BackgroundAmaryllidaceae alkaloids (AAs) are a large group of plant-specialized metabolites displaying an array of biological and pharmacological properties. Previous investigations on AA biosynthesis have revealed that all AAs share a common precursor, norbelladine, presumably synthesized by an enzyme catalyzing a Mannich reaction involving the condensation of tyramine and 3,4-dihydroxybenzaldehyde. Similar reactions have been reported. Specifically, norcoclaurine synthase (NCS) which catalyzes the condensation of dopamine and 4-hydroxyphenylacetaldehyde as the first step in benzylisoquinoline alkaloid biosynthesis.ResultsWith the availability of wild daffodil (Narcissus pseudonarcissus) database, a transcriptome-mining search was performed for NCS orthologs. A candidate gene sequence was identified and named norbelladine synthase (NBS). NpNBS encodes for a small protein of 19 kDa with an anticipated pI of 5.5. Phylogenetic analysis showed that NpNBS belongs to a unique clade of PR10/Bet v1 proteins and shared 41% amino acid identity to opium poppy NCS1. Expression of NpNBS cDNA in Escherichia coli produced a recombinant enzyme able to condense tyramine and 3,4-DHBA into norbelladine as determined by high-resolution tandem mass spectrometry.ConclusionsHere, we describe a novel enzyme catalyzing the first committed step of AA biosynthesis, which will facilitate the establishment of metabolic engineering and synthetic biology platforms for the production of AAs.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1570-4) contains supplementary material, which is available to authorized users.

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The type three secretion system (TTSS) locus of Aeromonas salmonicida subsp. salmonicida, located on the plasmid pAsa5, is known to be lost when the bacterium is grown at temperatures of 25 °C. The loss of the locus is due to the recombination of the insertion sequences flanking the TTSS region. However, the mechanism involved in this recombination is still elusive. Here, we analyzed 22 A. salmonicida subsp. salmonicida strains that had already lost their TTSS locus, and we systematically explored another 47 strains for their susceptibility to lose the same locus when grown at 25 °C. It appeared that strains from Europe were more prone to lose their TTSS locus compared to Canadian strains. More specifically, it was not possible to induce TTSS loss in Canadian strains that have AsaGEI2a, a genomic island, and prophage 3, or in Canadian strains without a genomic island. A comparative genomic approach revealed an almost perfect correlation between the presence of a cluster of genes, not yet characterized, and the susceptibility of various groups of strains to lose their locus. This cluster of genes encodes putative proteins with DNA binding capacity and phage proteins. This discovery creates new opportunities in the study of pAsa5 thermosensitivity.

Bloodstream infections caused by invasive, non-typhoidal Salmonella (iNTS) are a a major global health concern, particularly in Africa where the pathogenic variant of Salmonella Typhimurium sequence type (ST) 313 is...

Bloodstream infections caused by invasive, non-typhoidal salmonellae (iNTS) are a major global health concern. These infections are especially problematic in sub-Saharan Africa, where the sequence type (ST) 313 of invasive non-typhoidal Salmonella Typhimurium (iNTS) is dominant. Unlike S. Typhimurium strains that cause mild gastroenteritis, iNTS strains are resistant to multiple first-line antibiotics and have higher extraintestinal invasiveness, limiting current treatment options. Here, we performed multiple small molecule screens under infection-relevant conditions to reveal chemical sensitivities in ST313 as entry points to drug discovery to combat the clinical burden of iNTS. By screening the invasive ST313 sequence type under host-mimicking conditions, we identified the antimicrobial activity of the nucleoside analog 3’-azido-3’-deoxythymidine, which required bacterial thymidine kinase activity for its antimicrobial activity. In a parallel macrophage-based screening platform, we also identified three host-directed compounds (amodiaquine, berbamine, and indatraline) that significantly restricted intracellular replication of ST313 in macrophages without directly impacting bacterial viability. This work provides evidence that despite elevated invasiveness and multidrug resistance, iNTS S. Typhimurium remains susceptible to unconventional drug discovery approaches.