acid dehydrogenase. Expression of the permease is regulated in parallel with the two degradative enzymes, and all three functions are subject to catabolite repression. Regulatory mutants (putC) have constitutively high levels of all three activities, suggesting that all are regulated by a single mechanism.