Small RNA Deep-Sequencing Analyses Reveal a New Regulator of Virulence in Agrobacterium fabrum C58.

Novel ways of regulating Ti plasmid functions were investigated by studying small RNAs (sRNAs) that are known to act as posttranscriptional regulators in plant pathogenic bacteria. sRNA-seq analyses of Agrobacterium fabrum C58 allowed us to identify 1,108 small transcripts expressed in several growth conditions that could be sRNAs. A quarter of them were confirmed by bioinformatics or by biological experiments. Antisense RNAs represent 24% of the candidates and they are over-represented on the pTi (with 62% of pTi sRNAs), suggesting differences in the regulatory mechanisms between the essential and accessory replicons. Moreover, a large number of these pTi antisense RNAs are transcribed opposite to those genes involved in virulence. Others are 5'- and 3'-untranslated region RNAs and trans-encoded RNAs. We have validated, by rapid amplification of cDNA ends polymerase chain reaction, the transcription of 14 trans-encoded RNAs, among which RNA1111 is expressed from the pTiC58. Its deletion decreased the aggressiveness of A. fabrum C58 on tomatoes, tobaccos, and kalanchoe, suggesting that this sRNA activates virulence. The identification of its putative target mRNAs (6b gene, virC2, virD3, and traA) suggests that this sRNA may coordinate two of the major pTi functions, the infection of plants and its dissemination among bacteria.

Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains.

AIMS: The identification of a new compound active against Agrobacterium tumefaciens. METHODS AND RESULTS: The culture conditions of a newly isolated Bacillus subtilis strain, designed 14B, were optimized, as a first step, to produce its bacteriocin (termed Bac 14B) for the biocontrol of Agrobacterium spp., the causal agents of the crown gall disease. Bac 14B was then partially purified and biochemically characterized. Bacillus subtilis 14B was observed to produce an antibacterial compound having a protinaceous nature. As estimated by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE), the semi-purified bacteriocin substance was found to be a monomeric protein with a molecular weight of 21 kDa. While the latter's antimicrobial activity was completely stable during exposure to a temperature range of up to 100 degrees C for 2 h, its initial activity was totally lost at 121 degrees C for 20 min. The maximum bacteriocin production (4096 AU ml(-1)) was recorded after 96 h-incubation in an optimized Luria Bertani medium supplemented with 10 g l(-1) glucose, 15 g l(-1) K(2)HPO(4) and 5 g l(-1) MgSO(4) 7H(2)O at 30 degrees C in a shaking flask culture. Interestingly, the B. subtilis 14B culture supernatant that contained the bacteriocin under study was proved efficient in reducing both the percentage of galled plants and the number of galls in tomato. CONCLUSION: The findings revealed that B. subtilis 14B and its bacteriocin are efficient in reducing the percentage of infections in plants caused by Ag. tumefaciens. SIGNIFICANCE AND IMPACT OF THE STUDY: The results could be useful for the nurserymen who are particularly interested in the biocontrol of the crown gall disease.

Novel Ti plasmids in Agrobacterium strains isolated from fig tree and chrysanthemum tumors and their opinelike molecules.

Galls naturally induced on Fig and chrysanthemum plants by strains of Agrobacterium contained, in addition to other well-characterized opines such as nopaline, three tumor-specific opinelike molecules. These molecules were identified as deoxy-fructosyl-glutamine (dfg), deoxy-fructosyl-5-oxo-proline (dfop), and chrysopine (Chilton et al., unpublished). Strains isolated from Fig tree and chrysanthemum tumors harbored different and unrelated Ti plasmids as judged by hybridization with various vir and T-DNA probes. They also exhibited different opine-catabolic properties. The strains isolated from chrysanthemum plants (Chry strains) and Fig trees degraded chrysopine, but only the Chry strains used dfg and dfop. Remarkably, other strains of Agrobacterium catabolized these two molecules: dfg was degraded by most pathogenic and nonpathogenic Agrobacterium strains, and dfop by all Agrobacterium strains degrading the opine agropinic acid. These results have strong ecological and evolutionary inferences which fit previous speculation on the origin of opine-related functions.

