Carnitine is a quaternary amine compound found at high concentration in animal tissues, particularly muscle, and is most well studied for its contribution to fatty acid transport into mitochondria. In bacteria, carnitine is an important osmoprotectant, and can also enhance thermotolerance, cryotolerance and barotolerance. Carnitine can be transported into the cell or acquired from metabolic precursors, where it can serve directly as a compatible solute for stress protection or be metabolized through one of a few distinct pathways as a nutrient source. In this review, we summarize what is known about carnitine physiology and metabolism in bacteria. In particular, recent advances in the aerobic and anaerobic metabolic pathways as well as the use of carnitine as an electron acceptor have addressed some long-standing questions in the field. Received 5 January 2015 Accepted 17 March 2015Introduction Carnitine (c-trimethylamino-b-hydroxybutyric acid) ( Fig. 1) is a quaternary amine compound that can be produced by all domains of life, and was discovered in muscle extract in 1905 by Gulewitsch & Krimberg (1905) and Kutscher (1905). It was shown to be essential for larval development of the mealworm Tenebrio molitor and was originally designated vitamin B T based on this requirement. Later, it was discovered that carnitine can be synthesized in mammals and is now considered to be a quasi-nutrient or conditionally essential nutrient (Flanagan et al., 2010), as neonates have reduced biosynthesis and rely on placental transfer of carnitine in utero and exogenous sources after birth (Combs, 2012). Fifty years after the discovery of carnitine, it was demonstrated that assorted Gram-positive and Gram-negative bacteria could use carnitine in either aerobic or anaerobic environments for a variety of cellular functions, including as an electron acceptor, as a compatible solute to survive environmental insults or as a sole carbon, nitrogen and energy source. Bacterial carnitine metabolism was most recently reviewed in 1998 (Bieber, 1988;Bremer, 1983;Kleber, 1997;Rebouche & Seim, 1998) and the field has seen important advances. This review summarizes what we knew at the time of the previous reviews and emphasizes what we have learned since, including: (i) how anaerobic bacteria synthesize and utilize crotonobetaine and carnitine as final electron acceptors, (ii) the impact of carnitine degradation by the intestinal microbiota and the genes responsible for this anaerobic conversion, (iii) the genes involved in aerobic degradation of carnitine, and (iv) how carnitine as a compatible solute impacts survival within and outside of the host. Carnitine in the environmentRecent work makes it clear that while animals represent the most readily accessible source of carnitine, carnitine is often present and sometimes abundant in soil and natural waters. Quaternary ammonium compounds are abundant in a number of soil ecosystems, including comprising a quarter of the most abundant organic nitrogen compounds in the soil water of a subalpine gras...

Pseudomonas aeruginosa displays tremendous metabolic diversity, controlled in part by the abundance of transcription regulators in the genome. We have been investigating P. aeruginosa's response to the host, particularly changes regulated by the hostderived quaternary amines choline and glycine betaine (GB). We previously identified GbdR as an AraC family transcription factor that directly regulates choline acquisition from host phospholipids (via binding to plcH and pchP promoters), is required for catabolism of the choline metabolite GB, and is an activator that induces transcription in response to GB or dimethylglycine. Our goal was to characterize the GbdR regulon in P. aeruginosa by using genetics and chemical biology in combination with transcriptomics and in vitro DNA-binding assays. Here we show that GbdR activation regulates transcription of 26 genes from 12 promoters, 11 of which have measureable binding to GbdR in vitro. The GbdR regulon includes the genes encoding GB, dimethylglycine, sarcosine, glycine, and serine catabolic enzymes and the BetX and CbcXWV quaternary amine transport proteins. We characterized the GbdR consensus binding site and used it to identify that the recently characterized acetylcholine esterase gene, choE (PA4921), is also regulated by GbdR. The regulon member not directly controlled by GbdR is the secreted lipase gene lipA, which was also the only regulon member repressed under GbdR-activating conditions. Determination of the GbdR regulon provides deeper understanding of how GbdR links bacterial metabolism and virulence. Additionally, identification of two uncharacterized regulon members suggests roles for these proteins in response to choline metabolites.

