A distinct double positive IL-17A+/F+ T helper 17 cells induced inflammation leads to IL17 producing neutrophils in Type 1 reaction of leprosy patients.

Type 1 reactions (T1R) an inflammatory condition, of local skin patches in 30-40% leprosy patients during the course of MDT. IL-17A and IL-17F play an important role in regulating skin inflammation through neutrophils. In the present study, we have analyzed 18 of each T1R and Non-reactions (NR) patients through flow cytometry and qPCR. Interestingly we found that, CD3+CD4+ gated IL-17A+IL-17F+ cells were significantly high in T1R in both MLSA stimulated PBMCs and skin lesions as compared to NR leprosy patients. Hierarchical clustering analysis of gene expression showed that CXCL6, CXCL5, CCL20, CCL7, MMP13 and IL-17RB expression were significantly associated with IL-17A and IL-17F expression (Spearman r2 = 0.77 to 0.98), neutrophils and monocyte markers respectively. In this study, the inflammation noted in lesions of T1R is a different phenotype of Th17 which produce double positive IL-17A+IL17F+ and also contributes IL-17 producing neutrophils and thus would be useful for monitoring, diagnosis and treatment response before reactions episodes.

Mycobacterium tuberculosis cysteine biosynthesis genes mec+-cysO-cysM confer resistance to clofazimine.

The Mycobacterium tuberculosis mec+-cysO-cysM gene cluster was shown to be part of a novel cysteine biosynthesis pathway in vitro, but little is known about its essentiality or role in M. tuberculosis physiology. In this study, we generate a knock out of the mec+-cysO-cysM gene cluster in M. tuberculosis and show that the gene cluster is not essential under a variety of conditions, suggesting redundancy in pathways for cysteine biosynthesis in M. tuberculosis. The cysteine biosynthesis gene cluster is essential for resistance for clofazimine, a peroxide-producing anti-leprosy drug. Therefore, although under most conditions the pathway is not essential, it likely has an important role in defense against oxidative stress in M. tuberculosis.

Discovery of 16-Demethylrifamycins by Removing the Predominant Polyketide Biosynthesis Pathway in Micromonospora sp. Strain TP-A0468.

A number of strategies have been developed to mine novel natural products based on biosynthetic gene clusters and there have been dozens of successful cases facilitated by the development of genomic sequencing. During our study on biosynthesis of the antitumor polyketide kosinostatin (KST), we found that the genome of Micromonospora sp. strain TP-A0468, the producer of KST, contains other potential polyketide gene clusters, with no encoded products detected. Deletion of kst cluster led to abolishment of KST and the enrichment of several new compounds, which were isolated and characterized as 16-demethylrifamycins (referred to here as compounds 3 to 6). Transcriptional analysis demonstrated that the expression of the essential genes related to the biosynthesis of compounds 3 to 6 was comparable to the level in the wild-type and in the kst cluster deletion strain. This indicates that the accumulation of these compounds was due to the redirection of metabolic flux rather than transcriptional activation. Genetic disruption, chemical complementation, and bioinformatic analysis revealed that the production of compounds 3 to 6 was accomplished by cross talk between the two distantly placed polyketide gene clusters pks3 and M-rif This finding not only enriches the analogue pool and the biosynthetic diversity of rifamycins but also provides an auxiliary strategy for natural product discovery through genome mining in polyketide-producing microorganisms.IMPORTANCE Natural products are essential in the development of novel clinically used drugs. Discovering new natural products and modifying known compounds are still the two main ways to generate new candidates. Here, we have discovered several rifamycins with varied skeleton structures by redirecting the metabolic flux from the predominant polyketide biosynthetic pathway to the rifamycin pathway in the marine actinomycetes species Micromonospora sp. strain TP-A0468. Rifamycins are indispensable chemotherapeutics in the treatment of various diseases such as tuberculosis, leprosy, and AIDS-related mycobacterial infections. This study exemplifies a useful method for the discovery of cryptic natural products in genome-sequenced microbes. Moreover, the 16-demethylrifamycins and their genetically manipulable producer provide a new opportunity in the construction of novel rifamycin derivates to aid in the defense against the ever-growing drug resistance of Mycobacterium tuberculosis.

