Niche Construction and Exploitation by Agrobacterium: How to Survive and Face Competition in Soil and Plant Habitats.

Agrobacterium populations live in different habitats (bare soil, rhizosphere, host plants), and hence face different environmental constraints. They have evolved the capacity to exploit diverse resources and to escape plant defense and competition from other microbiota. By modifying the genome of their host, Agrobacterium populations exhibit the remarkable ability to construct and exploit the ecological niche of the plant tumors that they incite. This niche is characterized by the accumulation of specific, low molecular weight compounds termed opines that play a critical role in Agrobacterium 's lifestyle. We present and discuss the functions, advantages, and costs associated with this niche construction and exploitation.

Metagenomics revealed a quorum quenching lactonase QlcA from yet unculturable soil bacteria.

Quorum sensing (QS) is a signal mediated cell-cell communication system that couples bacterial cell density to a synchronized gene expression (Fuqua et al., 1994). Mostly, in Gram negative bacteria QS signals are N-acylhomoserine lactones (NAHLs) that coordinate important functions such as virulence and pathogenicity. QS signals or the elements involved in their production or perception could be targeted to disrupt QS, a phenomenon called Quorum quenching (QQ). QQ properties (chemicals and enzymes) are naturally found in various Living organisms, like bacteria (Rhodococcus and Commamonas), plants (carrot, soybean, pea seedling, chilli, garlic etc), and animals (human sera, pork kidney tissues). Consequently, various bacterial genes encoding for NAHL degrading enzymes, like NAHL lactonases (AiiA in Bacillus, AiiB and AttM in Agrobacterium tumefaciens) and acylase/-amidohydrolase (AiiD in Ralstonia) were identified (Givskov et al., 2006). In Pectobacterium carotovorum (causal agent of soft rot diseases) production of various virulence factors and cell wall maceration enzymes is QS dependant, and relies upon successful production, stability, emission and perception of NAHLs (C-8, oxo-C8 and C-10). Disruption of QS signalling by NAHL degrading bacteria, modified bacteria or plants expressing NAHL lactonases resulted in the reduced virulence of the pathogen (Faure et al., 2007). Until recently, investigations on QQ enzymes were carried out mostly on cultivable bacteria, that represent a tiny fraction of soil and root-associated bacteria. In this study, a metagenomics approach (Handelsman, 2004) was employed to access the hidden diversity of uncultivable soil bacteria that revealed a QQ enzyme, an NAHL lactonase, in these bacteria (Riaz et al., 2008).

[Violacein: a molecule of biological interest originating from the soil-borne bacterium Chromobacterium violaceum]. / La violacéine: une molécule d'intérêt biologique, issue de la bactérie tellurique Chromobacterium violaceum.

INTRODUCTION: Soil micro-organisms have evolved functions that allow them to withstand the strong competition for survival that characterizes most of their habitats. The production of antibiotic or antifungal compounds is one of these mechanisms. The relevant molecules often exhibit valuable therapeutic properties. EXEGESIS: Chromobacterium violaceum is a soil-borne bacterium producing a characteristic antibiotic termed violacein. It is part of a series of compounds released by C. violaceum to oppose competitors and predators in the soil and water environments. Violacein, and one of these compounds, i.e. structure FR901228, exhibit antiparasitic and antitumoral activities of potential medical interest. Genes involved in the synthesis of these compounds are available, the genome sequence of C. violaceum (strain ATCC 12472) being published. CONCLUSIONS: The above example, involving Chromobacterium, is not an exception: soil constitutes a reservoir of molecules, enzymatic activities and micro-organisms of biological interest, the study of which will undoubtedly lead to developments in fields as diverse as agronomy or animal and human therapeutics.

Comparison of the phenotypes and genotypes of biofilm and solitary epiphytic bacterial populations on broad-leaved endive.

