Cloning of a genetically unstable cytochrome P-450 gene cluster involved in degradation of the pollutant ethyl tert-butyl ether by Rhodococcus ruber.

Rhodococcus ruber (formerly Gordonia terrae) IFP 2001 is one of a few bacterial strains able to degrade ethyl tert-butyl ether (ETBE), which is a major pollutant from gasoline. This strain was found to undergo a spontaneous 14.3-kbp chromosomal deletion, which results in the loss of the ability to degrade ETBE. Sequence analysis of the region corresponding to the deletion revealed the presence of a gene cluster, ethABCD, encoding a ferredoxin reductase, a cytochrome P-450, a ferredoxin, and a 10-kDa protein of unknown function, respectively. The EthB and EthD proteins could be easily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were induced by ETBE in the wild-type strain. Upstream of ethABCD lies ethR, which codes for a putative positive transcriptional regulator of the AraC/XylS family. Transformation of the ETBE-negative mutant by a plasmid carrying the ethRABCD genes restored the ability to degrade ETBE. Complementation was abolished if the plasmid carried ethRABC only. The eth genes are located in a DNA fragment flanked by two identical direct repeats of 5.6 kbp. The ETBE-negative mutants carry a single copy of this 5.6-kbp repeat, suggesting that the 14.3-kbp chromosomal deletion resulted from a recombination between the two identical sequences. The 5.6-kbp repeat is a class II transposon carrying a TnpA transposase, a truncated form of the recombinase TnpR, and a terminal inverted repeat of 38 bp. The truncated TnpR is encoded by an IS3-interrupted tnpR gene.

Functions of S-layers.

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

Strategy for detection and identification of bacteria based on 16S rRNA genes in suspected cases of Whipple's disease.

The 16S ribosomal RNA (rRNA) gene of the phylogenetic subdivision containing gram-positive bacteria with a high G + C content was detected specifically in clinical specimens from patients suspected of having Whipple's disease. The primary structure of 16S rDNA amplified from clinical samples was determined by cloning and sequencing. Two sorts of sequences were identified: one corresponded exactly to the rRNA sequence of Tropheryma whippelii (GenBank accession no. M87484) while the other was related to that of members of the genus Corynebacterium. No sequence related to Mycobacterium spp. or Rhodococcus equi was observed. Exhaustive examination of negative specimens with broad-range eubacterial primers detected one sequence related to Enterobacteriaceae and another related to Enterococcus spp. To speed identification of T. whippelii, a nested amplification method was devised. A first amplification specific for the gram-positive bacteria subdivision was performed, followed by a second amplification with T. whippelii-specific primers. The amplified T. whippelii product was checked by digestion with AvaII, StuI, and PstI endonucleases. These techniques were applied to DNA extracted from seven intestinal biopsy samples, two cerebrospinal fluid samples and one articular fluid from patients suspected of having Whipple's disease. T. whippelii 16S rDNA was found in two of the biopsy samples, one of the cerebrospinal fluid samples and in the articular fluid.

Identification of Bartonella henselae and B. quintana 16s rDNA sequences by branch-, genus- and species-specific amplification.

Given the controversy surrounding the aetiology of cat scratch disease and the association of both Bartonella henselae and B. quintana with bacillary angiomatosis, a method for the direct detection in clinical samples of 16S rRNA from the Proteobacteria alpha subgroup was developed. The primary structure of amplified 16S rDNA was determined by cloning and sequencing. Three sequences were identified: one corresponded exactly to GenBank accession number M73229 (B. henselae); the second was related to, but distinct from, GenBank accession number Z11684 (referred to as 'B. henselae variant'); and a third sequence was identical with GenBank accession number M73228 (B. quintana). No sequence corresponding to Afipia spp. was found. To speed identification and reduce the cost of analysis, a nested amplification method for B. henselae and B. quintana was devised. These techniques were applied to DNA extracted from 30 unfixed lymph node biopsies, two liver biopsies and 36 node pus samples from patients with suspected cat scratch disease, and from 17 skin biopsies from AIDS patients with suspected bacillary angiomatosis. B. henselae or B. henselae variant sequences were found in 42 (62%) of 68 samples from suspected cat scratch disease. B. quintana was not associated with cat scratch disease, but a B. quintana sequence was found in seven (41%) of 17 samples from suspected bacillary angiomatosis patients. B. henselae 16S rDNA sequences were not found in bacillary angiomatosis specimens.

Cloning and expression of plasmid DNA sequences involved in Salmonella serotype typhimurium virulence.

A 22kb region of the 90kb virulence-associated plasmid, pIP1350, of Typhimurium strain C52 was cloned into the mobilizable vector pSUP202, yielding plasmid pIP1352. This recombinant plasmid restored full virulence to plasmidless strain C53 in a mouse model. Transposon Tn5 insertion mutagenesis demonstrated the existence of two DNA sequences in pIP1352 designated VirA and VirB, both of which are essential for the expression of virulence. A recombinant plasmid containing only the VirA and VirB regions markedly increased the virulence of the plasmidless strain C53, but did not confer full virulence. These results suggested that a third virulence-associated region might be present on pIP1352. Eleven proteins encoded by the 22kb insert sequence of pIP1352 were identified in Escherichia coli SE5000 maxicells. The VirA region encoded at least two proteins with apparent molecular weights of 71,000 and 28,000 and the VirB region encoded two proteins of 43,000 and 38,000.

