Genome Sequences of Diverse Human Cytomegalovirus Strains with Utility in Drug Screening and Vaccine Evaluation.

Cytomegalovirus displays genetic heterogeneity, which has implications for antiviral and vaccine development. Many studies have focused on laboratory isolates that have been extensively adapted for growth on fibroblasts. Here, we report whole-genome sequences for 10 human cytomegalovirus (HCMV) strains that readily grow on ARPE-19 human retinal pigment epithelial cells.

Reversible acetylation regulates acetate and propionate metabolism in Mycobacterium smegmatis.

Carbon metabolic pathways are important to the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. However, extremely little is known about metabolic regulation in mycobacteria. There is growing evidence for lysine acetylation being a mechanism of regulating bacterial metabolism. Lysine acetylation is a post-translational modification in which an acetyl group is covalently attached to the side chain of a lysine residue. This modification is mediated by acetyltransferases, which add acetyl groups, and deacetylases, which remove the acetyl groups. Here we set out to test whether lysine acetylation and deacetylation impact acetate metabolism in the model mycobacteria Mycobacterium smegmatis, which possesses 25 candidate acetyltransferases and 3 putative lysine deacetylases. Using mutants lacking predicted acetyltransferases and deacetylases we showed that acetate metabolism in M. smegmatis is regulated by reversible acetylation of acetyl-CoA synthetase (Ms-Acs) through the action of a single pair of enzymes: the acetyltransferase Ms-PatA and the sirtuin deacetylase Ms-SrtN. We also confirmed that the role of Ms-PatA in regulating Ms-Acs regulation depends on cAMP binding. We additionally demonstrated a role for Ms-Acs, Ms-PatA and Ms-SrtN in regulating the metabolism of propionate in M. smegmatis. Finally, along with Ms-Acs, we identified a candidate propionyl-CoA synthetase, Ms5404, as acetylated in whole-cell lysates. This work lays the foundation for studying the regulatory circuit of acetylation and deacetylation in the cellular context of mycobacteria.

Tat-dependent translocation of an F420-binding protein of Mycobacterium tuberculosis.

F(420) is a unique cofactor present in a restricted range of microorganisms, including mycobacteria. It has been proposed that F(420) has an important role in the oxidoreductive reactions of Mycobacterium tuberculosis, possibly associated with anaerobic survival and persistence. The protein encoded by Rv0132c has a predicted N-terminal signal sequence and is annotated as an F(420)-dependent glucose-6-phosphate dehydrogenase. Here we show that Rv0132c protein does not have the annotated activity. It does, however, co-purify with F(420) during expression experiments in M. smegmatis. We also show that the Rv0132c-F(420) complex is a substrate for the Tat pathway, which mediates translocation of the complex across the cytoplasmic membrane, where Rv0132c is anchored to the cell envelope. This is the first report of any F(420)-binding protein being a substrate for the Tat pathway and of the presence of F(420) outside of the cytosol in any F(420)-producing microorganism. The Rv0132c protein and its Tat export sequence are essentially invariant in the Mycobacterium tuberculosis complex. Taken together, these results show that current understanding of F(420) biology in mycobacteria should be expanded to include activities occurring in the extra-cytoplasmic cell envelope.

Identification of a lactate-quinone oxidoreductase in Staphylococcus aureus that is essential for virulence.

Staphylococcus aureus is an important human pathogen commonly infecting nearly every host tissue. The ability of S. aureus to resist innate immunity is critical to its success as a pathogen, including its propensity to grow in the presence of host nitric oxide (NO·). Upon exogenous NO· exposure, S. aureus immediately excretes copious amounts of L-lactate to maintain redox balance. However, after prolonged NO·-exposure, S. aureus reassimilates L-lactate specifically and in this work, we identify the enzyme responsible for this L-lactate-consumption as a L-lactate-quinone oxidoreductase (Lqo, SACOL2623). Originally annotated as Mqo2 and thought to oxidize malate, we show that this enzyme exhibits no affinity for malate but reacts specifically with L-lactate (K(M) = ∼330 µM). In addition to its requirement for reassimilation of L-lactate during NO·-stress, Lqo is also critical to respiratory growth on L-lactate as a sole carbon source. Moreover, Δlqo mutants exhibit attenuation in a murine model of sepsis, particularly in their ability to cause myocarditis. Interestingly, this cardiac-specific attenuation is completely abrogated in mice unable to synthesize inflammatory NO· (iNOS(-/-)). We demonstrate that S. aureus NO·-resistance is highly dependent on the availability of a glycolytic carbon sources. However, S. aureus can utilize the combination of peptides and L-lactate as carbon sources during NO·-stress in an Lqo-dependent fashion. Murine cardiac tissue has markedly high levels of L-lactate in comparison to renal or hepatic tissue consistent with the NO·-dependent requirement for Lqo in S. aureus myocarditis. Thus, Lqo provides S. aureus with yet another means of replicating in the presence of host NO·.