Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Low-copy-number T7 vectors for selective gene expression and efficient protein overproduction in Escherichia coli.

A set of low-copy-number vectors (pPD) has been constructed that permit selective gene expression and high-level protein overproduction in Escherichia coli, based on the bacteriophage T7 RNA polymerase/T7 promoter system. These plasmids carry a chloramphenicol resistance gene (cat) as a selective marker and an extended multiple cloning site for convenient gene cloning. Their replication is mediated by ori sequences derived from the low-copy-number vector pSC101. The efficient T7 gene 10 promoter present on these vectors allows selective and high-level transcription of cloned genes carrying their own translational initiation signals. In addition, low-copy-number T7 vectors were constructed that permit expression of genes lacking their own transcription and translation initiation elements by providing a ribosome binding site, an ATG start codon and a multiple cloning site devised for the cloning in all three reading frames. The pPD expression vectors were used to achieve high-level overproduction of the E. coli integral outer membrane protein Tsx, and the cytoplasmic enzymes beta-galactosidase (beta Gal) and UTP:alpha-D-glucose-1-phosphate uridylyltransferase (GalU). The characteristics of these low-copy-number T7 expression vectors should prove very useful for the cloning and high-level overexpression of genes whose gene products are deleterious to the E. coli host.

On the catalase-peroxidase gene, katG, of Mycobacterium leprae and the implications for treatment of leprosy with isoniazid.

The toxicity of the potent tuberculocidal agent, isoniazid, is mediated by the heme-containing enzyme, catalase-peroxidase, encoded by the katG gene. Although isoniazid has been used for the treatment of leprosy, it is shown here that the katG gene of Mycobacterium leprae is a pseudogene, which has probably been inactivated by multiple mutations. Inactive genes were detected by the polymerase chain reaction in several isolates of M. leprae, of different geographical origins, and attempts to complement an isoniazid-resistant strain of Mycobacterium smegmatis with the katG pseudogene were unsuccessful. Isoniazid is thus likely to be of no therapeutic benefit to leprosy patients.

[Effort phlebitis of the upper limb. Presentation of 2 cases and review of the literature]. / Phlébite d'effort du membre supérieur. Présentation de deux cas et revue de la littérature.

Effort phlebitis, which represented the principal aetiological form of deep vein thromboses of the upper limb, has become rare, while secondary forms, especially iatrogenic, are more frequent. The authors report two documented cases of effort phlebitis of the upper limb in two manual labourers aged 38 and 40 years. The aetiopathogenic and clinical features and clinical course of this disease are analysed.

Single amino acid substitutions affecting the substrate specificity of the Escherichia coli K-12 nucleoside-specific Tsx channel.

The Tsx protein from the Escherichia coli outer membrane is a channel-forming protein containing a nucleoside-specific binding site. The antibiotic albicidin enters the cell via this substrate-specific channel. Because albicidin is toxic for E. coli at a very low external substrate concentration, the Tsx channel is likely to contain a binding site for this antibiotic. To identify residues involved in the Tsx substrate-specific channel activity, we devised a selection scheme to isolate albicidin-resistant tsx mutants synthesizing Tsx proteins with defects in their nucleoside uptake function. We recovered seven distinct albicidin-resistant tsx alleles, six point mutations and a 39-base pair duplication. The mutants with a duplication of residues 21-33 of Tsx or with single amino acid substitutions of residue Gly28 (to Arg) and Ser217 (to Arg) are completely deficient in nucleoside uptake at a low substrate concentration. Substitutions of Phe27 to Leu, Gly28 to Glu, Gly239 to Asp, and Gly240 to Asp result in a Tsx protein partially defective in nucleoside transport. These mutant proteins still permit nonspecific diffusion of serine indicating that the mutations do not result in a block of the Tsx channel. Our results are discussed in terms of a model for the topological organization of the Tsx protein within the outer membrane of E. coli.

The Mycobacterium leprae genome: systematic sequence analysis identifies key catabolic enzymes, ATP-dependent transport systems and a novel polA locus associated with genomic variability.

