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Novel bioinformatics routines have been used to provide a more detailed definition of the proteome of Mycobacterium tuberculosis H37Rv. Over half of the current proteins result from gene duplication or domain shuffling events while one-sixth show no similarity to polypeptides described in other organisms. Prominent among the genes that appear to have been duplicated on numerous occasions are those involved in fatty acid metabolism, regulation of gene expression, and the unusually glycine-rich PE and PPE proteins. Protein similarity analysis, coupled with inspection of the genetic neighbourhood, was used to explore possible functional relatedness. This uncovered four large mce operons whose proteins may mediate initial interactions between the tubercle bacillus and host cells, together with a cluster of genes that might encode components of a structure required for secretion of ESAT-6 like proteins. Close linkage of the mmpL genes, encoding large membrane proteins, with those required for fatty acid metabolism suggests involvement in lipid transport. Compared to free-living bacteria, M. tuberculosis has a significantly smaller transport protein repertoire and this may reflect its intracellular lifestyle.

International audience; Functional studies have demonstrated a role for the Anopheles gambiae APL1A gene in resistance against the human malaria parasite, Plasmodium falciparum. Here, we exhaustively characterize the structure of the APL1 locus and show that three structurally different APL1A alleles segregate in the Ngousso colony. Genetic association combined with RNAi-mediated gene silencing revealed that APL1A alleles display distinct protective profiles against P. falciparum. One APL1A allele is sufficient to explain the protective phenotype of APL1A observed in silencing experiments. Epitope-tagged APL1A isoforms expressed in an in vitro hemocyte-like cell system showed that under assay conditions, the most protective APL1A isoform (APL1A(2)) localizes within large cytoplasmic vesicles, is not constitutively secreted, and forms only one protein complex, while a less protective isoform (APL1A(1)) is constitutively secreted in at least two protein complexes. The tested alleles are identical to natural variants in the wild A. gambiae population, suggesting that APL1A genetic variation could be a factor underlying natural heterogeneity of vector susceptibility to P. falciparum.

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette–Guérin (BCG) vaccines, Mycobacterium bovis BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene, glpD2 , is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding σ-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.

The 174-kb virulence plasmid pMUM001 inMycobacterium ulceransepidemic strain Agy99 harbors three very large and homologous genes that encode giant polyketide synthases (PKS) responsible for the synthesis of the lipid toxin mycolactone. Deeper investigation ofM. ulceransAgy99 resulted in identification of two types of spontaneous deletion variants of pMUM001 within a population of cells that also contained the intact plasmid. These variants arose from recombination between two 8-kb sections of the same plasmid sequence, resulting in the loss of a 65-kb region bearing two of the three mycolactone PKS genes. Investigation of nine diverseM. ulceransstrains by using PCR and Southern hybridization for eight pMUM001 gene sequences confirmed the presence of pMUM001-like elements (collectively called pMUM) in allM. ulceransstrains. Physical mapping of these plasmids revealed that likeM. ulceransAgy99, three strains had undergone major deletions in their mycolactone PKS loci. Online liquid chromatography-sequential mass spectrometry analysis of lipid extracts confirmed that strains with PKS deletions were unable to produce mycolactone or any related cometabolites. Interstrain comparisons of the plasmid gene sequences revealed greater than 98% nucleotide identity, and the phylogeny inferred from these sequences closely mimicked the phylogeny from a previous multilocus sequence typing study in which chromosomally encoded loci were used, a result that is consistent with the hypothesis thatM. ulceransdiverged from the closely related organismMycobacterium marinumby acquiring pMUM. Our results suggest that pMUM is a defining characteristic ofM. ulceransbut that in the absence of purifying selection, deletion of plasmid sequences and a corresponding loss of mycolactone production readily arise.

Everything that we need to know about Mycobacterium leprae, a close relative of the tubercle bacillus, is encrypted in its genome. Inspection of the 3.27 Mb genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus identified 1,605 genes encoding proteins and 50 genes for stable RNA species. Comparison with the genome sequence of Mycobacterium tuberculosis revealed an extreme case of reductive evolution, since less than half of the genome contains functional genes while inactivated or pseudogenes are highly abundant. The level of gene duplication was approximately 34% and, on classification of the proteins into families, the largest functional groups were found to be involved in the metabolism and modification of fatty acids and polyketides, transport of metabolites, cell envelope synthesis and gene regulation. Reductive evolution, gene decay and genome downsizing have eliminated entire metabolic pathways, together with their regulatory circuits and accessory functions, particularly those involved in catabolism. This may explain the unusually long generation time and account for our inability to culture the leprosy bacillus.