The discovery that biofilms are ubiquitous among the epiphytic microflora of leaves has prompted research about the impact of biofilms on the ecology of epiphytic microorganisms and on the efficiency of strategies to manage these populations for disease control and to ensure food safety. Biofilms are likely to influence the microenvironment and phenotype of the microorganisms they harbor. However, it is also important to determine whether there are differences in the types of bacteria within biofilms compared to those outside of biofilms so as to better target microorganisms via disease control strategies. Broad-leaved endive (Cichorium endivia var. latifolia) harbors biofilms containing fluorescent pseudomonads. These bacteria can cause considerable post-harvest losses when this plant is used for manufacturing minimally processed salads. To determine whether the population structure of the fluorescent pseudomonads in biofilms is different from that outside of biofilms on the same leaves, bacteria were isolated quantitatively from the biofilm and solitary components of the epiphytic population on leaves of field-grown broad-leaved endive. Population structure was determined in terms of taxonomic identities of the bacteria isolated, in terms of genotypic profiles, and in terms of phenotypic traits related to surface colonization and biofilm formation. The results illustrate that there are no systematic differences in the composition and structure of biofilm and solitary populations of fluorescent pseudomonads, in terms of either genotypic profiles or phenotypic profiles of the strains. However, Gram-positive bacteria tended to occur more frequently within biofilms than outside of biofilms. We suggest that leaf colonization by fluorescent pseudomonads involves a flux of cells between biofilm and solitary states. This would allow bacteria to exploit the advantages of these two types of existence; biofilms would favor resistance to stressful conditions, whereas solitary cells could foster spread of bacteria to newly colonizable sites on leaves as environmental conditions fluctuate.

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.

The Ti plasmid of Agrobacterium tumefaciens harbors an attM-paralogous gene, aiiB, also encoding N-Acyl homoserine lactonase activity.

The Agrobacterium tumefaciens C58 genome contains three putative N-acyl homoserine lactone (acyl-HSL) hydrolases, which are closely related to the lactonase AiiA of Bacillus. When expressed in Escherichia coli, two of the putative acyl-HSL hydrolases, AttM and AiiB, conferred the ability to degrade acyl-HSLs on the host. In Erwinia strain 6276, the lactonases reduced the endogenous acyl-HSL level and the bacterial virulence in planta.

Seasonal fluctuations and long-term persistence of pathogenic populations of Agrobacterium spp. in soils.

Short- and long-term persistence of pathogenic (i.e., tumor forming) agrobacteria in soil was investigated in six nursery plots with a history of high crown gall incidence. No pathogenic Agrobacterium strains were isolated in soil samples taken in fall and winter in any plots, but such strains were isolated from both bulk soils and weed rhizospheres (over 0.5 x 10(5) pathogenic CFU/g of bulk soil or rhizosphere) in three out of six plots in spring and summer. PCR amplifications of a vir sequence from DNA extracted from soil confirmed the presence of Ti plasmids in summer and their absence in fall and winter. The results indicate that strains that harbor a Ti plasmid had an unforeseen positive fitness versus Ti plasmid-free strains in soil and rhizosphere in spring and summer in spite of the apparent absence of tumor, and hence of opines. The gain of fitness occurred during a bloom of all cultivable agrobacteria observed only in conducive soils. An evolution of the pathogenic population was recorded during a 4-year period in one particularly conducive soil. In 1990, the pathogenic population in this soil consisted of only biovar 1 strains harboring both octopine- and nopaline-type Ti plasmids. In 1994, it consisted of only nopaline-type Ti plasmids equally distributed among biovar 1 and 2 strains. These results suggest that nopaline-type Ti plasmids conferred a better survival ability than octopine-type Ti plasmids to biovar 2 agrobacteria under the present field conditions.

Structure and characterization of the crown gall opines heliopine, vitopine and ridéopine.

