Enoyl-coenzyme A hydratase and antigen 85B of Mycobacterium habana are specifically recognized by antibodies in sera from leprosy patients.

Leprosy is an infectious disease caused by Mycobacterium leprae, which is a noncultivable bacterium. One of the principal goals of leprosy research is to develop serological tests that will allow identification and early treatment of leprosy patients. M. habana is a cultivable nonpathogenic mycobacterium and candidate vaccine for leprosy, and several antigens that cross-react between M. leprae and M. habana have been discovered. The aim of the present study was to extend the identification of cross-reactive antigens by identifying M. habana proteins that reacted by immunoblotting with antibodies in serum samples from leprosy patients but not with antibodies in sera from tuberculosis (TB) patients or healthy donors (HDs). A 28-kDa antigen that specifically reacted with sera from leprosy patients was identified. To further characterize this antigen, protein spots were aligned in two-dimensional polyacrylamide gels and Western blots. Spots cut out from the gels were then analyzed by mass spectrometry. Two proteins were identified: enoyl-coenzyme A hydratase (lipid metabolism; ML2498) and antigen 85B (Ag85B; mycolyltransferase; ML2028). These proteins represent promising candidates for the design of a reliable tool for the serodiagnosis of lepromatous leprosy, which is the most frequent form in Mexico.

Structural and functional characterization of the recR gene of Streptomyces.

The recR gene product is necessary for homologous recombination and recombinational DNA repair in eubacteria. We report the isolation and sequencing of the recR gene from Streptomyces coelicolor. It encodes a protein of 198 amino acids, with a predicted molecular mass of 22 kDa. The deduced amino acid sequence shows significant similarity to that of RecR proteins from other bacteria, including Escherichia coli and Bacillus subtilis. Like these, Streptomyces RecR contains potential helix-hairpin-helix, zinc finger and ATP-binding motifs, as well as the Toprim domain which is present also in topoisomerases of Types IA and II, primases and nucleases of the OLD family. The recR genes of Escherichia coli and Bacillus subtilis are immediately preceded by a small ORF (orf12 and orf107, respectively). An equivalent ORF (orf1) is also found in Streptomyces. S. lividans recR mutants, obtained either by insertional inactivation of recR or by deletion of the gene together with the preceding ORF, displayed increased sensitivity to DNA-damaging agents (such as UV light and methylmethanesulfonate), when compared with the wild-type strain. Both mutants could be complemented by the wild-type orflrecR genes and also by the recR gene alone. Based on these results, orf1 appears to be dispensable for the repair function of Streptomyces RecR. In studies of heterologous complementation, the B. subtilis recR region (orf107recR) was found to complement the S. lividans deltaorflrecR mutant, but the equivalent region from E. coli (orf12recR) could not. However, in the absence of orf107, B. subtilis recR was unable to restore the wild-type phenotype to the Streptomyces deletion mutant.

Characterization of IS1389, a new member of the IS3 family of insertion sequences isolated from Xanthomonas campestris pv. amaranthicola.

IS1389, a new insertion sequence belonging to the IS3 family, has been identified in Xanthomonas campestris pv. amaranthicola. The genome of this bacterium contains at least 11 copies of the element, whereas no hybridizing sequences were detected in other Xanthomonas species [X. axonopodis, X. fragaridae, X. phaseoli, and X. (Stenotrophomonas) maltophila]. Two nearly identical copies of the element (IS1389-A and IS1389-B) were characterized. According to analysis of sequence alignments and similar structural features, IS1389 belongs to the IS407 subgroup of the IS3 family, which duplicates 4 bp of target DNA upon insertion. IS1389-A was found in the proximity of the modification gene of the XamI restriction-modification system.

Establishment of a hybrid SalI-HgiDII type II restriction-modification system.

In the SalI system, endonuclease activity can be only achieved in the presence of a functional modification gene. Thus, the DNA methyltransferase is involved in the control of restriction. By fusion of the restriction gene of the SalI system to the modification gene of the isospecific HgiDII system a hybrid type II restriction-modification system was created. Although in the hybrid situation the level of endonuclease activity was significantly lower than in the natural system, the HgiDII modification enzyme clearly supports SalI restriction. The mechanism by which the two isospecific methyltransferases control restriction is currently under study.

Isolation and nucleotide sequence of the gene encoding the XamI DNA methyltransferase of Xanthomonas campestris pv. amaranthicola.

The gene (xamIM) encoding the DNA methyltransferase of the XamI restriction-modification system from Xanthomonas campestris pv. amaranithicola (M.XamI) has been cloned in Escherichia coli and its nucleotide sequence determined. The sequence predicts a protein of 527 amino acids that contains nine conserved motifs characteristic of DNA amino methyltransferases. In fact, M.XamI shows significant similarity with N6-adenine methyltransferases of the gamma group of amino methyltransferases, including M.SalI (from the isoschizomeric SalI restriction-modification system) and M.TaqI (the only N6-adenine methyltransferase for which a three-dimensional structure is available). M.XamI and M.SalI share two highly conserved regions within the C-terminal domain, one of which aligns with one of the DNA recognition loops proposed for M.TaqI. Analysis of the chromosomal DNA adjacent to xamIM led to the identification of an additional ORF (275 codons), downstream, in the same transcriptional orientation. Although some limited similarities between the SalI restriction enzyme and the product deduced from this ORF were found, the clone carrying xamIM did not express the expected endonuclease function.

Nucleic acid splicing events occur frequently during macronuclear development in the protozoan Oxytricha nova and involve the elimination of unique DNA.

During its life cycle, the hypotrichous ciliated protozoan Oxytricha nova transforms a copy of its chromosomal micronucleus into a transcriptionally active macronucleus which contains exclusively linear, gene-sized DNA molecules with an average size of about 2.2 kilobase pairs (kbp). The micronuclear precursors of two macronuclear DNA molecules have been examined. Each was found to contain at least five blocks of DNA sequences that are absent in the mature macronuclear DNA molecule. These blocks of sequences, referred to as internal eliminated sequences (IESs), must be removed by a nucleic acid breakage and joining process during development. The data obtained to date indicate that IESs are common and suggest that greater than 60,000 IES removal events occur during macronuclear development. Additional analyses indicate that IESs represent a portion of the unique micronuclear DNA sequences known to be eliminated during development. Comparisons of the sequences of IESs revealed common organizational features and some limited primary sequence homologies that suggest models for their developmental excision.

Gene-sized macronuclear DNA molecules are clustered in micronuclear chromosomes of the ciliate Oxytricha nova.

Following the sexual phase of its life cycle, the hypotrichous ciliate Oxytricha nova transforms a copy of its chromosomal micronucleus into a macronucleus containing short, linear DNA molecules with an average size of 2.2 kilobase pairs. In addition, more than 90% of the DNA sequences in the micronuclear genome are eliminated during this process. We have examined the organization of macronuclear DNA molecules in the micronuclear chromosomes. Macronuclear DNA molecules were found to be clustered and separated by less than 550 base pairs in two cloned segments of micronuclear DNA. Recombinant clones of two macronuclear DNA molecules that are adjacent in the micronucleus were also isolated and examined by DNA sequencing. The two macronuclear DNA molecules were found to be separated by only 90 base pairs in the micronuclear genome.