International audience; The physical map of the 3.6-megabase chromosome of Clostridium perfringens CPN50 was extended by positioning sites for the endonucleases SfiI and I-CeuI, and in parallel, the gene map was expanded by using a genome scanning strategy. This involved the cloning and sequencing of random chromosomal fragments, identification of the functions of the putative genes by database searches, and then hybridization analysis. The current gene map comprises almost 100 markers, many of which encode housekeeping functions while others are involved in sporulation or pathogenesis. Strikingly, most of the virulence genes were found to be confined to a 1,200-kb segment of the chromosome near oriC, while the pleiotropic regulatory locus, virRS, was situated toward the putative replication terminus. A comparison of the gene maps of three endospore-forming bacilli, C. perfringens, Clostridium beijerinckii, and Bacillus subtilis, revealed a similar order and distribution of key sporulation and heat shock genes which might reflect an ancient evolutionary relationship.

In the 1970s, Ionesco and Bouanchaud (1973) reported the first demonstration for a dostridial species that bacteriocin production was associated with the presence of a small plasmid. It was later shown that Clostridium perfringens CPN50 synthesized large quantities of the bacteriocin BCN5 after irradiation with ultraviolet light and that this bactericidal protein was secreted into the medium (Ionesco et al., 1974). Subsequent genetic studies revealed that the bacteriocinogenic plasmid, pIP404, was about 10 kb in size and could be mobilized by conjugation by certain cryptic plasmids commonly found in C. perfringens (Brefort et al., 1977). Several years later, pIP404 was adopted by our laboratory as a model system for studying DNA damage-inducible genes in the Clostridia and as a source of material for vector construction.

The nucleotide sequence of cosmid B1790, carrying the Rif-Str regions of the Mycobacterium leprae chromosome, has been determined. Twelve open reading frames were identified in the 36716bp sequence, representing 40% of the coding capacity. Five ribosomal proteins, two elongation factors and the beta and beta' subunits of RNA polymerase have been characterized and two novel genes were found. One of these encodes a member of the so-called ABC family of ATP-binding proteins while the other appears to encode an enzyme involved in repairing genomic lesions caused by free radicals. This finding may well be significant as M. leprae, an intracellular pathogen, lives within macrophages.

The bacteriocinogenic plasmid pIP404 from Clostridium perfringens was isolated and cloned in Escherichia coli, and its physical map was deduced. Expression of the bcn gene, encoding bacteriocin BCN5, is inducible by UV irradiation of C. perfringens and thus resembles the SOS-regulated bacteriocin genes of enteric bacteria. The location of bcn on pIP404 was established by a dot-blot procedure, using specific hybridization probes to analyze mRNA samples from induced and uninduced cultures. From the nucleotide sequence of its gene, the molecular weight of BCN5 was deduced to be 96,591, and a protein of this size was secreted by bacteriocin-producing cultures of C. perfringens. The primary structure of the protein suggests that it may function as an ionophore, since a hydrophobic domain, resembling those of the ionophoric colicins, is present at the COOH terminus. No bacteriocin activity could be detected in E. coli harboring plasmids bearing the bcn gene, even when the transcriptional and translational signals were replaced by those of lacZ. A possible explanation may be found in the unusual codon usage of the adenine-thymine-rich bcn gene, as this shows a preference for codons with a high adenine-plus-thymine content, especially in the wobble position. Many of the frequently used codons correspond to those recognized by minor tRNA species in E. coli. Consequently, bcn expression might be limited by tRNA availability in this bacterium.

The complete nucleotide sequence of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens has been determined. The plasmid genome comprises 10,207 bp and has a dA + dT content of 75%. Functions have been tentatively assigned to 6 of the 10 open reading frames and an origin-like region of repeated sequence identified. The codon usage of this extremely dA + dT rich plasmid is highly unusual and displays a pronounced preference for codons with the lowest dG + dC content. Only one of the genes from pIP404 was expressed at a significant level in Escherichia coli, suggesting that the atypical codon usage could represent a major obstacle to heterologous gene expression.

Clostridium perfringens strain CPN50 harbours a 10.2 kb plasmid known as pIP404 which, in addition to a set of UV-inducible genes involved in bacteriocin production, carries res, a gene probably encoding a site-specific recombinase. The RES protein is highly homologous to the resolvases of transposons from both Gram-negative and Gram-positive bacteria as well as enzymes involved in site-specific DNA inversion. A likely role for the RES protein would be to stabilize pIP404 by reducing the number of plasmid multimers resulting from homologous recombination. A putative resolution site for RES action was found overlapping the res promoter. Phylogenetic analysis of the primary structures of ten site-specific recombinases suggested a common descent and showed the RES protein to be closest to the resolvase encoded by Tn917 from Streptococcus faecalis.

The biosynthesis of yeast 5-aminolevulinate (ALA) synthase, a mitochondrial protein encoded by the nuclear HEM1 gene, has been studied in vitro in a cell-free translation system and in vitro in whole cells. In vitro translation of mRNA hybrid-selected by the cloned HEM1 gene, or of total RNA followed by immunoprecipitation with anti-(ALA synthase) antibody yielded a single polypeptide of higher molecular mass than the purified ALA synthase. This larger form, also seen in pulse-labeled cells, can be post-translationally processed by isolated mitochondria. These results show that the cytoplasmically made ALA synthase is synthesized with a cleavable extension which was estimated to be about 3.5 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The complete nucleotide sequence of the HEM1 gene and its flanking regions was determined. The 5′ ends of the HEM1 mRNAs map from - 76 to - 63 nucleotides upstream of the translation initiation codon. The open reading frame of 1644 base pairs encodes a protein of 548 amino acids with a calculated Mr of 59275. The predicted amino-terminal sequence of the protein is strongly basic (five basic and no acidic amino acids within the first 35 residues), rich in serine and threonine and must represent the transient presequence that targets this protein to the mitochondria. Comparison of deduced amino acid sequences indicates a clear homology between the mature yeast and chick embryo liver ALA synthases.

