SignificanceDiversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacteria, archaea, and their viruses. DGRs use a reverse transcriptase (RT)-mediated mechanism to diversify protein-encoding genes to facilitate adaptation of their hosts to changing environments. Here, we demonstrate that the Bordetella phage DGR-encoded RT uses the 3′-OH of a nicked template RNA to initiate reverse transcription, during which random nucleotides are incorporated when adenine residues in the template are copied into complementary DNA (cDNA). We further show that this mutated, covalently linked RNA-cDNA molecule is required for DGR-mediated sequence diversification, revealing a mechanism of accelerated evolution with broad practical applications.

VopF, a type III effector protein, has been identified as a contributory factor to the intestinal colonization of type III secretion system-positive, non-O1, non-O139 Vibrio cholerae strains. To gain more insight into the function of VopF, a yeast model was developed. Using this model, it was found that ectopic expression of VopF conferred toxicity in yeast. INTRODUCTIONVibrio cholerae, the aetiological agent of the diarrhoeal disease cholera, encompasses more than 200 serogroups, two of which (O1 and O139) are associated with cholera epidemics and pandemics. Studies on the pathogenesis of V. cholerae serogroup O1 and O139 have led to the identification of several critical virulence factors such as cholera toxin and the toxin co-regulated pilus. In addition, V. cholerae produces a major zinc-dependent metalloprotease known as haemagglutinin/protease. Strains belonging to the serogroups other than O1 and O139 are known collectively as the non-O1, non-O139 serogroup. Unlike their pathogenic counterparts O1 and O139, most non-O1, non-O139 strains are non-pathogenic and are found ubiquitously in the aquatic environment. Nevertheless, some members of this serogroup are capable of causing sporadic cases of moderate to severe gastroenteritis and extraintestinal infections in humans, despite the fact that the genes encoding toxin coregulated pilus and cholera toxin are absent, thus raising increasing concern in endemic areas. In contrast to O1 and O139 strains, non-O1, non-O139 strains employ a distinct virulence strategy that remains largely undefined. Recently, much work has been carried out in an effort to understand the biology in the context of pathogenesis and quorum sensing-mediated physiological events in these strains of V. cholerae (Chen et al., 2007;Dziejman et al., 2005;Joelsson et al., 2006;Raychaudhuri et al., 2006). A comparative genomic microarray analysis has suggested that these strains are quite divergent from O1 and O139 strains. Moreover, sequence data mining has also revealed that the genes encoding the type III secretion system (T3SS) in these strains are related to the T3SS 2 gene cluster of Vibrio parahaemolyticus (Dziejman et al., 2005). As non-O1, non-O139 strains are, by and large, devoid of cholera toxin and toxin co-regulated pilus, it is therefore conceivable that the T3SS could contribute towards the pathogenesis of these strains. In a continuing effort, Mekalanos and co-workers have characterized a T3SS effector molecule, VopF (NT01VC2350, WH2 motif domain protein), which has been posited to play a key role in enhancing the intestinal colonization of non-O1, non-O139 strains (Tam et al., 2007). Functionally, VopF belongs to the group of bacterial virulence factors that interfere with actin homeostasis. It is a protein of 530 aa, containing one formin homology 1 (FH1)-like domain and three WASP homology 2 (WH2) domains. A proline-rich motif bridges the second and third WH2 domains. The possible way in which VopF may promote intestinal colonization could either be by introducing a specific...

BackgroundTransposon mutagenesis is highly valuable for bacterial genetic and genomic studies. The transposons are usually delivered into host cells through conjugation or electroporation of a suicide plasmid. However, many bacterial species cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. For this reason, temperature-sensitive (ts) plasmids have also been developed for transposon mutagenesis, but prolonged incubation at high temperatures to induce ts plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive genetic changes. In addition, the ts phenotype of a plasmid is often strain- or species-specific, as it may become non-ts or suicidal in different bacterial species.ResultsWe have engineered several conditional suicide plasmids that have a broad host range and whose loss is IPTG-controlled. One construct, which has the highest stability in the absence of IPTG induction, was then used as a curable vector to deliver hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that these new tools can be used for efficient and regulatable transposon mutagenesis in Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa PAO1, we have used this method to generate a Tn5 insertion library with an estimated diversity of ~ 108, which is ~ 2 logs larger than the best transposon insertional library of PAO1 and related Pseudomonas strains previously reported.ConclusionWe have developed a number of IPTG-controlled conditional suicide plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly useful mutagenesis system has been developed. As the assay condition is mild, we believe that our methodology will have broad applications in microbiology research.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1319-0) contains supplementary material, which is available to authorized users.

30Background: Transposon mutagenesis is highly valuable for bacterial genetic and 31 genomic studies. The transposons are usually delivered into host cells through 32 conjugation or electroporation of a suicide plasmid. However, many bacterial species 33 cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. 34For this reason, temperature-sensitive (ts) plasmids have also been developed for 35 transposon mutagenesis, but prolonged incubation at high temperatures to induce ts 36 plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive 37 genetic changes. In addition, the ts phenotype of a plasmid is often strain-or species-38 specific, as it may become non-ts or suicidal in different bacterial species. 39 Results:We have engineered several conditional suicide plasmids that have a broad 40 host range and whose loss is IPTG-controlled. One construct, which has the highest 41 stability in the absence of IPTG induction, was then used as a curable vector to deliver 42 hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that 43 these new tools can be used for efficient and regulatable transposon mutagenesis in 44 Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa 45 PAO1, we have used this method to generate a Tn5 insertion library with an estimated 46 diversity of ~10 8 , which is ~2 logs larger than the best transposon insertional library of 47 PAO1 and related Pseudomonas strains previously reported. 48 Conclusion:We have developed a number of IPTG-controlled conditional suicide 49 plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly 50 useful mutagenesis system has been developed. As the assay condition is mild, we 51 believe that our methodology will have broad applications in microbiology research. 52 4 53 Background 54Transposon mutagenesis is a powerful technique for bacterial genetic and genomic 55 studies. One of the most widely used transposons is derived from Tn5. The Tn5 56 transposon contains two IS50 elements as inverted terminal repeats (Additional file 1: 57 Figure S1) [1, 2]. Both IS50 and Tn5 can be mobilized by their encoded transposase (Tnp) 58 protein, which recognizes two 19 base pair (bp) sequences at their ends, namely outside 59 end (OE) and inside end (IE), for transposition [2]. OE and IE differ by 7 bp (Additional 60 file 1: Figure S1). As Tn5 insertion is almost completely random, it can insert into any 61 gene in a bacterium. The native Tn5/IS50 is not very active, thus avoiding overt 62 deleterious effect on their hosts, but hyperactive mutants have been engineered as 63 genetic manipulation tools [2, 3]. The most active one contains a mosaic sequence of OE 64 and IE (mosaic end; ME) at the transposon termini and an engineered tnp gene encoding 65 a highly active transposase enzyme (Tnp H ), which together increase Tn5 transposition by 66 more than 1000-fold. 67Transposons for insertional mutagenesis are usually delivered into b...