The robust ability of Enterococcus faecalis to survive outside the host and to spread via oral-fecal transmission and its high degree of intrinsic and acquired antimicrobial resistance all complicate the treatment of hospital-acquired enterococcal infections. The conserved E. faecalis core genome serves as an important genetic scaffold for evolution of this bacterium in the modern health care setting and also provides interesting vaccine and drug targets. We used an innovative pooling/sequencing strategy to map a large collection of arrayed transposon insertions in E. faecalis OG1RF and generated an arrayed library of defined mutants covering approximately 70% of the OG1RF genome. Then, we performed high-throughput transposon sequencing experiments using this library to determine core genomic determinants of bile resistance in OG1RF. This collection is a valuable resource for comprehensive, functional enterococcal genomics using both traditional and high-throughput approaches and enables immediate recovery of mutants of interest.

The biosynthesis of wax esters in bacteria is accomplished by a unique pathway that combines a fatty alcohol and a fatty acyl coenzyme A substrate. Previous in vitro enzymatic studies indicated that two different enzymes could be involved in the synthesis of the required fatty alcohol in Marinobacter aquaeolei VT8. In this study, we demonstrate through a series of gene deletions and transcriptional analysis that either enzyme is capable of fulfilling the role of providing the fatty alcohol required for wax ester biosynthesis in vivo, but evolution has clearly selected one of these, a previously characterized fatty aldehyde reductase, as the preferred enzyme to perform this reaction under typical wax ester-accumulating conditions. These results complement previous in vitro studies and provide the first glimpse into the role of each enzyme in vivo in the native organism.T he global cycle of oil is of interest from the standpoints of both energy and the environment, as efforts by humankind to obtain this valuable resource can result in substantial releases of crude oil through incidents such as the Deepwater Horizon oil spill of 2010 in the Gulf of Mexico. We also note that crude oil from natural deposits is routinely released into aqueous environments, such as the oceans, by natural processes where geological reserves meet surface waters. These environments have allowed natural populations of organisms, such as marine bacteria, to evolve to utilize these supplies, rich in reduced carbon, for use as a biological fuel source. A primary focus related to oil-degrading marine bacteria is the oxidation of these oils to meet energy requirements of the living cell. Interestingly, for certain marine bacteria found to utilize and degrade oils, these bacteria are also capable of producing natural lipids that have economic values similar to those obtained from harvesting sperm whales prior to the late 20th century, even when the bacteria are grown on simple organic acids or carbohydrates. We selected the marine bacterium Marinobacter aquaeolei VT8, which was isolated from an oil well off the coast of Vietnam (1), as a model bacterial species to study metabolic processes in an oil-metabolizing and neutral lipid-accumulating bacteria. In addition to growing on long-chain hydrocarbons, M. aquaeolei VT8 also produces a natural hydrocarbon, the wax ester, when grown on simple citric acid cycle intermediates, such as succinate or citrate, as the sole carbon source (1-3), indicating that all of the precursors required for the biosynthesis of wax ester are indigenous to this strain.Biosynthesis of wax esters is accomplished by the combination of several different enzymes. The wax ester synthase/acyl-coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT) enzyme catalyzes the reaction of a fatty acyl-CoA substrate with a fatty alcohol (Fig. 1). While the fatty acyl-CoA utilized by the WS/ DGAT is proposed to come directly from the fatty acyl-CoA pool, the fatty alcohol is believed to be produced through the action of several reducta...

Immune tolerance to allografts has been pursued for decades as an important goal in transplantation. Administration of apoptotic donor splenocytes effectively induces antigen-specific tolerance to allografts in murine studies. Here we show that two peritransplant infusions of apoptotic donor leukocytes under short-term immunotherapy with antagonistic anti-CD40 antibody 2C10R4, rapamycin, soluble tumor necrosis factor receptor and anti-interleukin 6 receptor antibody induce long-term (≥1 year) tolerance to islet allografts in 5 of 5 nonsensitized, MHC class I-disparate, and one MHC class II DRB allele-matched rhesus macaques. Tolerance in our preclinical model is associated with a regulatory network, involving antigen-specific Tr1 cells exhibiting a distinct transcriptome and indirect specificity for matched MHC class II and mismatched class I peptides. Apoptotic donor leukocyte infusions warrant continued investigation as a cellular, nonchimeric and translatable method for inducing antigen-specific tolerance in transplantation.

Mycobacterium tuberculosis causes 10 million cases of active TB disease and over 1 million deaths worldwide each year. TB treatment is complex, requiring at least 6 months of therapy with a combination of antibiotics.

Transposon mutagenesis, in combination with parallel sequencing, is becoming a powerful tool for en-masse mutant analysis. A probability generating function was used to explain observed miniHimar transposon insertion patterns, and gene essentiality calls were made by transposon insertion frequency analysis (TIFA). TIFA incorporated the observed genome and sequence motif bias of the miniHimar transposon. The gene essentiality calls were compared to: 1) previous genome-wide direct gene-essentiality assignments; and, 2) flux balance analysis (FBA) predictions from an existing genome-scale metabolic model of Shewanella oneidensis MR-1. A three-way comparison between FBA, TIFA, and the direct essentiality calls was made to validate the TIFA approach. The refinement in the interpretation of observed transposon insertions demonstrated that genes without insertions are not necessarily essential, and that genes that contain insertions are not always nonessential. The TIFA calls were in reasonable agreement with direct essentiality calls for S. oneidensis, but agreed more closely with E. coli essentiality calls for orthologs. The TIFA gene essentiality calls were in good agreement with the MR-1 FBA essentiality predictions, and the agreement between TIFA and FBA predictions was substantially better than between the FBA and the direct gene essentiality predictions.