The replication functions of the bacteriocinogenic plasmid pIP404, from Clostridium perfringens, were localized to a 2.8-kb EcoRI-EcoRV fragment by cloning into a vector deficient for replication in Bacillus subtilis. This fragment contains two genes, cop and rep, which encode proteins and an 800-bp noncoding segment of complex structure consisting of multiple tandemly repeated sequences. The Cop protein is involved in copy number control, whereas the rep gene product is essential for plasmid replication. By deletion analysis the minimal origin of replication was defined as the rep gene plus most of the repeated sequences. A powerful promoter producing a 150-nucleotide RNA molecule, RNA1, that could act as an anti-sense RNA to the rep gene was detected in the "origin-like" region. In contrast to most other small plasmids of gram-positive bacteria, pIP404, and its derivatives, does not appear to replicate via a single stranded intermediate in either C. perfringens or B. subtilis.

Expression of a 4 kb segment of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens is inducible by UV-irradiation. DNA sequence analysis revealed that this region contains three genes: uviA, uviB and bcn encoding the bacteriocin BCN5. Biochemical studies with mRNAs showed that expression was controlled at the transcriptional level and that the genes were organized in two independent transcriptional units, uviAB and bcn, both directed by tandem promoters inducible by UV light. The bcn gene is transcribed from three promoters (P1, P2, P3) while transcription of uviAB is directed by two promoters (P4, P5). With the exception of P4, which bears some resemblance to the consensus eubacterial promoter sequence, none of these promoters was recognized in vitro by the major forms of RNA polymerase from C. perfringens, Bacillus subtilis or Escherichia coli. Promoters P1, P3 and P5, which show striking homology with each other, contain unusual sequences in the '-35' and '-10' regions known to be recognized by RNA polymerase and this might indicate positive control.

Mycobacterium bovis is the causative agent of tuberculosis in a range of animal species and man, with worldwide annual losses to agriculture of $3 billion. The human burden of tuberculosis caused by the bovine tubercle bacillus is still largely unknown. M. bovis was also the progenitor for the M. bovis bacillus Calmette–Guérin vaccine strain, the most widely used human vaccine. Here we describe the 4,345,492-bp genome sequence of M. bovis AF2122/97 and its comparison with the genomes of Mycobacterium tuberculosis and Mycobacterium leprae . Strikingly, the genome sequence of M. bovis is >99.95% identical to that of M. tuberculosis , but deletion of genetic information has led to a reduced genome size. Comparison with M. leprae reveals a number of common gene losses, suggesting the removal of functional redundancy. Cell wall components and secreted proteins show the greatest variation, indicating their potential role in host–bacillus interactions or immune evasion. Furthermore, there are no genes unique to M. bovis , implying that differential gene expression may be the key to the host tropisms of human and bovine bacilli. The genome sequence therefore offers major insight on the evolution, host preference, and pathobiology of M. bovis .

All 10 rRNA operons have been situated on the genome map of the anaerobic pathogen Clostridium perfringens. Four of these have been cloned and partially sequenced, and their transcriptional patterns in vivo and in vitro have been examined. Expression of rrnA, rrnB, and rrnE is directed by tandem promoters, P1 and P2, whereas rrnH is the only one to be expressed from a single promoter, which resembles P1. On inspection of the nucleotide sequences of the control regions, several sites which might be involved in the regulation of rrn expression were identified. These include a possible upstream activating region which could be recognized by the C. perfringens equivalent of the Escherichia coli Fis protein and a stringent response target site. Studies of maturation of 16S RNA identified two 5' cleavage sites and sequence analysis showed the dG+dC content of its gene, rrs, to be 52%, which is twice that of the genome.

Leprosy, a chronic human neurological disease, results from infection with the obligate intracellular pathogen Mycobacterium leprae, a close relative of the tubercle bacillus. Mycobacterium leprae has the longest doubling time of all known bacteria and has thwarted every effort at culture in the laboratory. Comparing the 3.27-megabase (Mb) genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus with that of Mycobacterium tuberculosis (4.41 Mb) provides clear explanations for these properties and reveals an extreme case of reductive evolution. Less than half of the genome contains functional genes but pseudogenes, with intact counterparts in M. tuberculosis, abound. Genome downsizing and the current mosaic arrangement appear to have resulted from extensive recombination events between dispersed repetitive sequences. Gene deletion and decay have eliminated many important metabolic activities including siderophore production, part of the oxidative and most of the microaerophilic and anaerobic respiratory chains, and numerous catabolic systems and their regulatory circuits.