The crown gall opines heliopine from tumors induced by octopine type Agrobacterium tumefaciens strains A6, A136(pTiB6-806), E9, A652 and 1590-1 and vitopine from tumor induced by grapevine strains S4 and T2 are identical to synthetic N2-(1'R-carboxyethyl)-L-glutamine. Tumors produced by strains S4 and T2 do not contain octopine or lysopine, but they do contain heliopine and the new opine ridéopine identified as N-(4'-aminobutyl)-D-glutamic acid. Grapevine strains S4 and T2 grow normally on tumor heliopine or synthetic heliopine and on tumor and synthetic ridéopine as well as on ridéopine lactam as sole carbon source. While octopine strains A6 and A136(pTiB6-806) do not grow on heliopine, mutant colonies do appear after a few weeks. Heliopine catabolism by octopine strains is not induced by octopine.

Acyl-homoserine lactone production is more common among plant-associated Pseudomonas spp. than among soilborne Pseudomonas spp.

A total of 137 soilborne and plant-associated bacterial strains belonging to different Pseudomonas species were tested for their ability to synthesize N-acyl-homoserine lactones (NAHL). Fifty-four strains synthesized NAHL. Interestingly, NAHL production appears to be more common among plant-associated than among soilborne Pseudomonas spp. Indeed, 40% of the analyzed Pseudomonas syringae strains produced NAHL which were identified most often as the short-chain NAHL, N-hexanoyl-L-homoserine lactone, N-(3-oxo-hexanoyl)-homoserine lactone, and N-(3-oxo-octanoyl)-L-homoserine lactone (no absolute correlation between genomospecies of P. syringae and their ability to produce NAHL could be found). Six strains of fluorescent pseudomonads, belonging to the species P. chlororaphis, P. fluorescens, and P. putida, isolated from the plant rhizosphere produced different types of NAHL. In contrast, none of the strains isolated from soil samples were shown to produce NAHL. The gene encoding the NAHL synthase in P. syringae pv. maculicola was isolated by complementation of an NAHL-deficient Chromobacterium mutant. Sequence analysis revealed the existence of a luxI homologue that we named psmI. This gene is sufficient to confer NAHL synthesis upon its bacterial host and has strong homology to psyI and ahlI, two genes involved in NAHL production in P. syringae pv. tabaci and P. syringae pv. syringae, respectively. We identified another open reading frame that we termed psmR, transcribed convergently in relation to psmI and partly overlapping psmI; this gene encodes a putative LuxR regulatory protein. This gene organization, with luxI and luxR homologues facing each other and overlapping, has been found so far only in the enteric bacteria Erwinia and Pantoea and in the related species P. syringae pv. tabaci.

Effect of crop rotation and soil cover on alteration of the soil microflora generated by the culture of transgenic plants producing opines.

The culture of transgenic Lotus corniculatus plants producing opines, which are bacterial growth substrates, leads to the selection of rhizospheric bacteria able to utilize these substrates. We have investigated the fate of the opine-utilizing community over time under different experimental conditions following elimination of selective pressure exerted by the transgenic plants. These plants were removed from the soil, which was either left unplanted or replanted with wild-type L. corniculatus or wheat plants. The density of opine-utilizing bacteria in the fallow soils remained essentially unchanged throughout the experiment, regardless of the soil of origin (soil planted with wild-type or transgenic plants). When wild-type Lotus plants were used to replace their transgenic counterparts, only the bacterial populations able to utilize the opines were affected. Long-term changes affecting the opine-utilizing bacterial community on Lotus roots was dependent upon the opine studied. The concentration of nopaline utilizers decreased, upon replacement of the transgenic plants, to a level similar to that of normal plants, while the concentration of mannopine utilizers decreased to levels intermediate between transgenic and normal plants. These data indicate that: (i) the opine-utilizing bacterial populations can be controlled in the rhizosphere via plant-exudate engineering; (ii) the interaction between the engineered plants and their root-associated micro-organisms is transgene specific; and (iii) alterations induced by the cultivation of transgenic plants may sometimes be persistent. Furthermore, opine-utilizing bacterial populations can be controlled by crop rotation. Therefore, favouring the growth of a rhizobacterium of agronomic interest via an opine-based strategy appears feasible.