Comparison between electrochemical and optoelectrochemical impedance measurements for detection of DNA hybridization.

The principles of the electrochemical and optoelectrochemical impedance measurements on bare electrolyte/dielectric/semiconductor structures are described. The analysis of the experimental curves allows access to several indications concerning the electrical behavior of such structures. The application of these techniques to follow the electrical behavior of structures modified with two biological systems was investigated. The antibody/antigen recognition did not change the surface charge and, therefore, did not affect the impedance curves with respect to the applied potential. By contrast, the hybridization of two complementary DNA strands on the surface of the structure induced a variation of flat band potential of the semiconductor leading to a shift of impedance curves along the potential axis. This means that it is possible to detect directly the DNA hybridization without the use of labeled probes. The use of light allows the surface to be probed locally. In the future, the application of this technique for direct detection of hybridization on DNA chips should be possible.

Rapid and accurate species and genomic species identification and exhaustive population diversity assessment of Agrobacterium spp. using recA-based PCR.

Agrobacteria are common soil bacteria that interact with plants as commensals, plant growth promoting rhizobacteria or alternatively as pathogens. Indigenous agrobacterial populations are composites, generally with several species and/or genomic species and several strains per species. We thus developed a recA-based PCR approach to accurately identify and specifically detect agrobacteria at various taxonomic levels. Specific primers were designed for all species and/or genomic species of Agrobacterium presently known, including 11 genomic species of the Agrobacterium tumefaciens complex (G1-G9, G13 and G14, among which only G2, G4, G8 and G14 still received a Latin epithet: pusense, radiobacter, fabrum and nepotum, respectively), A. larrymoorei, A. rubi, R. skierniewicense, A. sp. 1650, and A. vitis, and for the close relative Allorhizobium undicola. Specific primers were also designed for superior taxa, Agrobacterium spp. and Rhizobiaceace. Primer specificities were assessed with target and non-target pure culture DNAs as well as with DNAs extracted from composite agrobacterial communities. In addition, we showed that the amplicon cloning-sequencing approach used with Agrobacterium-specific or Rhizobiaceae-specific primers is a way to assess the agrobacterial diversity of an indigenous agrobacterial population. Hence, the agrobacterium-specific primers designed in the present study enabled the first accurate and rapid identification of all species and/or genomic species of Agrobacterium, as well as their direct detection in environmental samples.

Genomospecies identification and phylogenomic relevance of AFLP analysis of isolated and non-isolated strains of Frankia spp.

Amplified fragment length polymorphism (AFLP) was tested as an alternative to the DNA-DNA hybridization technique (DDH) to delineate genomospecies and the phylogenetic structure within the genus Frankia. Forty Frankia strains, including representatives of seven DDH genomospecies, were typed in order to infer current genome mispairing (CGM) and evolutionary genomic distance (EGD). The constructed phylogeny revealed the presence of three main clusters corresponding to the previously identified host-infecting groups. In all instances, strains previously assigned to the same genomospecies were grouped in coherent clusters. A highly significant correlation was found between DDH values and CGM computed from AFLP data. The species definition threshold was found to range from 0.071 to 0.098 mismatches per site, according to host-infecting groups, presumably as a result of large genome size differences. Genomic distances allowed new Frankia strains to be assigned to nine genomospecies previously determined by DDH. The applicability of AFLP for the characterization of uncultured endophytic strains was tested on experimentally inoculated plants and then applied to Alnus incana and A. viridis field nodules hosting culture refractory spore-positive (Sp+, that sporulate in planta) strains. Only 1.3% of all AFLP fragments were shown to be generated by the contaminant plant DNA and did not interfere with accurate genomospecies identification of strains. When applied to field nodules, the procedure revealed that Alnus Sp+ strains were bona fide members of the Alnus-Myrica host infecting group. They displayed significant genomic divergence from genomospecies G1 of Alnus infecting strains (i.e. Frankia alni) and thus may belong to another subspecies or genomospecies.

