Mycobacterium leprae RecA is structurally analogous but functionally distinct from Mycobacterium tuberculosis RecA protein

Mycobacterium leprae is closely related to Mycobacterium tuberculosis, yet causes a very different illness. Detailed genomic comparison between these two species of mycobacteria reveals that the decaying M. leprae genome contains less than half of the M. tuberculosis functional genes. The reduction of genome size and accumulation of pseudogenes in the M. leprae genome is thought to result from multiple recombination events between related repetitive sequences, which provided the impetus to investigate the recombination-like activities of RecA protein. In this study, we have cloned, over-expressed and purified M. leprae RecA and compared its activities with that of M. tuberculosis RecA. Both proteins, despite being 91% identical at the amino acid level, exhibit strikingly different binding profiles for single-stranded DNA with varying GC contents, in the ability to catalyze the formation of D-loops and to promote DNA strand exchange. The kinetics and the extent of single-stranded DNA-dependent ATPase and coprotease activities were nearly equivalent between these two recombinases. However, the degree of inhibition exerted by a range of ATP:ADP ratios was greater on strand exchange promoted by M. leprae RecA compared to its M. tuberculosis counterpart. Taken together, our results provide insights into the mechanistic aspects of homologous recombination and coprotease activity promoted by M. lepare RecA, and further suggests that it differs from the M. tuberculosis counterpart. These results are consistent with an emerging concept of DNA-sequence influenced structural differences in RecA nucleoprotein filaments and how these differences reflect on the multiple activities associated with RecA protein.     

Evidence of selection for protein introns in the recAs of pathogenic mycobacteria.

Protein introns are recently discovered genetic elements whose intervening sequences are removed from a precursor protein by an unusual protein splicing reaction. This involves the excision of a central spacer molecule, the protein intron, and the religation of the amino- and carboxy-terminal fragments of the precursor. The recA gene of Mycobacterium tuberculosis contains one such element and we now show that the other major mycobacterial pathogen, Mycobacterium leprae, also possesses a protein intron in its recA, although other mycobacterial recA genes do not. However, these two protein introns are different in size, sequence and location of insertion of their coding sequences into the recAs of M. tuberculosis and M. leprae, indicating that acquisition of the protein introns has occurred independently in the two species, and thus suggesting that there has been selection for splicing in the maturation of RecA in the pathogenic mycobacteria. The M. leprae protein intron provides an example of conditional protein splicing, splicing occurring in M. leprae itself but not when expressed in Escherichia coli, unlike most previously described protein introns. These observations suggest that protein introns may perform a function for their host, rather than being just selfish elements.     

Nucleotide sequence of the Mycobacterium leprae katG region.

Synthetic oligonucleotide primers based on the DNA sequence data of the Escherichia coli, Mycobacterium tuberculosis, and Mycobacterium intracellulare katG genes encoding the heme-containing enzyme catalase-peroxidase were used to amplify and analyze the Mycobacterium leprae katG region by PCR. A 1.6-kb DNA fragment, which hybridized to an M. tuberculosis katG probe, was obtained from an M. leprae DNA template. Southern hybridization analysis with a probe derived from the PCR-amplified fragment showed that the M. leprae chromosome contains only one copy of the putative katG sequence in a 3.4-kb EcoRI-BamHI DNA segment. Although the nucleotide sequence of the katG region of M. leprae was approximately 70% identical to that of the M. tuberculosis katG gene, no open reading frame encoding a catalase-peroxidase was detectable in the whole sequence. Moreover, two DNA deletions of approximately 100 and 110 bp were found in the M. leprae katG region, and they seemed to be present in all seven M. leprae isolates tested. These results strongly suggest that M. leprae lacks a functional katG gene and catalase-peroxidase activity.     

Genomic arrangement of a putative operon involved in maltose transport in the Mycobacterium tuberculosis complex and Mycobacterium leprae

A Mycobacterium bovis gene coding for a putative MalE maltose binding protein was cloned and its full-length sequence determined. Database searches revealed 99.9% identity with IpqY, encoding a putative sugar uptake protein from Mycobacterium tuberculosis strain H37Rv. The deduced protein product showed high sequence similarity to MalE-like proteins from a variety of bacterial species, including Mycobacterium leprae. Analysis of flanking database sequences from M. tuberculosis and M. leprae revealed the presence of malF-, malG- and malK-like genes. Comparison of these mycobacterial sequences with other maltose operons has allowed us to deduce a unique genomic arrangement of the genes involved in the uptake of maltose in members of the Mycobacterium tuberculosis complex and M. leprae.     