Virulence-associated plasmids of Salmonella serotype Typhimurium in experimental murine infection.

The growth pattern of Salmonella enterica subsp. enterica serotype Typhimurium (Typhimurium) was studied in mice to examine the role of the 60-Mdal virulence-associated plasmid in the pathogenesis of mouse typhoid. After repeated subcultures at 45 degrees C, isogenic variants harbouring the virulence-associated plasmid (strains C52, TM122 and TM332) or having lost this large plasmid (strains C53, TM123 and TM333) were obtained from three parental strains (strains C5, TM12 and TM33, respectively). Plasmid pIP1350, present in strain C52, was tagged by Tn10 and transferred by successive conjugations to strains C53, TM123 and TM333. The behaviour of these three Typhimurium lines was studied in C57BL/6, DBA2, B6D2 (C57BL/6 X DBA2 F1 hybrid) and OF1 mice after oral infection, subcutaneous injection into the hind footpad or intravenous inoculation. The kinetics of organ colonization were followed at intervals after injection by enumeration of viable bacteria in caecum, mesenteric or popliteal lymph node, spleen, liver, kidney and lung depending on the route of infection. Strains harbouring virulence plasmid and their cured derivatives did not differ significantly in their ability to colonize caecal content and to translocate to draining lymph nodes. Elimination of the virulence plasmid was correlated with a significant reduction in the ability of cured variants to colonize spleen and liver. Reintroduction of the virulence plasmid into plasmidless variants restored the virulence to the level originally observed. These data demonstrate that a 60-Mdal plasmid in Typhimurium strains is necessary to ensure colonization of spleen and liver of experimentally infected mice.

Molecular relationships between virulence plasmids of Salmonella serotypes typhimurium and dublin and large plasmids of other Salmonella serotypes.

All studied isolates of Salmonella serotypes abortusovis (16 strains), enteritidis (30 strains), paratyphi C (29 strains), and 2 out of 10 isolates of serotype newport harboured large 54-76-Kb plasmids. No such plasmids were found in the following serotypes: agona, bovismorbificans, heidelberg, infantis, panama, paratyphi A, paratyphi B, saintpaul, senftenberg and typhi. These plasmids and the virulence-associated plasmids of Salmonella serotypes typhimurium and dublin were compared at the molecular level. Plasmids from the same serotype usually showed similar HindIII endonuclease patterns. Plasmids from different serotypes displayed markedly different cleavage patterns. Using the 3H-labelled plasmid from serotype typhimurium strain C5 as a probe, nitrocellulose filter hybridization showed that all these plasmids shared homologous sequences distributed throughout the plasmid molecule. With the S1-nuclease method, all plasmids were 61 to 88% related to the virulence plasmid of serotype typhimurium strain C5. The large plasmids in Salmonella serotypes abortusovis, enteritidis, paratyphi C, newport and the virulence-associated plasmids in serotypes typhimurium and dublin thus constitute a single group of homology and represent a family of related plasmids. We suggest that this plasmid group may contribute to the pathogenic potential of host serotypes.

[Taxonomic position of Agrobacterium strains of hospital origin]. / Position taxonomique de souches de Agrobacterium d'origine hospitalière.

A collection of 31 strains received as Agrobacterium tumefaciens and A. radiobacter was subjected to detailed phenotypic and genomic studies. These strains were recovered from plants, soil, water and clinical specimens. Type strains of A. tumefaciens, A. radiobacter, A. rhizogenes and A. rubi were also included. The strains were tested for their ability to use 169 organic compounds as sources of carbon and energy. In addition, 11 conventional characters were studied for each strain. Relatedness among the strains was assessed by determining the extent of reassociation in heterologous DNA preparations. S1 nuclease and diethylaminoethyl-cellulose filters were used to separate reassociated from non-reassociated nucleotide sequences, and to determine the thermal stability of related nucleotide sequences. The resultant data revealed the following points: regardless of their phytopathogenic effects, 3-ketolactose-producing A. tumefaciens and A. radiobacter strains group into one species; this species contains 9 taxa which can be differentiated from each other by phenotypic and genomic characters; clinical isolates did not induce tumours on plants and clustered in three taxa of this species; the clinical and ecological significance of these organisms is not known; the present classification of the genus Agrobacterium is based on phytopathogenicity and does not reflect the phylogenetic relationships amongst these bacteria; as proposed previously by several workers, the genus Agrobacterium should be divided into 3 species on the basis of phenotypic and genomic characteristics. Different aspects of the classification and nomenclature of Agrobacterium are discussed.

[9 new Salmonella serotypes isolated in Africa and the Antilles]. / Neuf nouveaux sérotypes de salmonella isolés en Afrique et aux Antilles.

Description of nine new serotypes of Salmonella, three isolated in Senegal: S. tambacounda (I,3,I9:b:e,n,x) from Chiroptera, S. bargny (8,20:i:I,5) and S. fass (50:l,v:I,2) from horse; two recovered in Haute-Volta from patients with diarrhoea: S. tounouma (8,20:b:z6) and S. sya (47:b:z6); one isolated from a carrier in Antilles: S. gustavia (II:d:I,5); one from a snake in Tunisia: S. gafsa (I6:c:I,6); two from Madagascar: S. II I8:z4,z23:- isolated from a wild mammalian and S. III arizonae 6I:z52:z from a patient with diarrhoea.