Tn6603, a Carrier of Tn5053 Family Transposons, Occurs in the Chromosome and in a Genomic Island of Pseudomonas aeruginosa Clinical Strains.

Transposons of the Pseudomonasaeruginosa accessory gene pool contribute to phenotype and to genome plasticity. We studied local P. aeruginosa strains to ascertain the encroachment of mer-type res site hunter transposons into clinical settings and their associations with other functional modules. Five different Tn5053 family transposons were detected, all chromosomal. Some were solitary elements; one was in res of Tn1013#, a relative of a reported carrier of int-type res site hunters (class 1 integrons), but most were in res of Tn6603, a new Tn501-related transposon of unknown phenotype. Most of the Tn6603::Tn elements, and some Tn6603 and Tn6603::Tn elements found in GenBank sequences, were at identical sites in an hypothetical gene of P. aeruginosa genomic island PAGI-5v. The island in clonally differing strains was at either of two tRNALys loci, suggesting lateral transfer to these sites. This observation is consistent with the membership of the prototype PAGI-5 island to the ICE family of mobile genetic elements. Additionally, the res site hunters in the nested transposons occupied different positions in the Tn6603 carrier. This suggested independent insertion events on five occasions at least. Tn5053 family members that were mer-/tni-defective were found in Tn6603- and Tn501-like carriers in GenBank sequences of non-clinical Pseudomonas spp. The transposition events in these cases presumably utilized tni functions in trans, as can occur with class 1 integrons. We suggest that in the clinical context, P. aeruginosa strains that carry Tn6603 alone or in PAGI-5v can serve to disseminate functional res site hunters; these in turn can provide the requisite trans-acting tni functions to assist in the dissemination of class 1 integrons, and hence of their associated antibiotic resistance determinants.

Embedded elements in the IncPbeta plasmids R772 and R906 can be mobilized and can serve as a source of diverse and novel elements.

IncP plasmids are important contributors to bacterial adaptation. Their phenotypic diversity is due largely to accessory regions located in one or two specific parts of the plasmid. The accessory regions are themselves diverse, as judged from sequenced plasmids mostly isolated from non-clinical sources. To further understand the diversity, evolutionary history and functional attributes of the accessory regions, we compared R906 and R772, focusing on the oriV-trfA accessory region. These IncPß plasmids were from porcine and clinical sources, respectively. We found that the accessory regions formed potentially mobile elements, Tn510 (from R906) and Tn511 (from R772), that differed internally but had identical borders. Both elements appeared to have evolved from a TnAO22-like mer transposon that had inserted into an ancestral IncPß plasmid and then accrued additional transposable elements and genes from various proteobacteria. Structural comparisons suggested that Tn510 (and a descendent in pB10), Tn511 and the mer element in pJP4 represent three lineages that evolved from the same widely dispersed IncPß carrier. Functional studies on Tn511 revealed that its mer module is inactive due to a merT mutation, and that its aphAI region is prone to deletion. More significantly, we showed that by providing a suitable transposase gene in trans, the defective Tn510 and Tn511 could transpose intact or in part, and could also generate new elements (stable cointegrates and novel transposons). The ingredients for assisted transposition events similar to those observed here occur in natural microcosms, providing non-self-mobile elements with avenues for dispersal to new replicons and for structural diversification. This work provides an experimental demonstration of how the complex embedded elements uncovered in IncP plasmids and in other plasmid families may have been generated.

Mercury(II)-resistance transposons Tn502 and Tn512, from Pseudomonas clinical strains, are structurally different members of the Tn5053 family.

The mercury(II)-resistance transposons Tn502 and Tn512 were sequenced and shown to be members of the Tn5053 family. They are currently the sole representatives from the clinical setting and were obtained from geographically disparate Pseudomonas aeruginosa strains. The family is comprised of six novel transposons that display genetic and structural variability that has arisen in different ways. The mer and tni arms of the transposons can be differently combined, suggesting that chimeric interchanges have occurred, possibly mediated by the TniR resolvase. The mer modules within the mer arms are remnants of Tn21/Tn501-like transposons that inserted into a tni-containing ancestral transposon. The mer modules themselves are polymorphic and that of Tn502 is a new type. It includes the putative urf2M gene, the 3'-end of which expresses a protein and hence is a bone fide gene (tniM). Homologues of tniM occur beyond the Tn5053 family and include Tn21 tnpM. Other studies have implicated tniM and tnpM in transposition. Based on sequence and compositional differences, members of the environmentally widespread Tn5053 family constitute a different lineage from the related and clinically successful intI-containing Tn402 family.