Inventory of ABC proteins and their putative role in salt and drug tolerance in Debaryomyces hansenii.

ATP-binding cassette (ABC) is one of the largest superfamily of proteins, which are ubiquitously present, performing variety of cellular functions. These proteins as drug transporters have been enticing substantial consideration because of their clinical importance. The present study focuses on genome wide identification of ABC proteins of an important halotolerant yeast Debaryomyces hansenii and explores their role in salt and drug tolerance. Our bioinformatics analysis identified a total of 30 putative ABC protein-coding genes whose expression at transcript level was confirmed by qRT-PCR. Our comparative phylogenetic analysis of nucleotide binding domains of D. hansenii and topology prediction categorized these proteins into six subfamilies; ABCB/MDR, ABCC/MRP, ABCD/ALDP, ABCF/YEF3, ABCE/RLI, and ABCG/PDR based on the nomenclature adopted by the Human Genome Organization (HUGO). Further, our transmembrane domain (TMD) predictions suggest that out of 30 ABC proteins, only 22 proteins possess either two or one TMD and hence are considered as membrane localized ABC proteins. Notably, our transcriptional dynamics of ABC proteins encoding genes following D. hansenii cells treatment with different salts and drugs concentrations illustrated variable transcriptional response of some of the genes, pointing to their role in salt and drug tolerance. This study first time provides a comprehensive inventory of the ABC proteins of a haploid D. hansenii which will be helpful for exploring their functional relevance.

RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei.

Rifamycin and its derivatives are particularly effective against the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium leprae Although the biosynthetic pathway of rifamycin has been extensively studied in Amycolatopsis mediterranei, little is known about the regulation in rifamycin biosynthesis. Here, an in vivo transposon system was employed to identify genes involved in the regulation of rifamycin production in A. mediterranei U32. In total, nine rifamycin-deficient mutants were isolated, among which three mutants had the transposon inserted in AMED_0655 (rifZ, encoding a LuxR family regulator). The rifZ gene was further knocked out via homologous recombination, and the transcription of genes in the rifamycin biosynthetic gene cluster (rif cluster) was remarkably reduced in the rifZ null mutant. Based on the cotranscription assay results, genes within the rif cluster were grouped into 10 operons, sharing six promoter regions. By use of electrophoretic mobility shift assay and DNase I footprinting assay, RifZ was proved to specially bind to all six promoter regions, which was consistent with the fact that RifZ regulated the transcription of the whole rif cluster. The binding consensus sequence was further characterized through alignment using the RifZ-protected DNA sequences. By use of bionformatic analysis, another five promoters containing the RifZ box (CTACC-N8-GGATG) were identified, among which the binding of RifZ to the promoter regions of both rifK and orf18 (AMED_0645) was further verified. As RifZ directly regulates the transcription of all operons within the rif cluster, we propose that RifZ is a pathway-specific regulator for the rif cluster.IMPORTANCE To this day, rifamycin and its derivatives are still the first-line antituberculosis drugs. The biosynthesis of rifamycin has been extensively studied, and most biosynthetic processes have been characterized. However, little is known about the regulation of the transcription of the rifamycin biosynthetic gene cluster (rif cluster), and no regulator has been characterized. Through the employment of transposon screening, we here characterized a LuxR family regulator, RifZ, as a direct transcriptional activator for the rif cluster. As RifZ directly regulates the transcription of the entire rif cluster, it is considered a pathway-specific regulator for rifamycin biosynthesis. Therefore, as the first regulator characterized for direct regulation of rif cluster transcription, RifZ may provide a new clue for further engineering of high-yield industrial strains.

HTSstation: a web application and open-access libraries for high-throughput sequencing data analysis.

The HTSstation analysis portal is a suite of simple web forms coupled to modular analysis pipelines for various applications of High-Throughput Sequencing including ChIP-seq, RNA-seq, 4C-seq and re-sequencing. HTSstation offers biologists the possibility to rapidly investigate their HTS data using an intuitive web application with heuristically pre-defined parameters. A number of open-source software components have been implemented and can be used to build, configure and run HTS analysis pipelines reactively. Besides, our programming framework empowers developers with the possibility to design their own workflows and integrate additional third-party software. The HTSstation web application is accessible at http://htsstation.epfl.ch.