Cloning and Expression of Mycobacterium tuberculosis and Mycobacterium leprae Dihydropteroate Synthase in Escherichia coli

The genes for dihydropteroate synthase of Mycobacterium tuberculosis and Mycobacterium leprae were isolated by hybridization with probes amplified from the genomic DNA libraries. DNA sequencing revealed an open reading frame of 840 bp encoding a protein of 280 amino acids for M. tuberculosis dihydropteroate synthase and an open reading frame of 852 bp encoding a protein of 284 amino acids for M. leprae dihydropteroate synthase. The dihydropteroate synthases were expressed under control of the T5 promoter in a dihydropteroate synthase-deficient strain of Escherichia coli. Using three chromatography steps, we purified both M. tuberculosis and M. leprae dihydropteroate synthases to >98% homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed molecular masses of 29 kDa for M. tuberculosis dihydropteroate synthase and 30 kDa for M. leprae dihydropteroate synthase. Gel filtration of both enzymes showed a molecular mass of ca. 60 kDa, indicating that the native enzymes exist as dimers of two identical subunits. Steady-state kinetic parameters for dihydropteroate synthases from both M. tuberculosis and M. leprae were determined. Representative sulfonamides and dapsone were potent inhibitors of the mycobacterial dihydropteroate synthases, but the antimycobacterial agent p-aminosalicylate, a putative dihydropteroate synthase inhibitor, was a poor inhibitor of the enzymes.     

Genes for the protein antigens of the tuberculosis and leprosy bacilli

The lambda gt 11 expression vector permitted us to survey protein antigens of Mycobacterium leprae and Mycobacterium tuberculosis expressed in Escherichia coli. Using monoclonal antibodies, recombinant clones were detected producing three major antigens of M. tuberculosis and five major protein antigens of M. leprae. These recombinant antigens produced in E. coli should prove useful for diagnosis, epidemiology and possibly the development of recombinant mycobacterial vaccines.     

Extensive sequence homology between the mycobacterium leprae LSR (12 kDa) antigen and its Mycobacterium tuberculosis counterpart

The Mycobacterium leprae LSR (12 kDa) protein antigen has been reported to mimic whole cell M. leprae in T cell responses across the leprosy spectrum. In addition, B cell responses to specific sequences within the LSR antigen have been shown to be associated with immunopathological responses in leprosy patients with erythema nodosum leprosum. We have in the present study applied the M. leprae LSR DNA sequence as query to search for the presence of homologous genes within the recently completed Mycobacterium tuberculosis genome database (Sanger Centre, UK). By using the BLASTN search tool, a homologous M. tuberculosis open reading frame (336 bp), encoding a protein antigen of 12.1 kDa, was identified within the cosmid MTCY07H7B.25. The gene is designated Rv3597c within the M. tuberculosis H37Rv genome. Sequence alignment revealed 93% identity between the M. leprae and M. tuberculosis antigens at the amino acid sequence level. The finding that some B and T cell epitopes were localized to regions with amino acid substitutions may account for the putative differential responsiveness to this antigen in tuberculosis and leprosy.     

Lipid synthesis in mycobacteria: characterization of the biotin carboxyl carrier protein genes from Mycobacterium leprae and M. tuberculosis.

The causative agents of leprosy and tuberculosis, Mycobacterium leprae and Mycobacterium tuberculosis, have a lipid-rich cell envelope which contributes to virulence and antibiotic resistance. Acyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis, consists in mycobacteria of two subunits, one of which is biotinylated. Genes from M. leprae and M. tuberculosis encoding a biotinylated protein have been cloned and sequenced. Analysis of the derived protein sequences demonstrated the presence of biotin-binding sites and putative ATP-bicarbonate interactions sites, consistent with the proteins having a biotin carboxylase function as well as their being biotin carrier proteins.     

