A putative genomic island, PGI-1, in Ralstonia solanacearum biovar 2 revealed by subtractive hybridization

Ralstonia solanacearum biovar 2, a key bacterial pathogen of potato, has recently established in temperate climate waters. On the basis of isolates obtained from diseased (potato) plants, its genome has been assumed to be virtually clonal, but information on environmental isolates has been lacking. Based on differences in pulsed-field gel electrophoresis patterns, we compared the genomes of two biovar 2 strains with different life histories. Thus, genomic DNA of the novel environmental strain KZR-5 (The Netherlands) was compared to that of reference potato strain 715 (Bangladesh) by suppressive subtractive hybridization. Various strain-specific sequences were found, all being homologous to those found in the genome of reference potato strain 1609. Approximately 20% of these were related to genes involved in recombinational processes. We found a deletion of a 17.6-Kb region, denoted as a putative genomic island PGI-1, in environmental strain KZR-5. The deleted region was, at both extremes, flanked by a composite of two insertion sequence (IS) elements, identified as ISRso2 and ISRso3. The PGI-1 region contained open reading frames that putatively encoded a (p)ppGpp synthetase, a transporter protein, a transcriptional regulator, a cellobiohydrolase, a site-specific integrase/recombinase, a phage-related protein and seven hypothetical proteins. As yet, no phenotype could be assigned to the loss of PGI-1. The ecological behavior of strain KZR-5 was compared to that of reference strain 715. Strain KZR-5 showed enhanced tolerance to 4°C as compared to the reference strain, but was not affected in its virulence on tomato

Comparative Analysis of Acinetobacters: Three Genomes for Three Lifestyles

Acinetobacter baumannii is the source of numerous nosocomial infections in humans and therefore deserves close attention as multidrug or even pandrug resistant strains are increasingly being identified worldwide. Here we report the comparison of two newly sequenced genomes of A. baumannii. The human isolate A. baumannii AYE is multidrug resistant whereas strain SDF, which was isolated from body lice, is antibiotic susceptible. As reference for comparison in this analysis, the genome of the soil-living bacterium A. baylyi strain ADP1 was used. The most interesting dissimilarities we observed were that i) whereas strain AYE and A. baylyi genomes harbored very few Insertion Sequence elements which could promote expression of downstream genes, strain SDF sequence contains several hundred of them that have played a crucial role in its genome reduction (gene disruptions and simple DNA loss); ii) strain SDF has low catabolic capacities compared to strain AYE. Interestingly, the latter has even higher catabolic capacities than A. baylyi which has already been reported as a very nutritionally versatile organism. This metabolic performance could explain the persistence of A. baumannii nosocomial strains in environments where nutrients are scarce; iii) several processes known to play a key role during host infection (biofilm formation, iron uptake, quorum sensing, virulence factors) were either different or absent, the best example of which is iron uptake. Indeed, strain AYE and A. baylyi use siderophore-based systems to scavenge iron from the environment whereas strain SDF uses an alternate system similar to the Haem Acquisition System (HAS). Taken together, all these observations suggest that the genome contents of the 3 Acinetobacters compared are partly shaped by life in distinct ecological niches: human (and more largely hospital environment), louse, soil

The low-temperature-induced viable-but-nonculturable state affects the virulence of Ralstonia solanacearum biovar 2

van Overbeek. L. S.. Bergervoet. J. H. W.. Jacobs, F. H. H.. and van Elsas. J. D. 2004. The low-ternperature-induced viable-but-nonculturable state 2. Phytopathology affects the virulence of Ralstonia solanacearum biovar 94:463-469. The physiology and virulence of Ralstonia solanacerum biovar 2 strain 1609. kept in water at 4 and 20degreesC, were studied. At 20degreesC. total cell and plate count (colony forming units: CFU) numbers were similar, between log 5.03 and log 5.55 CFU,and log 5.03 and log 5.51 cells per ml. at days 0 and 132. respectively. However, CFU in the Cultures kept at 4degreesC dropped from log 6.78 CFU/ml at day 0 to below detection after 84 days. The presence of catalase in the agar resulted in higher CFU. and at day 84. log, 1.95 CFU/ml still was detectable. No colonies were observed at day 125. The presence of viable-but-nonculturable (VBNC) cells in the 4degreesC Cultures was confirmed using SYTO9 viability staining. Viable Cell numbers were log 1.77 higher than CFU on plates with catalase. At day 84 and after 125 days, log 3.70 viable cells per ml still were I present. Shifts in subpopulations differing in viability were found by flow cytometric sorting of 4degreesC-treated cells stained with SYTO9 (healthy) and propidium iodide (PI, compromised). The SYTO9-stained cell fractions dropped from 99 to 39%, and the PI-stained fractions increased from 0.7 to 33.3% between days 0 and 125. At 20degreesC. the SYTO9-stained fraction remained stable at 99% until day 132, SYTO9-stained cells sorted from 4degreesC Cultures at day 100 were injected into tomato plants. Upon incubation for 30 days, these plants did not show wilting. However. more than log 4.19 CFU and log 8.17 cells were recovered from these plants. Cells from colonies isolated from the nonwilted plants did not regain their virulence as demonstrated by Subsequent injection into several mew, sets of tomato plants. Cells from 4degreesC cultures injected at day 125 were not able to cause wilting of, or proliferate in, tomato plants. The threat posed by VBNC R. solanacearum cells upon incubation at 4degreesC was thus ephemeral because cells lost their capacity to cause disease after 125 days