Bacillus amyloliquefaciens Q-426 as a potential biocontrol agent against Fusarium oxysporum f. sp. spinaciae.

In recent years, Bacillus species have received considerable attention for the biological control of many fungal diseases. In this study, Bacillus amyloliquefaciens Q-426 was tested for its potential use against a variety of plant pathogens. Our screen for genes involved in the biosynthesis of antifungal agents revealed that the fen and bmy gene clusters are present in the Q-426 genome. Lipopeptides such as bacillomycin D, fengycin A, and fengycin B were purified from the bacterial culture broth and subsequently identified by ESI-mass spectrometry. The minimal inhibitory concentration of fengycin A against Fusarium oxysporum f. sp. spinaciae W.C. Snyder & H.N. Hansen O-27 was determined to be 31.25 µg ml(-1) . However, exposure of fungal cells to 50 µg ml(-1) of fengycin A did not allow permeation of fluorescein diacetate into the cytoplasm through the cell membrane. Moreover, leakage of intracellular inorganic cations, nucleic acid and protein were also not detected, indicating that the fungal cell membrane is not the primary target of action for fengycin A. Profound morphological changes were observed in the F. oxysporum strain and spore germination was completely inhibited, suggesting that 50 µg ml(-1) of fengycin A acts, at least, as a fungistatic agent.

Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions.

BACKGROUND: The PE and PPE multigene families of Mycobacterium tuberculosis comprise about 10% of the coding potential of the genome. The function of the proteins encoded by these large gene families remains unknown, although they have been proposed to be involved in antigenic variation and disease pathogenesis. Interestingly, some members of the PE and PPE families are associated with the ESAT-6 (esx) gene cluster regions, which are regions of immunopathogenic importance, and encode a system dedicated to the secretion of members of the potent T-cell antigen ESAT-6 family. This study investigates the duplication characteristics of the PE and PPE gene families and their association with the ESAT-6 gene clusters, using a combination of phylogenetic analyses, DNA hybridization, and comparative genomics, in order to gain insight into their evolutionary history and distribution in the genus Mycobacterium. RESULTS: The results showed that the expansion of the PE and PPE gene families is linked to the duplications of the ESAT-6 gene clusters, and that members situated in and associated with the clusters represent the most ancestral copies of the two gene families. Furthermore, the emergence of the repeat protein PGRS and MPTR subfamilies is a recent evolutionary event, occurring at defined branching points in the evolution of the genus Mycobacterium. These gene subfamilies are thus present in multiple copies only in the members of the M. tuberculosis complex and close relatives. The study provides a complete analysis of all the PE and PPE genes found in the sequenced genomes of members of the genus Mycobacterium such as M. smegmatis, M. avium paratuberculosis, M. leprae, M. ulcerans, and M. tuberculosis. CONCLUSION: This work provides insight into the evolutionary history for the PE and PPE gene families of the mycobacteria, linking the expansion of these families to the duplications of the ESAT-6 (esx) gene cluster regions, and showing that they are composed of subgroups with distinct evolutionary (and possibly functional) differences.

The RNA polymerase III-dependent family of genes in hemiascomycetes: comparative RNomics, decoding strategies, transcription and evolutionary implications.

We present the first comprehensive analysis of RNA polymerase III (Pol III) transcribed genes in ten yeast genomes. This set includes all tRNA genes (tDNA) and genes coding for SNR6 (U6), SNR52, SCR1 and RPR1 RNA in the nine hemiascomycetes Saccharomyces cerevisiae, Saccharomyces castellii, Candida glabrata, Kluyveromyces waltii, Kluyveromyces lactis, Eremothecium gossypii, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica and the archiascomycete Schizosaccharomyces pombe. We systematically analysed sequence specificities of tRNA genes, polymorphism, variability of introns, gene redundancy and gene clustering. Analysis of decoding strategies showed that yeasts close to S.cerevisiae use bacterial decoding rules to read the Leu CUN and Arg CGN codons, in contrast to all other known Eukaryotes. In D.hansenii and C.albicans, we identified a novel tDNA-Leu (AAG), reading the Leu CUU/CUC/CUA codons with an unusual G at position 32. A systematic 'p-distance tree' using the 60 variable positions of the tRNA molecule revealed that most tDNAs cluster into amino acid-specific sub-trees, suggesting that, within hemiascomycetes, orthologous tDNAs are more closely related than paralogs. We finally determined the bipartite A- and B-box sequences recognized by TFIIIC. These minimal sequences are nearly conserved throughout hemiascomycetes and were satisfactorily retrieved at appropriate locations in other Pol III genes.