Comparative studies of the cell structures of Mycobacterium leprae and M. tuberculosis using the electron microscopy freeze-substitution technique

The cell envelope and cytoplasmic architecture of the Mycobacterium leprae Thai-53 strain were examined using the freeze-substitution technique of electron microscopy and compared with those of the M. tuberculosis H37Rv strain. Both strains had similarly multilayered envelope architectures composed of an electron-translucent layer, a peptidoglycan layer and the plasma membrane, from outside to inside. A comparison of the structures of these two mycobacteria revealed that the M. leprae cell was smaller in size and had a thinner peptidoglycan layer than the M. tuberculosis cell. The cell widths measured on electron micrographs were 0.44 microm for M. tuberculosis and 0.38 microm for M. leprae. The peptidoglycan layer of M. leprae was 4-5 nm, while the corresponding layer of M. tuberculosis was 10-15 nm.     

Unique structural features of the peptidoglycan of Mycobacterium leprae

The peptidoglycan structure of Mycobacterium spp. has been investigated primarily with the readily cultivable Mycobacterium smegmatis and Mycobacterium tuberculosis and has been shown to contain unusual features, including the occurrence of N-glycolylated, in addition to N-acetylated, muramic acid residues and direct cross-linkage between meso-diaminopimelic acid residues. Based on results from earlier studies, peptidoglycan from in vivo-derived noncultivable Mycobacterium leprae was assumed to possess the basic structural features of peptidoglycans from other mycobacteria, other than the reported replacement of l-alanine by glycine in the peptide side chains. In the present study, we have analyzed the structure of M. leprae peptidoglycan in detail by combined liquid chromatography and mass spectrometry. In contrast to earlier reports, and to the peptidoglycans in M. tuberculosis and M. smegmatis, the muramic acid residues of M. leprae peptidoglycan are exclusively N acetylated. The un-cross-linked peptide side chains of M. leprae consist of tetra- and tripeptides, some of which contain additional glycine residues. Based on these findings and genome comparisons, it can be concluded that the massive genome decay in M. leprae does not markedly affect the peptidoglycan biosynthesis pathway, with the exception of the nonfunctional namH gene responsible for N-glycolylmuramic acid biosynthesis.     

Characterization and taxonomic implications of the rRNA genes of Mycobacterium leprae.

The number of rRNA genes of Mycobacterium leprae was determined by restriction analysis of M. leprae total chromosomal DNA. A single set of rRNA genes was found. This set was subcloned from a cosmid library of M. leprae DNA into pUC13 and was characterized by restriction analysis and hybridization with Escherichia coli rRNA genes. The 16S, 23S, and 5S genes of M. leprae were clustered on a 5.3-kilobase DNA fragment. On one hand, restriction analysis of the set of rRNA genes showed the uniqueness of M. leprae among mycobacteria, but on the other hand, it suggested that M. leprae strains of several origins are very much alike. Quantitative hybridization studies between M. leprae rDNA and total DNA of various bacteria demonstrated a close relatedness between M. leprae and corynebacteria, nocardia, and mycobacteria, especially Mycobacterium tuberculosis.     

Comparative mycobacterial genomics

Genomics is providing us with a mass of information about the biochemistry, physiology and pathogenesis of Mycobacterium tuberculosis and Mycobacterium leprae. Comparison of the two genome sequences is mutually enriching and indicates that the M. leprae genome appears to have undergone shrinkage and large-scale gene inactivation, which may account for the exceptionally slow growth of this organism.     

Characterization of DNA strand transfer promoted by Mycobacterium smegmatis RecA reveals functional diversity with Mycobacterium tuberculosis RecA

The RecA-like proteins constitute a group of DNA strand transfer proteins ubiquitous in eubacteria, eukarya, and archaea. However, the functional relationship among RecA proteins is poorly understood. For instance, Mycobacterium tuberculosis RecA is synthesized as a large precursor, which undergoes an unusual protein-splicing reaction to generate an active form. Whereas the precursor was inactive, the active form promoted DNA strand transfer less efficiently compared to EcRecA. Furthermore, gene disruption studies have indicated that the frequencies of allele exchange are relatively lower in Mycobacterium tuberculosis compared to Mycobacterium smegmatis. The mechanistic basis and the factors that contribute to differences in allele exchange remain to be understood. Here, we show that the extent of DNA strand transfer promoted by the M. smegmatis RecA in vitro differs significantly from that of M. tuberculosis RecA. Importantly, M. smegmatis RecA by itself was unable to promote strand transfer, but cognate or noncognate SSBs rendered it efficient even when added prior to RecA. In the presence of SSB, MsRecA or MtRecA catalyzed strand transfer between ssDNA and varying lengths of linear duplex DNA with distinctly different pH profiles. The factors that were able to suppress the formation of DNA networks greatly stimulated strand transfer reactions promoted by MsRecA or MtRecA. Although the rate and pH profiles of dATP hydrolysis catalyzed by MtRecA and MsRecA were similar, only MsRecA was able to couple dATP hydrolysis to DNA strand transfer. Together, these results provide insights into the functional diversity in DNA strand transfer promoted by RecA proteins of pathogenic and nonpathogenic species of mycobacteria.     

