SummaryThe RD1 genomic region is present in virulent strains of Mycobacterium tuberculosis (MTB), missing from the vaccine strain M. bovis BCG, and its importance to virulence has been established experimentally. Based on in silico analysis, it has been suggested that RD1 may encode a novel secretion system, but the mechanism by which this region affects virulence is unknown. Here we examined mutants disrupted in five individual RD1 genes. Both in vitro and in vivo , each mutant displayed an attenuated phenotype very similar to a mutant missing the entire RD1 region. Genetic complementation of individual genes restored virulence. Attenuated mutants could multiply within THP-1 cells, but they were unable to spread to uninfected macrophages. We also examined export of two immunodominant RD1 proteins, CFP-10 and ESAT-6. Export of these proteins was greatly reduced or abolished in each attenuated mutant. Again, genetic complementation restored a wild-type phenotype. Our results indicate that RD1 genes work together to form a single virulence determinant, and argue that RD1 encodes a novel specialized secretion system that is required for pathogenesis of MTB.

Region of difference (RD1) genes are present in virulent Mycobacterium tuberculosis but not the vaccine strain M. bovis bacille Calmette-Guérin (BCG). The deletion of RD1 from M. tuberculosis produces an attenuation strikingly like that of BCG, which suggests the use of RD1 mutant strains for improvement of the tuberculosis (TB) vaccine. We performed long-term murine infection with M. tuberculosis H37Rv: Delta RD1 and BCG. Mice infected with H37Rv: Delta RD1 gained less weight than did BCG-infected control mice, and, after >1 year, their lungs harbored many more bacteria and displayed significant levels of inflammation. This difference in virulence has important implications for the pursuit of strains lacking RD1 in the development of the TB vaccine.

One potentially important type of flux from standing-decaying marshgrass is the production and release of ascospores. The most extensive measurements of ascospore release from the principal marshgrass (Spartina alterniflora, smooth cordgrass) of saltmarshes of the eastern coastal United States involved an arbitrary, weeklong period of wet incubation of leaf-blade samples. We examined the possibility that shorter incubations would yield higher estimates of hourly rates of ascospore release, testing wet incubations of 3 to 71 h, using standing-decaying leaf blades of smooth cordgrass from low on living shoots and high on dead shoots, incubations of 31 h appeared to be optimal. Species compositions of ascospores expelled from the two leaf types were distinctly different: high leaves yielded primarily a Mycosphaerella species or Phaeosphaeria halima; low leaves yielded primarily Phaeosphaeria spartinicola or the Mycosphaerella species. All of these species consistently exhibited high coefficients of variation (>100%) for their mean rates of release of ascospores. Only the Mycosphaerella species on high leaves gave evidence of a delayed onset of ascospore expulsion during incubation, and this evidence was equivocal. Grand mean rates of ascospore release for P. spartinicola and the Mycosphaerella species were, respectively, 106 and 238 spores cm -2 abaxial leaf area h-1.Key Words ascomycetes; ascospore-expulsion rate; marshgrass; Mycosphaerella; Phaeosphaeria.Flow to secondary production by ascomycetous fungi is the primary destiny of dead shoots of smooth cordgrass (Spartina alterniflora Loisel), the highly photosynthetically productive grass of eastern USA saltmarshes (Newell, 1996;Newell and Porter, 2000), prior to movement into the marsh's trophic relay (Kneib, 1997). Newell and Wasowski (1995) attempted to measure one form of ascomycetous production from smooth cordgrass, as rate of output of meiospores (ascospores). They recorded the rate of deposition of ascospores onto target coverslips for wetted, naturally-decaying leaf blades. They used one arbitrarily chosen standard incubation period (168 h) for the ascospore-releasing blades. Newell and Wasowski (1995) counted ascospores only for the two species of ascomycetes (Phaeosphaeria spartinicola Leuchtmann and Buergenerula spartinae Kohlm. & Gessner) that they detected by direct-microscopic enumeration as the major producers of mature ascomata in naturally decaying blades low on partially living shoots. It was subsequently discovered that a prominent ascospore-producing species in standing-decaying smoothcordgrass blades is Mycosphaerella sp. 2 (of Kohlmeyer and Kohlmeyer, 1979), which has smaller, more cryptic ascomata than the ascomata of P, spartinicola with which they are often mixed (Newell and Porter, 2000). We report here our attempt to improve interpretability of the ascospore-capture technique as used with saltmarsh grasses; the resultant data add to the currently meager Corresponding author. E-mail, newell(~uga.edu definition of the spatiotemporal patte...