The SXT/R391 Family of Integrative Conjugative Elements Is Composed of Two Exclusion Groups

Conjugative elements often encode entry exclusion systems that convert host cells into poor recipients for identical or similar elements. The diversity of exclusion systems within families of conjugative elements has received little attention. We report here the most comprehensive study to date of the diversity of exclusion determinants within a single family of conjugative elements. Unexpectedly, our analyses indicate that there are only two exclusion groups among the diverse members of the SXT/R391 family of integrative conjugative elements

Determinants of Entry Exclusion within Eex and TraG Are Cytoplasmic▿ †

We report here functional and topological analyses of TraG and Eex, the donor and recipient cell inner membrane proteins that mediate entry exclusion in the SXT/R391 family of integrative conjugative elements. We found that the exclusion-determining regions of the Eex variants EexS (SXT) and EexR (R391) are located in distinct yet overlapping regions of the proteins. Unexpectedly, the carboxyl-terminal regions of TraG and Eex, which contain the residues essential for exclusion activity and specificity, were found to localize in the cell cytoplasm. These observations suggest that complex topological rearrangements of conjugative proteins must occur during mating to enable these domains to interact

SXT-Related Integrating Conjugative Element in New World Vibrio cholerae

SXT-related integrating conjugative elements (ICEs) became prevalent in Asian Vibrio cholerae populations after V. cholerae O139 emerged. Here, we describe an SXT-related ICE, ICEVchMex1, in a Mexican environmental V. cholerae isolate. Identification of ICEVchMex1 represents the first description of an SXT-related ICE in the Western Hemisphere. The significant differences between the SXT and ICEVchMex1 genomes suggest that these ICEs have evolved independently

Triosephosphate isomerase is dispensable in vitro yet essential for Mycobacterium tuberculosis to establish infection

Triosephosphate isomerase (TPI) catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). This reaction is required for glycolysis and gluconeogenesis, and tpi has been predicted to be essential for growth of Mycobacterium tuberculosis. However, when studying a conditionally regulated tpi knockdown mutant, we noticed that depletion of TPI reduced growth of M. tuberculosis in media containing a single carbon source but not in media that contained both a glycolytic and a gluconeogenic carbon source. We used such two-carbon-source media to isolate a tpi deletion (Delta tpi) mutant. The Delta tpi mutant did not survive with single carbon substrates but grew like wild-type (WT) M. tuberculosis in the presence of both a glycolytic and a gluconeogenic carbon source. C-13 metabolite tracing revealed the accumulation of TPI substrates in Delta tpi and the absence of alternative triosephosphate isomerases and metabolic bypass reactions, which confirmed the requirement of TPI for glycolysis and gluconeogenesis in M. tuberculosis. The Delta tpi strain was furthermore severely attenuated in the mouse model of tuberculosis, suggesting that M. tuberculosis cannot simultaneously access sufficient quantities of glycolytic and gluconeogenic carbon substrates to establish infection in mice

Glucose Phosphorylation Is Required for <em>Mycobacterium tuberculosis</em> Persistence in Mice

<div><p><em>Mycobacterium tuberculosis</em> (Mtb) is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of glucose phosphorylation, the first reaction in glycolysis. We discovered that Mtb expresses two functional glucokinases. Mtb required the polyphosphate glucokinase PPGK for normal growth on glucose, while its second glucokinase GLKA was dispensable. ¹³C-based metabolomic profiling revealed that both enzymes are capable of incorporating glucose into Mtb's central carbon metabolism, with PPGK serving as dominant glucokinase in wild type (wt) Mtb. When both glucokinase genes, <em>ppgK</em> and <em>glkA</em>, were deleted from its genome, Mtb was unable to use external glucose as substrate for growth or metabolism. Characterization of the glucokinase mutants in mouse infections demonstrated that glucose phosphorylation is dispensable for establishing infection in mice. Surprisingly, however, the glucokinase double mutant failed to persist normally in lungs, which suggests that Mtb has access to glucose <em>in vivo</em> and relies on glucose phosphorylation to survive during chronic mouse infections.</p> </div

Rv0650 (GLKA) can mediate growth on glucose.

<p>(A) Growth of <i>ΔppgK</i> and <i>ΔppgK</i> complemented with <i>ppgK</i> on an integrative plasmid or with <i>glkA</i> on an episomal plasmid in carbon defined media with 0.4% glucose. (B, C) Growth of <i>ΔglkA</i> (B) and <i>ΔppgKΔglkA</i> (C) in carbon defined media with 0.4% glycerol (squares), 0.4% glucose (circles) or no carbon (asterisks). Open circles depict growth of the <i>ΔppgKΔglkA</i> mutant complemented with <i>ppgK</i> in glucose containing medium. (D) Glucose dose response in liquid media of wt, <i>ΔglkA</i> and <i>ΔppgK ΔglkA</i>. (E, F) Growth of wt (E) and <i>ΔppgKΔglkA</i> (F) in carbon defined media with 0.2% acetate and 0.2% acetate+0.2% glucose. Data represent two to three independent experiments.</p

Gluokinases are dispensable for trehalose metabolism and survival during starvation.

<p>(A) Growth in carbon defined media with 0.4% trehalose. (B) Survival of wt, <i>ΔglkA</i>, <i>ΔppgK</i>, and <i>ΔppgKΔglkA</i> in PBS. (C) Survival of <i>ΔppgKΔglkA</i> in carbon defined media containing 0.4% glucose or no carbon.</p

The glucokinase double mutant is hypersusceptible to hydrogen peroxide, but attenuated in phagocyte oxidase deficient mice.

<p>(A) Susceptibility to low pH, reactive nitrogen intermediates and hydrogen peroxide. Strains were exposed to pH 4.5 for 6 days, to 3 mM NaNO₂ at pH 5.5 for 3 days and to 5 mM H₂O₂ for 4 hrs and bacterial survival was determined by plating CFU. Data are means ± s.d. of triplicate cultures. Hypersusceptibility of <i>ΔppgKΔglkA</i> to 5 mM H₂O₂ was demonstrated in three independent experiments. (B) Bacterial titers in lungs of gp96^phox−/− mice infected with wt and <i>ΔppgKΔglkA</i>. Data are means ± s.d. from three mice per time point per group and representative of two independent experiments.</p