Tn502 and Tn512 are res site hunters that provide evidence of resolvase-independent transposition to random sites.

In this study, we report on the transposition behavior of the mercury(II) resistance transposons Tn502 and Tn512, which are members of the Tn5053 family. These transposons exhibit targeted and oriented insertion in the par region of plasmid RP1, since par-encoded components, namely, the ParA resolvase and its cognate res region, are essential for such transposition. Tn502 and, under some circumstances, Tn512 can transpose when par is absent, providing evidence for an alternative, par-independent pathway of transposition. We show that the alternative pathway proceeds by a two-step replicative process involving random target selection and orientation of insertion, leading to the formation of cointegrates as the predominant product of the first stage of transposition. Cointegrates remain unresolved because the transposon-encoded (TniR) recombination system is relatively inefficient, as is the host-encoded (RecA) system. In the presence of the res-ParA recombination system, TniR-mediated (and RecA-mediated) cointegrate resolution is highly efficient, enabling resolution both of cointegrates involving functional transposons (Tn502 and Tn512) and of defective elements (In0 and In2). These findings implicate the target-encoded accessory functions in the second stage of transposition as well as in the first. We also show that the par-independent pathway enables the formation of deletions in the target molecule.

Topological characterization of an inner membrane (1-->3)-beta-D-glucan (curdlan) synthase from Agrobacterium sp. strain ATCC31749.

The crdS gene of Agrobacterium sp. strain ATCC31749 encodes the curdlan synthase (CrdS) protein based on the homology of the derived CrdS protein sequence with those of beta-glycosyl transferases with repetitive action patterns (Stasinopoulos et al. [1999] Glycobiology, 9, 31-41). Here we show that chemical (NTG) mutagenesis of crdS abolishes curdlan production and the induced mutations can be complemented by a cloned crdS amplicon, thus providing genetic confirmation that crdS is essential for curdlan production. When expressed in the native Agrobacterium or in Escherichia coli, the largely hydrophobic CrdS protein exhibited an Mr of approximately 60 kDa (compared with the predicted mass of 73,121 Da) and was located in the inner membrane of both bacteria. By analyzing reciprocal fusions between crdS and the reporter genes, lacZ and phoA, and assessing the sensitivity of CrdS in spheroplasts to proteinase K, CrdS was shown to be an integral membrane protein with seven transmembrane helices and an Nout-Cin disposition. A central large and relatively hydrophilic cytoplasmic region carries the substrate-binding and catalytic D,D,D35QxxRW motif. The amino acid sequence of this domain of CrdS was threaded onto the 3D structure of the comparable domain of the SpsA protein, a member of the family GT-2 glycosyl transferases, and enabled the identification of corresponding amino acids involved in binding UDP in CrdS. Analysis of Agrobacterium membrane preparations using blue native-PAGE provided preliminary evidence that CrdS occurs in multimeric protein complexes of approximately 420 kDa and approximately 500 kDa.

Cloning and characterization of the phosphatidylserine synthase gene of Agrobacterium sp. strain ATCC 31749 and effect of its inactivation on production of high-molecular-mass (1-->3)-beta-D-glucan (curdlan).

Genes involved in the production of the extracellular (1-->3)-beta-glucan, curdlan, by Agrobacterium sp. strain ATCC 31749 were described previously (Stasinopoulos et al., Glycobiology 9:31-41, 1999). To identify additional curdlan-related genes whose protein products occur in the cell envelope, the transposon TnphoA was used as a specific genetic probe. One mutant was unable to produce high-molecular-mass curdlan when a previously uncharacterized gene, pss(AG), encoding a 30-kDa, membrane-associated phosphatidylserine synthase was disrupted. The membranes of the mutant lacked phosphatidylethanolamine (PE), whereas the phosphatidylcholine (PC) content was unchanged and that of both phosphatidylglycerol and cardiolipin was increased. In the mutant, the continued appearance of PC revealed that its production by this Agrobacterium strain is not solely dependent on PE in a pathway controlled by the Pss(AG) protein at its first step. Moreover, PC can be produced in a medium lacking choline. When the pss(AG)::TnphoA mutation was complemented by the intact pss(AG) gene, both the curdlan deficiency and the phospholipid profile were restored to wild-type, demonstrating a functional relationship between these two characteristics. The effect of the changed phospholipid profile could occur through an alteration in the overall charge distribution on the membrane or a specific requirement for PE for the folding into or maintenance of an active conformation of any or all of the structural proteins involved in curdlan production or transport.