Emergence of species-specific transporters during evolution of the hemiascomycete phylum.

We have traced the evolution patterns of 2480 transmembrane transporters from five complete genome sequences spanning the entire Hemiascomycete phylum: Saccharomyces cerevisiae, Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica. The use of nonambiguous functional and phylogenetic criteria derived from the TCDB classification system has allowed the identification within the Hemiascomycete phylum of 97 small phylogenetic transporter subfamilies comprising a total of 355 transporters submitted to four distinct evolution patterns named "ubiquitous," "species specific," "phylum gains and losses," or "homoplasic." This analysis identifies the transporters that contribute to the emergence of species during the evolution of the Hemiascomycete phylum and may aid in establishing novel phylogenetic criteria for species classification.

Characterization of phylogenetically distant members of the adenylate cyclase family from mycobacteria: Rv1647 from Mycobacterium tuberculosis and its orthologue ML1399 from M. leprae.

Analysis of the genome sequence of Mycobacterium tuberculosis H37Rv has identified 16 genes that are similar to the mammalian adenylate and guanylate cyclases. Rv1647 was predicted to be an active adenylate cyclase but its position in a phylogenetically distant branch from the other enzymes characterized so far from M. tuberculosis makes it an interestingly divergent nucleotide cyclase to study. In agreement with its divergence at the sequence level from other nucleotide cyclases, the cloning, expression and purification of Rv1647 revealed differences in its biochemical properties from the previously characterized Rv1625c adenylate cyclase. Adenylate cyclase activity of Rv1647 was activated by detergents but was resistant to high concentrations of salt. Mutations of substrate-specifying residues to those present in guanylate cyclases failed to convert the enzyme into a guanylate cyclase, and did not alter its oligomeric status. Orthologues of Rv1647 could be found in M. leprae, M. avium and M. smegmatis. The orthologue from M. leprae (ML1399) was cloned, and the protein was expressed, purified and shown biochemically to be an adenylate cyclase, thus representing the first adenylate cyclase to be described from M. leprae. Importantly, Western-blot analysis of subcellular fractions from M. tuberculosis and M. leprae revealed that the Rv1647 and ML1399 gene products respectively were expressed in these bacteria. Additionally, M. tuberculosis was also found to express the Rv1625c adenylate cyclase, suggesting that multiple adenylate cyclase proteins may be expressed simultaneously in this organism. These results suggest that class III cyclase-like gene products probably have an important role to play in the physiology and perhaps the pathology of these medically important bacteria.

Macro-array and bioinformatic analyses reveal mycobacterial 'core' genes, variation in the ESAT-6 gene family and new phylogenetic markers for the Mycobacterium tuberculosis complex.

To better understand the biology and the virulence determinants of the two major mycobacterial human pathogens Mycobacterium tuberculosis and Mycobacterium leprae, their genome sequences have been determined recently. In silico comparisons revealed that among the 1439 genes common to both M. tuberculosis and M. leprae, 219 genes code for proteins that show no similarity with proteins from other organisms. Therefore, the latter 'core' genes could be specific for mycobacteria or even for the intracellular mycobacterial pathogens. To obtain more information as to whether these genes really were mycobacteria-specific, they were included in a focused macro-array, which also contained genes from previously defined regions of difference (RD) known to be absent from Mycobacterium bovis BCG relative to M. tuberculosis. Hybridization of DNA from 40 strains of the M. tuberculosis complex and in silico comparison of these genes with the near-complete genome sequences from Mycobacterium avium, Mycobacterium marinum and Mycobacterium smegmatis were undertaken to answer this question. The results showed that among the 219 conserved genes, very few were not present in all the strains tested. Some of these missing genes code for proteins of the ESAT-6 family, a group of highly immunogenic small proteins whose presence and number is variable among the genomically highly conserved members of the M. tuberculosis complex. Indeed, the results suggest that, with few exceptions, the 'core' genes conserved among M. tuberculosis H37Rv and M. leprae are also highly conserved among other mycobacterial strains, which makes them interesting potential targets for developing new specific anti-mycobacterial drugs. In contrast, the genes from RD regions showed great variability among certain members of the M. tuberculosis complex, and some new specific deletions in Mycobacterium canettii, Mycobacterium microti and seal isolates were identified and further characterized during this study. Together with the distribution of a particular 6 or 7 bp micro-deletion in the gene encoding the polyketide synthase pks15/1, these results confirm and further extend the revised phylogenetic model for the M. tuberculosis complex recently presented.