Polyphasic characterization of xanthomonads pathogenic to members of the Anacardiaceae and their relatedness to species of Xanthomonas.

We have used amplified fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA) and DNA-DNA hybridization for genotypic classification of Xanthomonas pathovars associated with the plant family Anacardiaceae. AFLP and MLSA results showed congruent phylogenetic relationships of the pathovar mangiferaeindicae (responsible for mango bacterial canker) with strains of Xanthomonas axonopodis subgroup 9.5. This subgroup includes X. axonopodis pv. citri (synonym Xanthomonas citri). Similarly, the pathovar anacardii, which causes cashew bacterial spot in Brazil, was included in X. axonopodis subgroup 9.6 (synonym Xanthomonas fuscans). Based on the thermal stability of DNA reassociation, consistent with the AFLP and MLSA data, the two pathovars share a level of similarity consistent with their being members of the same species. The recent proposal to elevate X. axonopodis pv. citri to species level as X. citri is supported by our data. Therefore, the causal agents of mango bacterial canker and cashew bacterial spot should be classified as pathovars of X. citri, namely X. citri pv. mangiferaeindicae (pathotype strain CFBP 1716) and X. citri pv. anacardii (pathotype strain CFBP 2913), respectively. Xanthomonas fuscans should be considered to be a later heterotypic synonym of Xanthomonas citri.

Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal regions.

Chromosomes and Ti plasmids of 41 Agrobacterium strains, belonging to biovars 1, 2, 3, and Agrobacterium rubi species were characterized by the restriction fragment length polymorphism of PCR-amplified DNAs. Profiles that were obtained by the analysis of the amplified 16S rDNA confirmed the grouping of the strains according to their species. Higher polymorphism was detected in the intergenic spacer between the 16S rDNA and 23S rDNA genes, allowing efficient discrimination of strains. Identification of most strains was possible, and the genetic relatednesses of Agrobacterium strains could be estimated. The analysis of the plasmid Ti encoded regions between the tmr and nos genes, and the virA and virB2 genes, allowed fingerprinting of Ti plasmids. Genomic typing by the rapid PCR-RFLP method is thus shown to be useful for an independent identification of strains and of the conjugative Ti plasmids.

Novel tellurite-amended media and specific chromosomal and Ti plasmid probes for direct analysis of soil populations of Agrobacterium biovars 1 and 2.

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K(2)TeO(3) was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K(2)TeO(3) concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K(2)TeO(3)-amended medium. The bona fide agrobacterium colonies growing on media amended with K(2)TeO(3) were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10(3) to 10(4) agrobacteria. g of dry soil(-1) in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.

Comparison of Randomly Amplified Polymorphic DNA with Amplified Fragment Length Polymorphism To Assess Genetic Diversity and Genetic Relatedness within Genospecies III of Pseudomonas syringae.

Recently, DNA pairing analyses showed that Pseudomonas syringae pv. tomato and related pathovars, including P. syringae pv. maculicola, form a genomic species (Pseudomonas tomato) (L. Gardan, H. L. Shafik, and P. A. D. Grimont, p. 445-448, in K. Rudolph, T. J. Burr, J. W. Mansfield, D. Stead, A. Vivian, and J. von Kietzell, ed., Pseudomonas syringae Pathovars and Related Pathogens, 1997). The genetic diversity of 23 strains belonging to this genomic species and 4 outgroup strains was analyzed with randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphic (AFLP) techniques. Simple boiling of P. syringae cells was suitable for subsequent DNA amplification to obtain reliable patterns in RAPD and AFLP analyses. In general, the grouping of P. syringae strains by both analysis techniques corresponded well with the classification obtained from an RFLP analysis of ribosomal DNA operons, DNA pairing studies, and an analysis of pathogenicity data. However, two strains of P. syringae pv. maculicola produced distinct DNA patterns compared to the DNA patterns of other P. syringae pv. maculicola strains; these patterns led us to assume that horizontal transfer of DNA could occur between bacterial populations. Both techniques used in this study have high discriminating power because strains of P. syringae pv. tomato and P. syringae pv. maculicola which were indistinguishable by other techniques, including pathogenicity tests on tomato, were separated into two groups by both RAPD and AFLP analyses. In addition, data analysis showed that the AFLP method was more efficient for assessing intrapathovar diversity than RAPD analysis and allowed clear delineation between intraspecific and interspecific genetic distances, suggesting that it could be an alternative to DNA pairing studies. However, it was not possible to distinguish the two races of P. syringae pv. tomato on the basis of an analysis of the data provided by either the AFLP or RAPD technique.

