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Quorum sensing (QS) allows many common bacterial pathogens to coordinate group behaviors such as virulence factor production, host colonization, and biofilm formation at high population densities. This cell–cell signaling process is regulated by N-acyl L-homoserine lactone (AHL) signals, or autoinducers, and LuxR-type receptors in Gram-negative bacteria. SdiA is an orphan LuxR-type receptor found in Escherichia, Salmonella, Klebsiella, and Enterobacter genera that responds to AHL signals produced by other species and regulates genes involved in several aspects of host colonization. The inhibition of QS using non-native small molecules that target LuxR-type receptors offers a non-biocidal approach for studying, and potentially controlling, virulence in these bacteria. To date, few studies have characterized the features of AHLs and other small molecules capable of SdiA agonism, and no SdiA antagonists have been reported. Herein, we report the screening of a set of AHL analogs to both uncover agonists and antagonists of SdiA and to start to delineate structure–activity relationships (SARs) for SdiA:AHL interactions. Using a cell-based reporter of SdiA in Salmonella enterica serovar Typhimurium, several non-natural SdiA agonists and the first set of SdiA antagonists were identified and characterized. These compounds represent new chemical probes for exploring the mechanisms by which SdiA functions during infection and its role in interspecies interactions. Moreover, as SdiA is highly stable when produced in vitro, these compounds could advance fundamental studies of LuxR-type receptor:ligand interactions that engender both agonism and antagonism.

Dopaminergic neurotransmission in the striatum has been widely implicated in the reinforcing properties of substances of abuse. However, the striatum is functionally heterogeneous, and previous work has mostly focused on psychostimulant drugs. Therefore, we investigated how dopamine within striatal sub-regions modulates alcohol-directed behaviour in rats. We assessed the effects of infusion of the dopamine receptor antagonist alpha-flupenthixol into the shell and core of the nucleus accumbens (NAcc) and the dorsolateral striatum (DLS) on responding for alcohol under fixed ratio 1 (FR1) and progressive ratio (PR) schedules of reinforcement. Bilateral infusion of alpha-flupenthixol into the NAcc shell reduced responding for alcohol under both the FR1 (15 μg/side) and the PR schedule (3.75 - 15 μg/side) of reinforcement. Infusion of alpha-flupenthixol into the NAcc core (7.5 - 15 μg/side) also decreased responding for alcohol under both schedules. By contrast, alpha-flupenthixol infusion into the DLS did not affect FR1 responding, but reduced responding under the PR schedule (15 μg/side). The decreases in responding were related to earlier termination of responding during the session, whereas the onset and rate of responding remained largely unaffected. Together, these data suggest that dopamine in the NAcc shell is involved in the incentive motivation for alcohol, whereas DLS dopamine comes into play when obtaining alcohol requires high levels of effort. In contrast, NAcc core dopamine appears to play a more general role in alcohol reinforcement. In conclusion, dopaminergic neurotransmission acts in concert in sub-regions of the striatum to modulate different aspects of alcohol-directed behaviour. This article is protected by copyright. All rights reserved.

Targeted mutagenesis increases the activity and alters the regioselectivity of flavin-dependent halogenases., Flavin-dependent halogenases are potentially valuable biocatalysts for the regioselective halogenation of aromatic compounds. These enzymes, utilising benign inorganic halides, offer potential advantages over traditional non-enzymatic halogenation chemistry that often lacks regiocontrol and requires deleterious reagents. Here we extend the biocatalytic repertoire of the tryptophan halogenases, demonstrating how these enzymes can halogenate a range of alternative aryl substrates. Using structure guided mutagenesis we also show that it is possible to alter the regioselectivity as well as increase the activity of the halogenases with non-native substrates including anthranilic acid; an important intermediate in the synthesis and biosynthesis of pharmaceuticals and other valuable products.

The lipopeptides ramoplanin from Actinoplanes sp. ATCC 33076 and enduracidin produced by Streptomyces fungicidicus are effective antibiotics against a number of drug-resistant Gram-positive pathogens. While these two antibiotics share a similar cyclic peptide structure, comprising 17 amino acids with an N-terminal fatty acid side chain, ramoplanin has a di-mannose moiety that enduracidin lacks. The mannosyl substituents of ramoplanin enhance aqueous solubility, which was important in the development of ramoplanin as a potential treatment for Clostridium difficile infections. In this study we have determined the function of the putative mannosyltransferase encoded by ram29 from the ramoplanin biosynthetic gene cluster. Bioinformatics revealed that Ram29 is an integral membrane protein with a putative DxD motif that is suggested to bind to, and activate, a polyprenyl phosphomannose donor and an extracytoplasmic C-terminal domain that is predicted to bind the ramoplanin aglycone acceptor. The ram29 gene was cloned into the tetracycline inducible plasmid pMS17 and integrated into the genome of the enduracidin producer S. fungicidicus. Induction of ram29 expression in S. fungicidicus resulted in the production of monomannosylated enduracidin derivatives, which are not present in the WT strain. Tandem MS analysis showed that mannosylation occurs on the Hpg11 residue of enduracidin. In addition to confirming the function of Ram29, these findings demonstrate how the less common, membrane-associated, polyprenyl phosphosugar-dependent glycosyltransferases can be used in natural product glycodiversification. Such a strategy may be valuable in future biosynthetic engineering approaches aimed at improving the physico-chemical and biological properties of bioactive secondary metabolites including antibiotics.

Additional file 1. Additional methods, figures and tables.

Additional file 1. Additional methods, figures and tables.