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Staphylococcus aureus is one of the principal causative agents of bacteremia which can progress to sepsis. Rapid diagnostic tests for identification and antibiotic resistance profiling of S. aureus would improve patient outcomes and antibiotic stewardship, but existing methods require a lengthy culture step to obtain enough material for testing. Complexity of the host matrix, where pathogenic microbes are often present, also interferes with many diagnostic methods. Here, we describe a straightforward and rapid method for enriching viable S. aureus using bio-orthogonal, or "click," chemistry methods. Bacteria labeled in this manner can potentially be cultured, interrogated using molecular methods for pathogen identification, or used to test antibiotic susceptibility.

Transcription and expression regulation of some individual cel genes (cel5A, cel5I, cel5D and cel44O) of Clostridium cellulolyticum were investigated. Unlike the cip-cel operon, these genes are transcribed as monocistronic units of transcription, except cel5D. The location of the transcription initiation sites was determined using RT-PCR and the mRNA 5'-end extremities were detected using primer extension experiments. Similarly to the cip-cel operon, cel5A and cel5I expressions are regulated by a carbon catabolite repression mechanism, whereas cel44O and cel5D expressions do not seem to be submitted to this regulation. The role of the putative transcriptional regulator GlyR2 in the regulation of cel5D expression was investigated. The recombinant protein GlyR2 was produced and was shown to bind in vitro to the cel5D and glyR2 promoter regions, suggesting that besides regulating its own expression, GlyR2 may regulate cel5D expression. To test this hypothesis in vivo, an insertional glyR2 mutant was generated and the effect of this disruption on cel5D expression was evaluated. Levels of cel5D mRNAs in the mutant were 16 fold lower than that of the wild-type strain suggesting that GlyR2 acts as an activator of cel5D expression.

Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Mtb-infected macrophages are likely to be sequestered inside the hypoxic environments of the granuloma and differentiate into lipid-loaded macrophages that contain triacylglycerol (TAG)-filled lipid droplets which may provide a fatty acid-rich host environment for Mtb. We report here that human peripheral blood monocyte-derived macrophages and THP-1 derived macrophages incubated under hypoxia accumulate Oil Red O-staining lipid droplets containing TAG. Inside such hypoxic, lipid-loaded macrophages, nearly half the Mtb population developed phenotypic tolerance to isoniazid, lost acid-fast staining and accumulated intracellular lipid droplets. Dual-isotope labeling of macrophage TAG revealed that Mtb inside the lipid-loaded macrophages imports fatty acids derived from host TAG and incorporates them intact into Mtb TAG. The fatty acid composition of host and Mtb TAG were nearly identical suggesting that Mtb utilizes host TAG to accumulate intracellular TAG. Utilization of host TAG by Mtb for lipid droplet synthesis was confirmed when fluorescent fatty acid-labeled host TAG was utilized to accumulate fluorescent lipid droplets inside the pathogen. Deletion of the Mtb triacylglycerol synthase 1 (tgs1) gene resulted in a drastic decrease but not a complete loss in both radiolabeled and fluorescent TAG accumulation by Mtb suggesting that the TAG that accumulates within Mtb is generated mainly by the incorporation of fatty acids released from host TAG. We show direct evidence for the utilization of the fatty acids from host TAG for lipid metabolism inside Mtb. Taqman real-time PCR measurements revealed that the mycobacterial genes dosR, hspX, icl1, tgs1 and lipY were up-regulated in Mtb within hypoxic lipid loaded macrophages along with other Mtb genes known to be associated with dormancy and lipid metabolism.

Mycobacterium tuberculosis (Mtb) is known to produce wax esters (WE) when subjected to stress. However, nothing is known about the enzymes involved in biosynthesis of WE and their role in mycobacterial dormancy. We report that two putative Mtb fatty acyl-CoA reductase genes (fcr) expressed in E. coli display catalytic reduction of fatty acyl-CoA to fatty aldehyde and fatty alcohol. Both enzymes (FCR1/Rv3391) and FCR2/Rv1543) showed a requirement for NADPH as the reductant, a preference for oleoyl-CoA over saturated fatty acyl-CoA and were inhibited by thiol-directed reagents. We generated Mtb gene-knockout mutants for each reductase. Metabolic incorporation of( 14)C-oleate into fatty alcohols and WE was severely diminished in the mutants under dormancy-inducing stress conditions that are thought to be encountered by the pathogen in the host. The fatty acyl-CoA reductase activity in cell lysates of the mutants under nitric oxide stress was significantly reduced when compared with the wild type. Complementation restored the lost activity completely in the Δfcr1 mutant and partially in the Δfcr2 mutant. WE synthesis was inhibited in both Δfcr mutants. The Δfcr mutants exhibited faster growth rates, an increased uptake of (14)C-glycerol suggesting increased permeability of the cell wall, increased metabolic activity levels and impaired phenotypic antibiotic tolerance under dormancy-inducing combined multiple stress conditions. Complementation of the mutants did not restore the development of antibiotic tolerance to wild-type levels. Transcript analysis of Δfcr mutants showed upregulation of genes involved in energy generation and transcription, indicating the inability of the mutants to become dormant. Our results indicate that the fcr1 and fcr2 gene products are involved in WE synthesis under in vitro dormancy-inducing conditions and that WE play a critical role in reaching a dormant state. Drugs targeted against the Mtb reductases may inhibit its ability to go into dormancy and therefore increase susceptibility of Mtb to currently used antibiotics thereby enhancing clearance of the pathogen from patients.