Evidence for a cluster of genes on chromosome 17q11-q21 controlling susceptibility to tuberculosis and leprosy in Brazilians.

The region of conserved synteny on mouse chromosome 11/human 17q11-q21 is known to carry a susceptibility gene(s) for intramacrophage pathogens. The region is rich in candidates including NOS2A, CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL5/RANTES, CCR7, STAT3 and STAT5A/5B. To examine the region in man, we studied 92 multicase tuberculosis (627 individuals) and 72 multicase leprosy (372 individuals) families from Brazil. Multipoint nonparametric analysis (ALLEGRO) using 16 microsatellites shows two peaks of linkage for leprosy at D17S250 (Z(lr) score 2.34; P=0.01) and D17S1795 (Z(lr) 2.67; P=0.004) and a single peak for tuberculosis at D17S250 (Z(lr) 2.04; P=0.02). Combined analysis shows significant linkage (peak Z(lr) 3.38) at D17S250, equivalent to an allele sharing LOD score 2.48 (P=0.0004). To determine whether one or multiple genes contribute, 49 informative single nucleotide polymorphisms were typed in candidate genes. Family-based allelic association testing that was robust to family clustering demonstrated significant associations with tuberculosis susceptibility at four loci separated by intervals (NOS2A-8.4 Mb-CCL18-32.3 kb-CCL4-6.04 Mb-STAT5B) up to several Mb. Stepwise conditional logistic regression analysis using a case/pseudo-control data set showed that the four genes contributed separate main effects, consistent with a cluster of susceptibility genes across 17q11.2.

The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria.

BACKGROUND: The genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide. RESULTS: Finished and unfinished genome sequencing data of 98 publicly available microbial genomes has been analyzed for the presence of orthologs of the ESAT-6 loci. The multiple duplicates of the ESAT-6 gene cluster found in the genome of M. tuberculosis H37Rv are also conserved in the genomes of other mycobacteria, for example M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses of the resulting sequences have established the duplication order of the gene clusters and demonstrated that the gene cluster known as region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an ortholog could be found in the genomes of Corynebacterium diphtheriae and Streptomyces coelicolor. CONCLUSIONS: Comparative genomic analysis revealed that the presence of the ESAT-6 gene cluster is a feature of some high-G+C Gram-positive bacteria. Multiple duplications of this cluster have occurred and are maintained only within the genomes of members of the genus Mycobacterium.

Massive gene decay in the leprosy bacillus.

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.

Comparison of the UDP-N-acetylmuramate:L-alanine ligase enzymes from Mycobacterium tuberculosis and Mycobacterium leprae.

In the peptidoglycan of Mycobacterium leprae, L-alanine of the side chain is replaced by glycine. When expressed in Escherichia coli, MurC (UDP-N-acetyl-muramate:L-alanine ligase) of M. leprae showed K(m) and V(max) for L-alanine and glycine similar to those of Mycobacterium tuberculosis MurC, suggesting that another explanation should be sought for the presence of glycine.

Characterization of a Haemophilus ducreyi mutant deficient in expression of cytolethal distending toxin.

