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.

One of the cellular consequences of nitrosative stress is alkylation damage to DNA. To assess whether nitrosative stress is registered on the genome of Mycobacterium tuberculosis, mutants lacking an alkylation damage repair and reversal operon were constructed. Although hypersensitive to the genotoxic effects of N-methyl-N-nitro-N-nitrosoguanidine in vitro, the mutants displayed no phenotype in vivo, suggesting that permeation of nitrosative stress to the level of cytotoxic DNA damage is restricted.During the course of an infection, Mycobacterium tuberculosis is expected to sustain significant levels of nitrosative stress. Nitrate reductase activity provides endogenous nitrosative stress through the production of ⅐ NO (33). Although expressed under aerobic and anaerobic conditions, this activity is required at elevated levels during metabolic adaptation of the organism to microaerophilia (31) and may serve a respiratory function within the burgeoning granuloma (32). Exogenous nitrosative stress increases significantly after the onset of acquired immunity via the production of ⅐ NO by activated macrophages (4). The reaction of ⅐ NO with O 2 produces nitrous anhydride (3), which nitrosates amines and amides to produce compounds that are metabolically activated to form potent DNA alkylating agents (10,13,15,30). As such, alkylation damage of DNA can be considered a downstream sensor of the level of nitrosative stress to which a cell is exposed (Fig. 1).In order to assess the permeation of nitrosative stress to this level in M. tuberculosis, we set out to develop mutant strains that would be defective in dealing with the consequences of alkylation damage. Alkylation of DNA can form cytotoxic (Nalkylation) or promutagenic (O-alkylation) lesions (30, 34). Since M. tuberculosis contains an operon of genes predicted to be involved in the repair (Rv1317c) and reversal (Rv1316c) of such lesions (5, 9, 16), we constructed deletion mutants in this operon by two independent strategies (Fig. 2) and investigated the growth characteristics of the mutant strains in vitro and in vivo. The SID-H and BS-SK strains both lacked the region of Adl that contains the acceptor site (in AdaA) for methyl groups from methyl phosphotriesters and regulates the adaptive response (24), as well as the entire AlkA domain. They also lacked the region of Ogt containing the acceptor site for alkyl groups from O-alkylated bases (29).The mutant strains grew normally in vitro (data not shown) but were hypersensitive to the genotoxic effects of the alkylating agent N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG) (Fig. 3). The viability of both the H37Rv and SID-H strains was reduced in a dose-dependent manner by lower concentrations of MNNG, but the mutant strain was ca. 100-fold more sensitive than the wild type to the cytotoxic effects of this compound (Fig. 3A). The BS-SK strain was found to be similarly hypersensitive to the cytotoxic effects of MNNG (data not shown). The damage-hypersensitive phenotype of strain SID-H was consistent with loss of the DNA...

To gain more insight into the structure of the African horsesickness virus (AHSV) core particle, we have cloned, partially characterized and expressed the two major core proteins, VP3 and VP7, of AHSV-9. VP7 was found to be highly conserved amongst different serotypes. The VP3 and VP7 genes were subsequently expressed in insect cells by means of recombinant baculoviruses. VP7 was synthesized to very high levels and aggregated into distinctive crystals. Co-expression of VP3 and VP7 resulted in the intracellular formation of core-like particles which structurally resembled empty AHSV cores.African horsesickness is a highly infectious non-contagious disease of equines and in horses the mortality rate exceeds 90 %. The aetiological agent, African horsesickness virus (AHSV), belongs to the genus Orbivirus in the family Reoviridae. Like bluetongue virus (BTV), the prototype orbivirus, the AHSV virion is composed of seven structural proteins organized into a double-layered capsid containing the 10 double-stranded (ds) RNA segments of the viral genome (Bremer, 1976 ;Roy et al., 1994). The icosahedral core is composed of two major proteins, VP3 and VP7, and three minor structural proteins (VP1, 4 and 6). It has been shown that VP7 trimers constitute the surface of the BTV core : hence the prominent knob-like protrusions which overlay a scaffold of VP3 (Prasad et al., 1992 ;Grimes et al., 1995). The outer capsid layer is composed of the two major proteins, VP2 and VP5, of which VP2 is the serotype-specific antigen (Burrage et al

An assay modeled on a known polymorphism in the PE_PGRS9 gene of Mycobacterium tuberculosis was designed to assess the mutability of a sequence containing interspersed PGRS repeats. Application of the assay in Mycobacterium smegmatis revealed sequence plasticity: in addition to recapitulating the mutation on which it was based, other mutations likely mediated by replication slippage between PGRS repeats were detected. However, the mutation rates argued against marked hypermutability of such sequences in mycobacteria.One of the defining features of the Mycobacterium tuberculosis genome is the presence of the large PE and PPE gene families, which together comprise ϳ10% of its coding capacity and are characterized by a GϩC content of ϳ80% (26), which is markedly higher than the average 65.6% GϩC content across the genome (8). The PE_PGRS subfamily of the 100-member PE family of M. tuberculosis H37Rv comprises ϳ63 members, whose hallmark feature is the presence of multiple PGRS (polymorphic GC-rich sequence) elements in their 3Ј coding regions that encode a novel, C-terminal, Gly-Ala-rich "PGRS domain" characterized by numerous repeats of GGA and GGN residues (7). The function of PE_PGRS proteins in M. tuberculosis has been the subject of considerable interest and debate (7). Interaction of PE_PGRS proteins with the immune system is well documented (1,5,6,11,13), with the most widely studied member, PE_PGRS33 (Rv1818c), being shown to be a cell surface-associated antigen (5). Differential expression of PE_PGRS genes has been observed under a variety of experimental conditions (1,3,10,14,16,29,34), suggesting a possible specialization of function within the family, and studies with M. marinum have provided evidence of the involvement of PE_PGRS genes in mycobacterial virulence (27).There is substantial evidence implicating PE_PGRS elements in genome plasticity and strain evolution. The earliest report of allelic diversity in a PE_PGRS gene was for the Rv0746 genes (PE_PGRS9) of M. tuberculosis H37Rv and M. bovis BCG Pasteur (8) (Fig. 1A). A subsequent investigation of PE_PGRS variability at the protein level was strongly indicative of allelic diversity within and between species (1). Genome-wide sequence comparisons similarly revealed significant allelic variation in PE_PGRS genes between M. tuberculosis H37Rv and CDC1551 (15). More recently, sequence analysis of PE_PGRS33 in 123 clinical isolates of M. tuberculosis identified 25 different variations, including 9 deletions, 3 insertions, a deletion/insertion, and 12 single-nucleotide polymorphisms, attesting to its polymorphic character (33).In spite of the evidence for allelic diversity, at least in some PE and PPE members, it is not known whether the encoding genes are inherently unstable and prone to mutation at significantly elevated rates. To assess the mutability of PGRS-containing sequences in mycobacteria, we developed a reporter assay for detecting mutational events within such sequences, and in this paper we describe the results of its application in Mycoba...