In the framework of the mycobacterial genome sequencing project, a continuous 37,049 bp sequence from the Mycobacterium leprae chromosome has been determined. Computer analysis revealed 10 complete open reading frames, and nine of their products show similarity to known proteins. Seven of these were identified as the enzyme isocitrate lyase, two P-type ATPase cation transporters, two AMP-binding proteins, the ribosomal protein S1, and DNA polymerase I. Interestingly, the polA gene, encoding DNA polymerase, is flanked by two inverted copies of a new class of the M. leprae specific repetitive sequence, RLEP, and this structure resembles a transposable element. A second copy of this element was found at another locus in the genome, but the two copies were not present in equal amounts and could not be found in all isolates of M. leprae. This is the first evidence for genomic variability in the leprosy bacillus and might ultimately be useful for developing a molecular test capable of distinguishing between strains of M. leprae.

Homing events in the gyrA gene of some mycobacteria.

The A subunit of DNA gyrase in Mycobacterium leprae, unlike its counterpart in Mycobacterium tuberculosis, is produced by protein splicing as its gene, gyrA, harbors a 1260-bp in-frame insertion encoding an intein, a putative homing endonuclease. Analysis of the gyrA locus from different mycobacterial species revealed the presence of inteins in Mycobacterium flavescens, Mycobacterium gordonae and Mycobacterium kansasii but not in 10 other pathogenic or saprophytic mycobacteria. In all four cases where intein coding sequences were found, they were localized in the same position in gyrA, immediately downstream of the codon for the key active-site residue Tyr-130. The intein products were similar, but not identical, in sequence and the splice junctions displayed all the features found in other polypeptides known to be produced by protein splicing from a precursor protein. Paired motifs, found in homing endonucleases encoded by some group I RNA introns, and inteins showing endonuclease activity, were present in the gyrA inteins as were other intein-specific signatures. Some strains of M. flavescens, M. gordonae, and M. kansasii were shown by PCR analysis to have inteinless gyrA genes, in contrast to the situation in M. leprae where all the isolates possessed insertions in gyrA. Sequencing of the corresponding regions revealed that, although the GyrA protein sequence was conserved, the nucleotide sequences differed in gyrA genes with and without inteins, suggesting that the homing endonuclease displays sequence specificity.

Identification of a segment of the Escherichia coli Tsx protein that functions as a bacteriophage receptor area.

The Escherichia coli outer membrane protein Tsx functions as a nucleoside-specific channel and serves as the receptor for colicin K and a number of T-even-type bacteriophages, including phage T6. To identify those segments of the Tsx protein that are important for its phage receptor function, we devised a selection and screening procedure which allowed us to isolate phage-resistant strains synthesizing normal amounts of Tsx. Three different Tsx-specific phages (T6, Ox1, and H3) were employed for the selection of phage-resistant derivatives of a strain expressing a tsx(+)-lacZ+ operon fusion, and 28 tsx mutants with impaired phage receptor function were characterized. Regardless of the Tsx-specific phage used for the initial mutant selection, cross-resistance against a set of six different Tsx phages invariably occurred. With one exception, these mutant Tsx proteins could still serve as a colicin K receptor. DNA sequence analysis of 10 mutant tsx genes revealed the presence of four distinct tsx alleles: two point mutations, an 18-bp deletion, and a 27-bp tandem duplication. In three isolates, Asn-249 was replaced by a Lys residue (tsx-504), and in four others, residue Asn-254 was replaced by Lys (tsx-505). The deletion (tsx-506; one isolate) removed six amino acids (residue 239 to residue 244) from the 272-residue Tsx polypeptide chain, and the DNA duplication (tsx-507; two isolates) resulted in the addition of nine extra amino acids (residue 229 to residue 237) to the Tsx protein. In contrast to the wild-type Tsx protein and the other mutant Tsx proteins the Tsx-507 protein was cleaved by trypsin when intact cells were treated with this protease. The Tsx proteins encoded by the four tsx alleles still functioned in deoxyadenosine uptake in vivo, demonstrating that their nucleoside-specific channel activity was not affected by the alterations that caused the loss of their phage receptor function. HTe changes in the Tsx polypeptide that confer resistance against the Tsx-specific phages are clustered in a small region near the carboxy terminus of Tsx. Our results are discussed in terms of a model for the topological organization of the carboxy-terminal end of the Tsx protein within the outer membrane.