Population Heterogeneity of Agrobacterium tumefaciens in Galls of Populus L. from a Single Nursery.

This study focused on the natural crown gall infections occurring in a Leuce poplar nursery. Soil effects on crown gall frequency were detected, indicating that contamination was due to a resident Agrobacterium tumefaciens population, which was present before seedling plantation. The crown gall frequency on poplar progenies varied from 3 to 67%, indicating the feasibility of improvement in crown gall resistance. Of 129 tumor isolates, 128 were pathogenic. These isolates were of biotype 1 or 2. Biochemical, serological, and antibiotic resistance typing results concurred, indicating the presence of four biotype 1 and two biotype 2 resident subpopulations. No significant change was noticed in the relative proportions of subpopulations from one year to another. Pathogenic subpopulations both in vitro and in planta were susceptible to Kerr K84 (P. B. New and A. Kerr, J. Appl. Bacteriol. 90:172-179, 1972). In addition, no serological cross-reactions were found to occur between K84 and the pathogenic subpopulations.

Conditions for natural transformation of Ralstonia solanacearum.

The development of competence allowing natural transformation of Ralstonia solanacearum was found to occur during exponential growth and not in response to any excreted factors. Linear DNAs were effectively integrated by recombination requiring a minimum of 50 bp of homologous DNA. Therefore, DNA from other genera and species were ineffective.

Engineering root exudation of Lotus toward the production of two novel carbon compounds leads to the selection of distinct microbial populations in the rhizosphere.

The culture of opine-producing transgenic Lotus plants induces the increase in the rhizosphere of bacterial communities that are able to utilize these molecules as sole carbon source. We used transgenic Lotus plants producing two opines, namely mannopine and nopaline, to characterize the microbial communities directly influenced by the modification of root exudation. We showed that opine-utilizers represent a large community in the rhizosphere of opine-producing transgenic Lotus. This community is composed of at least 12 different bacterial species, one third of which are able to utilize the opine mannopine and two thirds the opine nopaline. Opine utilizers are diverse, belonging to the Gram-positive and -negative bacteria. We described two novel mannopine-utilizing species, Rhizobium and Duganella spp., and five novel nopaline-utilizing species, Duganella, Afipia, Phyllobacterium, Arthrobacter, and Bosea spp. Although opine utilizers mostly belong to the alpha-Proteobacteria, Rhizobiaceae family, there is little overlap between the populations able to utilize each of the two opines produced by the plants. Noticeably, in the rhizosphere of transgenic Lotus, only the opine mannopine favors the growth of Agrobacterium tumefaciens, the bacterium from which opines have been characterized. The diversity of opine utilizers from the rhizosphere of Lotus plants is greater than that observed from any other environment. Therefore, transgenic plants with engineered exudation constitute an excellent tool to isolate and characterize specific microbial populations.

Direct conjugal transfers of Ti plasmid to soil microflora.

