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.

zPicture: dynamic alignment and visualization tool for analyzing conservation profiles.

Comparative sequence analysis has evolved as an essential technique for identifying functional coding and noncoding elements conserved throughout evolution. Here, we introduce zPicture, an interactive Web-based sequence alignment and visualization tool for dynamically generating conservation profiles and identifying evolutionarily conserved regions (ECRs). zPicture is highly flexible, because critical parameters can be modified interactively, allowing users to differentially predict ECRs in comparisons of sequences of different phylogenetic distances and evolutionary rates. We demonstrate the application of this module to identify a known regulatory element in the HOXD locus, in which functional ECRs are difficult to discern against the highly conserved genomic background. zPicture also facilitates transcription factor binding-site analysis via the rVista tool portal. We present an example of the HBB complex when zPicture/rVista combination specifically pinpoints to two ECRs containing GATA-1, NF-E2, and TAL1/E47 binding sites that were identified previously as transcriptional enhancers. In addition, zPicture is linked to the UCSC Genome Browser, allowing users to automatically extract sequences and gene annotations for any recorded locus. Finally, we describe how this tool can be efficiently applied to the analysis of nonvertebrate genomes, including those of microbial organisms.

Characterization of the fibronectin-attachment protein of Mycobacterium avium reveals a fibronectin-binding motif conserved among mycobacteria.

Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Evidence suggests that the initial portal of infection by M. avium is often the gastrointestinal tract. However, the mechanism by which the M. avium crosses the epithelial barrier is unclear. A possible mechanism is suggested by the ability of M. avium to bind fibronectin, an extracellular matrix protein that is a virulence factor for several extracellular pathogenic bacteria which bind to mucosal surfaces. To further characterize fibronectin binding by M. avium, we have cloned the M. avium fibronectin-attachment protein (FAP). The M. avium FAP (FAP-A) has an unusually large number of Pro and Ala residues (40% overall) and is 50% identical to FAP of both Mycobacterium leprae and Mycobacterium tuberculosis. Using recombinant FAP-A and FAP-A peptides, we show that two non-continuous regions in FAP-A bind fibronectin. Peptides from these regions and homologous sequences from M. leprae FAP inhibit fibronectin binding by both M. avium and Mycobacterium bovis Bacillus Calmette-Guerin (BCG). These regions have no homology to eukaryotic fibronectin-binding proteins and are only distantly related to fibronectin-binding peptides of Gram-positive bacteria. Nevertheless, these fibronectin-binding regions are highly conserved among the mycobacterial FAPs, suggesting an essential function for this interaction in mycobacteria infection of their metazoan hosts.

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.

A putative FAD-binding domain in a distinct group of oxidases including a protein involved in plant development.

Using methods for database screening with individual protein sequences and alignment blocks, a conserved domain is delineated in a group of proteins including several FAD-dependent oxidases. Two motifs within this domain resemble phosphate-binding loops and may be directly involved in FAD binding. These motifs can be readily distinguished from previously described nucleotide-binding sites using a method for database screening with position-dependent weight matrices derived from alignment blocks. Unexpectedly, this group of known and predicted FAD-dependent oxidases includes the product of the DIMINUTO gene, which is involved in Arabidopsis development, and its homologues from man and Mycobacterium leprae.