An inducible, nondegradative phytoalexin resistance mechanism in Dictyostelium discoideum is suppressed by mutations that alter membrane sterol composition.

Pretreatment of Dictyostelium discoideum amoebae with a sublethal concentration of the pea phytoalexin pisatin was shown to induce nondegradative resistance to subsequent challenges with inhibitory concentrations. An alteration of membrane sterol composition either with the azasterol A25822B or by mutations in nysC that confer resistance to the polyene antibiotic nystatin suppressed the induction of pisatin resistance. Wild-type cells grown on pisatin medium acquired resistance to nystatin; however, after transfer to nystatin medium, they lost their pisatin resistance phenotype but remained nystatin resistant. To account for this asymmetry in the induction and maintenance of cross-resistance after growth on pisatin and nystatin media, we propose a model in which the two resistance phenotypes are governed by distinct mechanisms. This model presumes that growth on pisatin induces membrane alterations that predispose cells to acquire nystatin resistance but that the pisatin-induced membrane alterations are not maintained in the absence of pisatin.     

Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function

A recA deletion mutant of Mycobacterium smegmatis has been isolated by homologous recombination using a sacB counterselection strategy. Deletion of the recA gene from the chromosome was demonstrated by Southern hybridizations and by polymerase chain reaction (PCR). Western analysis using anti-RecA antibodies confirmed that the RecA protein was not made by the mutant strain. The recA deletion strain exhibited enhanced sensitivity to UV irradiation and failed to undergo homologous recombination. The results obtained from the recombination assays suggest that in wild-type M. smegmatis the majority of colonies arise from single cross-over homologous recombination events with only a very minor contribution from random integrations. The deficiencies in UV survival and recombination were complemented by introduction of the cloned M. smegmatis recA gene. Overexpression of RecA was found to be toxic in the absence of recX , which is found downstream of and co-transcribed with recA and is thus also affected by the deletion of recA . The M. smegmatis recA deletion strain was also complemented by the M. tuberculosis recA gene with or without its intein; most importantly, the frequency of double cross-over homologous recombination events was identical regardless of whether the M. tuberculosis recA gene contained or lacked the intein. Thus, the low frequency of homologous recombination observed in M. tuberculosis is not due to the presence of an intein-coding sequence in its recA gene per se .     

TheNeurospora crassa erg3 gene encodes a protein with sequence homology to both yeast sterol C-14 reductase and chicken lamin B receptor

Theerg3 gene ofNeurospora crassa was sequenced (EMBL accession no. X77955) and found to encode a protein of 490 amino acid residues with significant homology to the yeast sterol biosynthetic enzyme C-14 reductase (39% identity) and also to the C-tenninal region in the sequence reported for the chicken lamin B receptor (41% identity). The possibility that a single protein may possess both lamin B receptor and sterol C-14 reductase functions might account for non-sterol-biosynthetic effects of mutations in sterol biosynthesis genes and of inhibitors of sterol biosynthetic enzymes.     

Unravelling the pleiotropic role of the MceG ATPase in Mycobacterium smegmatis

The Mce systems are complex ABC transporters that are encoded by different numbers of homologous operons in Actinobacteria. While the four Mce systems of Mycobacterium tuberculosis are all energized by a single ATPase, MceG, each system appears to import different fatty acids or sterols. To explore if this behaviour can be extended to saprophytic mycobacteria, whose more complex genomes encode more Mce systems, we have identified and characterized the MceG orthologue of Mycobacterium smegmatis. This bacterium relies on MceG to energize its six Mce systems that contribute to a variety of cellular functions including sterol uptake and cell envelope maintenance. In the absence of MceG, M. smegmatis was not able to utilize cholesterol or phytosterols as carbon sources implying that this ATPase is necessary to energize the Mce4‐sterol transport system. Other phenotypic alterations observed in the ΔMceG mutant, such as cell envelope modifications, suggest a pleiotropic functionality of the Mce systems that are particularly important for stress responses. Several ΔMceG phenotypes were recapitulated in a strain lacking only the unique C‐terminal region of MceG, suggesting an important functional or regulatory function for this domain.     

The functions of OmpATb,a pore-forming protein of Mycobacterium tuberculosis

The functions of OmpATb, the product of the ompATb gene of Mycobacterium tuberculosis and a putative porin, were investigated by studying a mutant with a targeted deletion of the gene, and by observing expression of the gene in wild-type M. tuberculosis H37Rv by real-time polymerase chain reaction (PCR) and immunoblotting. The loss of ompATb had no effect on growth under normal conditions, but caused a major reduction in ability to grow at reduced pH. The gene was substantially upregulated in wild-type bacteria exposed to these conditions. The mutant was impaired in its ability to grow in macrophages and in normal mice, although it was as virulent as the wild type in mice that lack T cells. Deletion of the ompATb gene reduced permeability to several small water-soluble substances. This was particularly evident at pH 5.5; at this pH, uptake of serine was minimal, suggesting that, at this pH, OmpATb might be the only functioning porin. These data indicate that OmpATb has two functions: as a pore-forming protein with properties of a porin, and in enabling M. tuberculosis to respond to reduced environmental pH. It is not known whether this second function is related to the porin-like activity at low pH or involves a completely separate role for OmpATB. The involvement with pH is likely to contribute to the ability of M. tuberculosis to overcome host defence mechanisms and grow in a mammalian host.     

Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B

Entry into host macrophages and evasion of intracellular destruction mechanisms, including phagosome-lysosome fusion, are critical elements of Mycobacterium tuberculosis (Mtb) pathogenesis. To achieve this, the Mtb genome encodes several proteins that modify host signaling pathways. PtpA, a low-molecular weight tyrosine phosphatase, is a secreted Mtb protein of unknown function. The lack of tyrosine kinases in the Mtb genome suggests that PtpA may modulate host tyrosine phosphorylated protein(s). We report that a genetic deletion of ptpA attenuates Mtb growth in human macrophages, and expression of PtpA-neutralizing antibodies simulated this effect. We identify VPS33B, a regulator of membrane fusion, as a PtpA substrate. VPS33B and PtpA colocalize in Mtb-infected human macrophages. PtpA secretion combined with active-phosphorylated VPS33B inhibited phagosome-lysosome fusion, a process arrested in Mtb infections. These results demonstrate that PtpA is essential for Mtb intracellular persistence and identify a key host regulatory pathway that is inactivated by Mtb.