The bacterial species in soil that can receive a Ti plasmid by conjugation from Agrobacterium spp. were investigated. In order to have direct access to the potential reservoir of Ti plasmid amongst soil microflora, the conjugal system consisting of a multiply auxotrophic derivative of C58 (ST-96-4) and a derivative of pTiC58Delta(acc)R (pSTiEGK) containing a triple antibiotic-resistance cassette in traM was used to transfer the Ti plasmid in a complex soil microflora used as the recipient. Numerous transconjugants were obtained by this method but none was identified as Agrobacterium. This could be explained by the low density of Agrobacterium in the tested soil. As indicated by analysis of the ribosomal gene rrs, transconjugants recovered directly from soil were found to be new bacterial species which appeared to be closely related to Sinorhizobium spp.

IS292: a novel insertion element from Agrobacterium.

A new insertion sequence, IS292, located in the 6b gene of a nopaline-type Agrobacterium strain (X88-292) isolated from poplar was identified and sequenced. IS292 is 2494 bp long, has 21 bp inverted terminal repeats with two mismatches, and generates 10 bp direct repeats upon integration. No sequence similarity was found between IS292 and other insertion elements associated with Agrobacterium, but it shows strong similarity with ISR/1 from Rhizobium leguminosarum bv. viciae. The occurrence of IS292-like sequences in various Agrobacterium isolates, especially different Agrobacterium strains isolated from the same biotope, was demonstrated by DNA hybridization.

Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction.

In order to develop a rapid and specific detection test for bacteria in soil, we improved a method based on the polymerase chain reaction (PCR). Each step of the protocol, including direct lysis of cells, DNA purification, and PCR amplification, was optimized. To increase the efficiency of lysis, a step particularly critical for some microorganisms which resist classical techniques, we used small soil samples (100 mg) and various lytic treatments, including sonication, microwave heating, and thermal shocks. Purification of nucleic acids was achieved by passage through up to three Elutip d columns. Finally, PCR amplifications were optimized via biphasic protocols using booster conditions, lower denaturation temperatures, and addition of formamide. Two microorganisms were used as models: Agrobacterium tumefaciens, which is naturally absent from the soil used and was inoculated to calibrate the validity of the protocol, and Frankia spp., an actinomycete indigenous to the soil used. Specific primers were characterized either in the plasmid-borne vir genes for A. tumefaciens or in the variable regions of the 16S ribosomal gene for Frankia spp. Specific detection of the inoculated A. tumefaciens strain was routinely obtained when inocula ranged from 10(7) to 10(3) cells. Moreover, the strong correlation we observed between the size of the inocula and the results of the PCR reactions permitted assessment of the validity of the protocol in enumerating the number of microbial cells present in a soil sample. This allowed us to estimate the indigenous population of Frankia spp. at 0.2 x 10(5) genomes (i.e., amplifiable target sequences) per g of soil.

Relationship between spatial and genetic distance in Agrobacterium spp. in 1 cubic centimeter of soil.

The spatial and genetic unit of bacterial population structure is the clone. Surprisingly, very little is known about the spread of a clone (spatial distance between clonally related bacteria) and the relationship between spatial distance and genetic distance, especially at very short scale (microhabitat scale), where cell division takes place. Agrobacterium spp. Biovar 1 was chosen because it is a soil bacterial taxon easy to isolate. A total of 865 microsamples 500 microm in diameter were sampled with spatial coordinates in 1 cm(3) of undisturbed soil. The 55 isolates obtained yielded 42 ribotypes, covering three genomic species based on amplified ribosomal DNA restriction analysis (ARDRA) of the intergenic spacer 16S-23S, seven of which contained two to six isolates. These clonemates (identical ARDRA patterns) could be found in the same microsample or 1 cm apart. The genetic diversity did not change with distance, indicating the same habitat variability across the cube. The mixing of ribotypes, as assessed by the spatial position of clonemates, corresponded to an overlapping of clones. Although the population probably was in a recession stage in the cube (10(3) agrobacteria g(-1)), a high genetic diversity was maintained. In two independent microsamples (500 microm in diameter) at the invasion stage, the average genetic diversity was at the same level as in the cube. Quantification of the microdiversity landscape will help to estimate the probability of encounter between bacteria under realistic natural conditions and to set appropriate sampling strategies for population genetic analysis.