Comparative genomics of metabolic pathways in Mycobacterium species: gene duplication, gene decay and lateral gene transfer

The genus Mycobacterium comprises significant pathogenic species that infect both humans and animals. One species within this genus, Mycobacterium tuberculosis, is the primary killer of humans resulting from bacterial infections. Five mycobacterial genomes belonging to four different species (M. tuberculosis, Mycobacterium bovis, Mycobacterium leprae and Mycobacterium avium ssp. paratuberculosis) have been sequenced to date and another 14 mycobacterial genomes are at various stages of completion. A comparative analysis of the gene products of key metabolic pathways revealed that the major differences among these species are in the gene products constituting the cell wall and the gene families encoding the acidic glycine-rich (PE/PPE/PGRS) proteins. Mycobacterium leprae has evolved by retaining a minimal gene set for most of the gene families, whereas M. avium ssp. paratuberculosis has acquired some of the virulence factors by lateral gene transfer.     

SOS induction in mycobacteria: analysis of the DNA-binding activity of a LexA-like repressor and its role in DNA damage induction of the recA gene from Mycobacterium smegmatis

The protein encoded by the lexA gene from Mycobacterium leprae was overproduced in Escherichia coli . The recombinant protein bound to the promoter regions of the M. leprae lexA , M. leprae recA and M. smegmatis recA genes at sites with the sequences 5'-GAACACATGTTT and 5'-GAACAGGTGTTC, which belong to the 'Cheo box' family of binding sites recognized by the SOS repressor from Bacillus subtilis . Gel mobility shift assays were used to confirm that proteins with the same site specificity of DNA binding are also present in Mycobacterium tuberculosis and M. smegmatis . Complex formation was impaired by mutagenic disruption of the dyad symmetry of the M. smegmatis recA Cheo box. LexA binding was also inhibited by preincubation of the M. smegmatis and M. tuberculosis extracts with anti- M. leprae LexA antibodies, suggesting that the mycobacterial LexA proteins are functionally conserved at the level of DNA binding. Finally, exposure of M. smegmatis to DNA-damaging agents resulted in induction of the M. smegmatis recA promoter with concomitant loss of DNA binding of LexA to its Cheo box, confirming that this organism possesses the key regulatory elements of a functional SOS induction system.     

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.     

Nitric oxide and Mycobacterium leprae pathogenicity

Leprosy is an old, still dreaded infectious disease caused by the obligate intracellular bacterium Mycobacterium leprae. During the infectious process, M. leprae is faced with the host macrophagic environment, where the oxidative stress and NO release, combined with low pH, low pO2, and high pCO2, contribute to limit the growth of the bacilli. Comparative genomics has unraveled massive gene decay in M. leprae, linking the strictly parasitic lifestyle with the reductive genome evolution. Compared with Mycobacterium tuberculosis and Mycobacterium bovis, the leprosy bacillus has lost most of the genes involved in the detoxification of reactive oxygen and nitrogen species. The very low reactivity of the unique truncated hemoglobin retained by M. leprae could account for the susceptibility of this exceptionally slow-growing microbe to NO.     

ATP content of Mycobacterium tuberculosis grown in vivo and in vitro

In order to determine the reason for the slow growth of Mycobacterium leprae either in a host or in vitro, the growth characteristics of Mycobacterium tuberculosis were studied. The ATP content of in vitro-grown M. tuberculosis was about 520 pg/10(6) viable organisms. The ATP levels from in vivo-derived organisms obtained from liver and spleen of mice was about 130 pg (in cases of chronic infection) and about 270 pg (in cases of acute infection). When the in vivo-derived organisms were inoculated into culture medium, the growth rates for both types of organisms, acute as well as chronic infection, were the same and the maximum growth was reached during the fifth subculture. Although the maximum ATP content for both types of organism was the same, it was attained during the 4th subculture for organisms obtained during acute infection and during the 6th subculture for those obtained during chronic infection. The comparison between the ATP content of M. leprae and of M. tuberculosis indicates the reason for the slow growth of M. leprae.