Model plants for studying the interaction between Methylobacterium mesophilicum and Xylella fastidiosa

Over the last few years, endophytic bacterial communities associated with citrus have been studied as key components interacting with Xylella fastidiosa. In this study, we investigated the possible interaction between the citrus endophyte Methylobacterium mesophilicum SR1.6/6 and X. fastidiosa in model plants such as Catharanthus roseus (Madagaskar periwinkle) and Nicotiana clevelandii (Clevelands tobacco). The aim of this study was to establish the fate of M. mesophilicum SR1.6/6 after inoculation of C. roseus and N. clevelandii plants, using PCR-DGGE (polymerase chain reaction - denaturing gradient gel electrophoresis) and plating techniques. Shifts in the indigenous endophytic bacterial communities were observed in plants inoculated with strain SR1.6/6, using specific primers targeting alpha- and beta-Proteobacteria. Cells of strain SR1.6/6 were observed in a biofilm structure on the root and hypocotyl surfaces of in vitro seedlings inoculated with M. mesophilicum SR1.6/6. This emphasizes the importance of these tissues as main points of entrance for this organism. The results showed that C. roseus and N. clevelandii could be used as model plants to study the interaction between M. mesophilicum and X. fastidiosa

The complete nucleotide sequence and environmental distribution of the cryptic, conjugative, broad-host-range plasmid pIPO2 isolated from bacteria of the wheat rhizosphere

Tauch A, Schneiker-Bekel S, Selbitschka W, et al. The complete nucleotide sequence and environmental distribution of the cryptic, conjugative, broad-host-range plasmid pIPO2 isolated from bacteria of the wheat rhizosphere. MICROBIOLOGY-SGM. 2002;148 (Pt 6):1637-1653.Plasmid pIPO2 is a cryptic, conjugative, broad-host-range plasmid isolated from the wheat rhizosphere. it efficiently self-transfers between alpha, beta and gamma Proteobacteria and has a mobilizing/retromobilizing capacity for IncQ plasmids. The complete nucleotide sequence of pIPO2 is presented on the basis of its mini-Tn5::luxABtet-tagged derivative, pIPO2T. The pIPO2 sequence is 39815 bp long and contains at least 43 complete ORFs. Apart from a suite of ORFs with unknown function, all of the genes carried on pIPO2 are predicted to be involved in plasmid replication, maintenance and conjugative transfer. The overall organization of these genes is different from previously described plasmids, but is similar to the genetic organization seen in pSB102, a conjugative plasmid recently isolated from the bacterial community of the alfalfa rhizosphere. The putative conjugative transfer region of pIPO2 covers 23 kb and contains the genes required for DNA processing (Dtr) and mating pair formation (Mpf). The organization of these transfer genes in pIPO2 is highly similar to the genetic organization seen in the environmental plasmid pSB102 and in pXF51 from the plant pathogen Xylella fastidiosa. Plasmids pSB102 and pXF51 have recently been proposed to form a new family of environmental broad-host-range plasmids. Here it is suggested that pIPO2 is a new member of this family. The proposed Mpf system of pIPO2 shares high amino acid sequence similarity with equivalent VirB proteins from the type IV secretion system of Brucella spp. Sequence information was used to design primers specific for the detection of pIPO2. Environmental DNA from a range of diverse habitats was screened by PCR with these primers. Consistently positive signals for the presence of pIPO2 were obtained from a range of soil-related habitats, including the rhizospheres of young wheat plants, of field-grown oats and of grass (all gramineous plants), as well as from the rhizosphere of tomato plants. These data add to the growing evidence that plasmids carry advantageous genes with as yet undefined functions in plant-associated communities