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that kills HeLa, HEp-2, and other human epithelial cells in vitro. H. ducreyi CDT activity is encoded by a three-gene cluster (cdtABC), and antibody to the cdtC gene product can neutralize CDT activity in vitro (L. D. Cope, S. R. Lumbley, J. L. Latimer, J. Klesney-Tait, M. K. Stevens, L. S. Johnson, M. Purven, R. S. Munson, Jr., T. Lagergard, J. D. Radolf, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 94:4056-4061, 1997). Culture supernatant fluid from a recombinant Escherichia coli strain containing the H. ducreyi cdtABC gene cluster readily killed both HeLa cells and HaCaT keratinocytes and had a modest inhibitory effect on the growth of human foreskin fibroblasts. Insertional inactivation of the cdtC gene in this recombinant E. coli strain eliminated the ability of this strain to kill HeLa cells and HaCaT keratinocytes. This mutated H. ducreyi cdtABC gene cluster was used to construct an isogenic H. ducreyi cdtC mutant. Monoclonal antibodies against the H. ducreyi CdtA, CdtB, and CdtC proteins were used to characterize protein expression by this cdtC mutant. Culture supernatant fluid from this H. ducreyi cdtC mutant did not detectably affect any of the human cells used in this study. The presence of the wild-type H. ducreyi cdtC gene in trans in this H. ducreyi mutant restored its ability to express a CDT that killed both HeLa cells and HaCaT keratinocytes. The isogenic H. ducreyi cdtC mutant was shown to be as virulent as its wild-type parent strain in the temperature-dependent rabbit model for experimental chancroid. Lack of expression of the H. ducreyi CdtC protein also did not affect the ability of this H. ducreyi mutant to survive in the skin of rabbits.

The Nramp1 protein and its role in resistance to infection and macrophage function.

Susceptibility to infectious diseases is under genetic control in humans. Animal models provide an ideal tool to study the genetic component of susceptibility and to identify candidate genes that can then be tested for association or linkage studies in human populations from endemic areas of disease. The Nramp1 gene was isolated by positional cloning the host resistance locus Bcg/Ity/Lsh, and mutations at this locus impair the resistance of mice to infections with intracellular parasites, such as Salmonella, Leishmania, and Mycobacterium. Allelic variants at the human Nramp1 homologue have recently been found to be associated with susceptibility to tuberculosis and leprosy in humans. The Nramp1 protein is an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages. After phagocytosis, Nramp1 is targeted to the membrane of the microbe-containing phagosome, where it may modify the intraphagosomal milieu to affect microbial replication. Although the biochemical mechanism of action of Nramp1 at that site remains unknown, Nramp homologues have been identified in many other animal species and actually define a protein family conserved from bacteria to humans. Some of these homologues have been shown to be divalent cation transporters. Recently, a second member of the mammalian Nramp family, Nramp2, was discovered and shown to be mutated in animal models of iron deficiency. The Nramp2 protein was subsequently shown to be the major transferrin-independent iron uptake system of the intestine. Together, these results suggest that Nramp1 may control intracellular microbial replication by actively removing iron or other divalent cations from the phagosomal space.

Gene organization in the trxA/B-oriC region of the Streptomyces coelicolor chromosome and comparison with other eubacteria.

The gene organization was determined in the trxA/B-rnpA region of the Streptomyces coelocolor chromosome, near to the origin of replication, oriC. Previously, we showed that the trxA and trxB genes, coding for thioredoxin and thioredoxin reductase, respectively, occur in S. coelicolor as a gene cluster and are contained on a cosmid H24 that carries oriC and several genes involved in DNA replication. Here we show that the trxA/B locus is positioned approx. 9.4kb from oriC, present the nucleotide sequence of the trxA/B-rnpA region and use sequence analysis to identify the nature of the intervening genes. Seven open reading frames were found, all oriented in the same direction, five of which were identified as the S. coelicolor homologs of SpoIIIJ, Jag, GidB, Soj and SpoOJ in Bacillus subtilis and which have been ascribed different functions in this and other bacteria for either DNA replication, chromosomal partitioning or morphological development. The arrangement of the genes coding for the above five proteins in the trxA/B-rnpA region in S. coelicolor resembles that in Mycobacterium leprae, Mycobacterium tuberculosis, B. subtilis and Pseudomonas putida, and supports the view that many of the genes necessary for development and cell division in bacteria are organized in a similar fashion. In B. subtilis and P. putida, however, the trxA/B genes are not present in the above gene arrangement.