A highly selectable and highly transferable Ti plasmid to study conjugal host range and Ti plasmid dissemination in complex ecosystems.

A conjugal donor system, ST2, was constructed to study the conjugal dissemination of a Ti plasmid to wild-type recipient bacteria in vitro and in situ. The system consisted of a polyauxotrophic derivative of C58 harboring a hyperconjugative and highly selectable Ti plasmid, pSTiEGK, which was constructed by inserting a multiple antibiotic resistance cassette in the traM- mcpA region of pTiC58Delta accR. ST2 transfers pSTiEGK constitutively at frequencies up to 10(-1) to plasmidless Agrobacterium recipients. The host range of pSTiEGK includes all the known genomic species of Agrobacterium, indigenous soil agrobacteria and some Rhizobium and Phyllobacterium spp. All transconjugants became pathogenic upon acquisition of the Ti plasmid and were also able to transfer pSTiEGK by conjugation. This host range was indistinguishable from that of its wild-type parent pTiC58, and therefore pSTiEGK constitute a valid proxy to study the dissemination of Ti plasmids directly in the environment. Transconjugants can be selected on a combination of four antibiotics, which efficiently prevents the growth of the indigenous microbiota present in complex environments. The transfer of pSTiEGK to members of the genus Agrobacterium was affected primarily by the plasmid content of the recipient strain (10(3)- to 10(5)-fold reduction), e.g., the presence of incompatible plasmids. As a consequence, a species should be considered permissive to Ti transfer whenever one permissive isolate is found.

Ti plasmids from Agrobacterium characterize rootstock clones that initiated a spread of crown gall disease in Mediterranean countries.

Crown gall caused by Agrobacterium is one of the predominant diseases encountered in rose cultures. However, our current knowledge of the bacterial strains that invade rose plants and the way in which they spread is limited. Here, we describe the integrated physiological and molecular analyses of 30 Agrobacterium isolates obtained from crown gall tumors and of several reference strains. Characterization was based on the determination of the biovar, analysis of 16S ribosomal DNA restriction fragment length polymorphisms by PCR (PCR-RFLP), elucidation of the opine type, and PCR-RFLP analysis of genes involved in virulence and oncogenesis. This study led to the classification of rose isolates into seven groups with common chromosome characteristics and seven groups with common Ti plasmid characteristics. Altogether, the rose isolates formed 14 independent groups, with no specific association of plasmid- and chromosome-encoded traits. The predominant Ti plasmid characteristic was that 16 of the isolates induced the production of the uncommon opine succinamopine, while the other 14 were nopaline-producing isolates. With the exception of one, all succinamopine Ti plasmids belonged to the same plasmid group. Conversely, the nopaline Ti plasmids belonged to five groups, one of these containing seven isolates. We showed that outbreaks of disease provoked by the succinamopine-producing isolates in different countries and nurseries concurred with a common origin of specific rootstock clones. Similarly, groups of nopaline-producing isolates were associated with particular rootstock clones. These results strongly suggest that the causal agent of crown gall disease in rose plants is transmitted via rootstock material.

Molecular tools for the characterisation of antibiotic-resistant bacteria.

This review will discuss a number of molecular tools which are currently used as well as some innovative approaches for the characterisation of antibiotic-resistant bacterial strains. Various methods involved in the detection and characterisation of genes and mutations associated with antibiotic resistance and that are used for strain typing as part of epidemiological studies, are described. Furthermore, a few examples are discussed in which the results of both gene and strain characterisation are combined to investigate the underlying mechanism of the spread of antibiotic resistance. Some of the available molecular techniques are heavily supported by the existence of databases on the Internet. These databases either contain a fast growing amount of sequence information or a large number of allelic or fingerprint profiles. The current progress in applied DNA technology and the ongoing projects on the elucidation of the whole genomic sequence of bacterial species have lead and will further lead to the development and application of sophisticated new strategies for the analysis of antibiotic resistant bacterial strains.