Extracellular vesicles are highly abundant in seminal fluids and have a known role enhancing sperm function. Clinical pregnancy rates after IVF treatment are improved after female exposure to seminal fluid. Seminal fluid extracellular vesicles (SF-EVs) are candidate enhancers, however, whether SF-EVs interact with cells from the endometrium and modulate the implantation processes is unknown. Here, we investigated whether SF-EVs interact with endometrial stromal cells (ESCs) and enhance decidualisation, a requisite for implantation. SF-EVs, isolated from human seminal fluid (n = 11) by ultracentrifugation, were characterised by nanoparticle tracking analysis and Western blotting, and purified using size exclusion chromatography. Non-decidualised and decidualised primary ESCs (n = 5) were then treated with SF-EVs. Binding of bio-maleimide-labelled SF-EVs was detected by flow cytometry and fluorescence microscopy. Prolactin and IGFBP-1 protein levels in culture media were also analysed after single and multiple SF-EV exposure. SF-EVs size ranged from 50 to 300 nm, and they expressed exosomal markers (ALIX, SYNTENIN-1, CD9 and CD81). SF-EVs bound to non-decidualised and decidualised ESCs at similar levels. ESCs prolactin secretion was increased after single (p = 0.0044) and multiple (p = 0.0021) SF-EV exposure. No differences were found in IGFBP-1 protein levels. In conclusion, SF-EVs enhance in vitro ESC decidualisation and increase secretion of prolactin, an essential hormone in implantation. This elucidates a novel role of SF-EVs on endometrial receptivity. Abbreviations: ECACC: European Collection of Authenticated Cell Cultures; ESCs: endometrial stromal cells; EVs: extracellular vesicles; FCS: foetal calf serum; HRP: horse-radish peroxidase; IFNγ: interferon-gamma; IGF: insulin-like growth factor; IGFBP-1: insulin-like growth factor binding protein 1; IVF: in vitro fertilisation; MVB: multivesicular bodies; NTA: nanoparticle tracking analysis; PRLR−/−: homozygous prolactin receptor knockout; RT: room temperature; SF-EVs: seminal fluid extracellular vesicles; STR: short tandem repeat; TGFβ: transforming growth factor β; uNK: uterine natural killer

BackgroundThe power and simplicity of visual colony counting have made it the mainstay of microbiological analysis for more than 130 years. A disadvantage of the method is the long time required to generate visible colonies from cells in a sample. New rapid testing technologies generally have failed to maintain one or more of the major advantages of culture-based methods.Principal FindingsWe present a new technology and platform that uses digital imaging of cellular autofluorescence to detect and enumerate growing microcolonies many generations before they become visible to the eye. The data presented demonstrate that the method preserves the viability of the microcolonies it detects, thus enabling generation of pure cultures for microbial identification. While visual colony counting detects Escherichia coli colonies containing about 5×106 cells, the new imaging method detects E. coli microcolonies when they contain about 120 cells and microcolonies of the yeast Candida albicans when they contain only about 12 cells. We demonstrate that digital imaging of microcolony autofluorescence detects a broad spectrum of prokaryotic and eukaryotic microbes and present a model for predicting the time to detection for individual strains. Results from the analysis of environmental samples from pharmaceutical manufacturing plants containing a mixture of unidentified microbes demonstrate the method's improved test turnaround times.ConclusionThis work demonstrates a new technology and automated platform that substantially shortens test times while maintaining key advantages of the current methods.

Pathogens must metabolize host-derived compounds during infection and properly regulate the responsible pathways. Carnitine is a common eukaryotic-associated quaternary amine compound that can be catabolized by Pseudomonas aeruginosa. Here we expand on our understanding of how this metabolic pathway is regulated and provide details on how carnitine catabolism is intertwined with glycine betaine catabolism at the level of transcriptional control.