FbpA, a novel multifunctional Listeria monocytogenes virulence factor.

Listeria monocytogenes is a Gram-positive intracellular bacterium responsible for severe opportunistic infections in humans and animals. Signature-tagged mutagenesis (STM) was used to identify a gene named fbpA, required for efficient liver colonization of mice inoculated intravenously. FbpA was also shown to be required for intestinal and liver colonization after oral infection of transgenic mice expressing human E-cadherin. fbpA encodes a 570-amino-acid polypeptide that has strong homologies to atypical fibronectin-binding proteins. FbpA binds to immobilized human fibronectin in a dose-dependent and saturable manner and increases adherence of wild-type L. monocytogenes to HEp-2 cells in the presence of exogenous fibronectin. Despite the lack of conventional secretion/anchoring signals, FbpA is detected using an antibody generated against the recombinant FbpA protein on the bacterial surface by immunofluorescence, and in the membrane compartment by Western blot analysis of cell extracts. Strikingly, FbpA expression affects the protein levels of two virulence factors, listeriolysin O (LLO) and InlB, but not that of InlA or ActA. FbpA co-immunoprecipitates with LLO and InlB, but not with InlA or ActA. Thus, FbpA, in addition to being a fibronectin-binding protein, behaves as a chaperone or an escort protein for two important virulence factors and appears as a novel multifunctional virulence factor of L. monocytogenes.

A new oxygen-regulated operon in Escherichia coli comprises the genes for a putative third cytochrome oxidase and for pH 2.5 acid phosphatase (appA)

The Escherichia coli acid phosphatase gene appA is expressed in response to oxygen deprivation and is positively controlled by the product of appR (katF) which encodes a putative new sigma transcription-initiation factor. However, transcription of appA from its nearest promoter (P1) did not account for total pH 2.5 acid phosphatase expression and was not subject to regulation. The cloned region upstream of appA was extended and analyzed by insertions of transposon TnphoA and by fusions with lacZ. It contains two new genes, appC and appB, which both encode extracytoplasmic proteins. appC and appB are expressed from a promoter (P2) lying just upstream of appC. Both genes are regulated by oxygen, as is appA, and by appR gene product exactly as previously shown for appA. Analysis of the nucleotide sequence and of the origins of transcription have confirmed that the P2-appC-appB- (ORFX)-P1-appA region is organized on the chromosome as an operon transcribed clockwise from P2 and that P1 is a minor promoter for appA alone. Genes appC and appB encode proteins of Mr 58,133 and 42,377, respectively, which have the characteristics of integral membrane proteins. The deduced amino acid sequences of appC and appB show 60% and 57% homology, respectively, with subunits I and II of the E. coli cytochrome d oxidase (encoded by genes cydA and cydB). The notion that the AppC and AppB proteins constitute a new cytochrome oxidase or a new oxygen-detoxifying system is supported by the observation of enhanced sensitivity to oxygen of mutants lacking all three genes, cyo (cytochrome o oxidase), cyd (cytochrome d oxidase) and appB, compared to that of cyo cyd double mutants.

Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis.

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.

Gene arrangement and organization in a approximately 76 kb fragment encompassing the oriC region of the chromosome of Mycobacterium leprae.

A continuous 75627 bp segment of the Mycobacterium leprae chromosome spanning the oriC region was sequenced. The gene order at this locus was similar to that found in the replication origin region of many other prokaryotes, particularly Mycobacterium tuberculosis and Streptomyces coelicolor. As in the case of several Gram-positive bacteria, essential genes involved in basic cellular functions, such as DNA or RNA metabolism (dnaA, dnaB, dnaN, gyrB, gyrA, pcnB, recF, rnpA, ssb), cell wall synthesis (ponA, pbpA) and probably cell division (gidB, rodA) were found. Strikingly, the gidA gene was absent from this part of the genome and there was no rRNA operon near oriC. The gyrA gene harbours an intein coding sequence indicating that protein splicing is required to produce the mature A subunit of DNA gyrase. Among the many other noteworthy features were ORFs encoding putative serine/threonine protein kinases and a protein phosphatase, three tRNA genes, one M. leprae-specific repetitive element and a glnQ pseudogene.