Vilma A. Stanisich

New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region

Medical microbiology is extremely reliant on the culture of bacteria from clinical specimens and their subsequent identification by biochemical and phenotypic characteristics for the diagnosis of disease. Following determination of the structure of DNA by Watson & Crick (1953), studies in bacteriology have seen a major shift from functional to molecular techniques for identifying bacteria (Towner & Cockayne, 1993). This review will deal with the 16s-23s spacer region of the rRNA operon (Fig. 1) and its use in the identification of micro-organisms at the species and strain (typing) levels.

The Properties and Host Range of Male-specific Bacteriophages of Pseudomonas aeruginosa

Summary: Three Pseudomonas aeruginosa phages specific for bacteria harbouring the P-group plasmid RP1 have been isolated, and their properties compared with those of a previously described sex-specific phage, PRR1 (Olsen & Shipley, 1973). These phages are distinguishable from each other by various criteria, although in terms of host range to FP+ and RP+ lines of P. aeruginosa pao they comprise broadly two groups. Thus all the phages infect bacteria harbouring any of a group of plasmids with similar properties to those of RP1, but whereas the filamentous phage Pf3 is specific for this group, the host ranges of PRR1, PR3 and PR4 are considerably wider. Nevertheless, with one exception, this does not extend to plasmids isolated outside the United Kingdom, which suggests that all these plasmids share a common ancestry even though by other criteria they constitute three fairly discrete subgroups. Of the plasmids that fail to allow phage propagation, three, when present in the same cell as RP1, reduce its susceptibility to phage infection. This inhibition may reflect a relationship between these elements, similar to that found among plasmids of Enterobacteria. A correlation is observed between the susceptibility of bacteria to phage infection and their ability to mediate plasmid transfer, such that these phages can conveniently be used to isolate both derepressed or transfer-defective mutants of various R factors.

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)-β-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.

2 Identification and Analysis of Plasmids at the Genetic Level

Publisher Summary Many bacteria of diverse type and habitat are known to harbour plasmid DNA. This observation lends credence to the view that these elements are ubiquitous among prokaryotes and are likely to be detected in any species in which a thorough search is made. In recent years, the demonstration of plasmids in an increasing variety of bacteria has been striking and is directly the result of the development of techniques that allow physical demonstration, isolation, and molecular characterization of plasmid DNA. Thus, plasmids in bacteria in which genetic analysis is currently limited or impossible are as amenable to detailed molecular study as those from the genetically well-characterized bacteria. Such investigations are satisfactory where the primary concern is molecular characterization, comparison, and in vitro manipulation of plasmids for specific purposes, such as the construction of cloning vectors.

Location and characterization of two functions on RP1 that inhibit the fertility of the IncW plasmid R388.

Two fertility-inhibition functions which reduce R388 (IncW) transfer were detected on RP1 (60 kb, IncP). The respective genes, fiwA and fiwB, were mapped by transposon insertion mutagenesis to the regions between coordinates 32.8 to 31.7 kb (fiwA), and 59.8 to 0.8 kb (fiwB). The fiwA function occurs in a non-essential region of RP1 whereas fiwB is straddled by essential plasmid-maintenance and host-range determinants and apparently coincides (or overlaps) with the gene for tellurite-resistance.

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.

Characterization of Pseudomonas Mercury-resistance transposon Tn502, which has a preferred insertion site in RP1

Tn502mer differs in size and restriction map from the well-characterized Tn501mer. It also differs in its preferential and high-frequency insertion into the 6 kb PstI-C region of RP1. The affinity for this region is perpetuated in pVS76, a clone of RP1 PstI-C in pBR322. Restriction mapping of independent pVS76::Tn502 derivatives revealed that Tn502 inserted at the same site (or small region) in PstI-C corresponding to the 35 kb coordinate in RP1. Insertion occurred in both orientations, but one was preferred. When PstI-C was deleted from RP1, acquisition of Tn502 was reduced and the sites of insertion randomized.

Location of a function on RP1 that fertility inhibits Inc W plasmids.

Two fertility inhibition (Fi+) functions which reduce R388 (Inc W) transfer were detected on RP1 (Inc P). Neither function affected R388 -mediated surface exclusion but they could be distinguished by their effect on pilus production. One of the functions was located in the 6.5-kb Pst1 -C region of RP1, part or all of which also occurs on six Fi+ but not two Fi- Inc P plasmids studied.

Embedded elements in the IncPβ 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 IncPβ 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 IncPβ 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 IncPβ 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.

Characterization of a Tra 2 function of RP1 that affects growth of Pseudomonas aeruginosa PAO and surface exclusion in Escherichia coli K12

pVS438, a clone of part of the Tra 2 region of RP1 in RSF1010, confers two unusual phenotypes: poor growth (Slo+) in Pseudomonas aeruginosa PAO and surface exclusion (Sfx+) in Escherichia coli K12. Both of these phenotypes were found to be encoded by a 1.8-kb fragment of RP1 (from 25.9-27.7 kb) that spans the traB gene. However, whether both phenotypes, neither, or only Slo+ is expressed by this fragment depends on its location and orientation in RSF1010. In pVS438, where this fragment occurs in the SmR locus of RSF1010, expression of the Sfx+ phenotype is due to augmented transcription from the two promoters that cotranscribe the SuRSmR genes. When augmentation is abolished by insertion of Tn5 between these promoters and the cloned fragment, or by insertion of the fragment elsewhere in RSF1010, a Slo+Sfx- phenotype results. DNA that confers only the Slo+ phenotype was mapped to the 26.2-26.8 kb region of RP1 between traE and traB and the designation, traS, given to the gene responsible. Despite the recognition of a traS+ (Slo+) component of DNA within that encoding the Slo+ and Sfx+ phenotypes, this gene seems nevertheless to be responsible for the Sfx+ phenotype since hydroxylamine-induced Slo- mutants of pVS438 are usually also Sfx-. These apparently conflicting observations and the precise interplay between the Slo+, Sfx+, and TraB+ phenotypes were not resolved. Finally, traS is not essential for plasmid transfer since pVS438 and a Slo-Sfx- derivative of it can both equally complement an RP1tra-deletion mutant of part of the Tra 2 region.