Characterization of plasmid-borne and chromosome-encoded traits of Agrobacterium biovar 1, 2, and 3 strains from France.

We collected 111 Agrobacterium isolates from galls of various origins (most of them from France) and analyzed both their plasmid-borne and chromosome-encoded traits. Phenotypic analysis of these strains allowed their classification in three phena which exactly matched the delineation of biovars 1, 2, and 3. A fourth phenon was identified which comprises three atypical strains. The phenotypic analysis has also allowed us to identify 12 additional characteristics which could be used to identify the three biovars of Agrobacterium. Our results also suggest that biovar 1 and 2 represent distinct species. Analysis of plasmid-borne traits confirmed that tartrate utilization is a common feature of biovar 3 strains (now named Agrobacterium vitis) and of Agrobacterium grapevine strains in general. Among pathogenic strains of Agrobacterium, several exhibited unusual opine synthesis and degradation patterns, and one strain of biovar 3 induced tumors containing vitopine and a novel opine-like molecule derived from putrescine. We have named this compound ridéopine.

Lupine leghemoglobin I: expression in transgenic Lotus and tobacco tissues.

The proximal parts of the promoters of the genes for symbiotic-type hemoglobins are generally conserved, but the promoter of the lbI gene of lupine (LulbI) shows some unusual structural features. It lacks typical organ-specific elements characteristic of all the leghemoglobin gene promoters described thus far. We have analysed its functional activity in transgenic Lotus corniculatus. A fusion construct between the lbI promoter and the GUS reporter gene was expressed mainly in the central zone of the root nodule, but the product was also detected in the non-nodule root zone and in roots in tissue culture. In roots of transgenic tobacco, the activity of the promoter was only 24% lower than in Lotus nodules. LulbI promoter activity was also detected in tobacco leaves. Lupine hemoglobin I has a higher sequence identity to symbiotic-type hemoglobins and thus it groups within the "Class II" hemoglobins.

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.

Auxin production is a common feature of most pathovars of Pseudomonas syringae.

We investigated indole-3-acetic acid (IAA) production by 57 pathovars of Pseudomonas syringae and related species. Most of those analyzed produced IAA, especially in the presence of tryptophan. Eight strains produced high IAA concentrations in the absence of Trp. The iaaM and iaaH genes of P. savastanoi pv. savastanoi were detected in a limited number of strains only, including the eight above-mentioned strains. Thus, IAA synthesis in most assayed strains of P. syringae and related species does not involve genes highly similar to iaaM and iaaH. In contrast, the iaaL gene encoding an IAA-lysine synthase was detected in most pathovars, and was often found on plasmids.

A T-DNA gene required for agropine biosynthesis by transformed plants is functionally and evolutionarily related to a Ti plasmid gene required for catabolism of agropine by Agrobacterium strains.

The mechanisms that ensure that Ti plasmid T-DNA genes encoding proteins involved in the biosynthesis of opines in crown gall tumors are always matched by Ti plasmid genes conferring the ability to catabolize that set of opines on the inducing Agrobacterium strains are unknown. The pathway for the biosynthesis of the opine agropine is thought to require an enzyme, mannopine cyclase, coded for by the ags gene located in the T(R) region of octopine-type Ti plasmids. Extracts prepared from agropine-type tumors contained an activity that cyclized mannopine to agropine. Tumor cells containing a T region in which ags was mutated lacked this activity and did not contain agropine. Expression of ags from the lac promoter conferred mannopine-lactonizing activity on Escherichia coli. Agrobacterium tumefaciens strains harboring an octopine-type Ti plasmid exhibit a similar activity which is not coded for by ags. Analysis of the DNA sequence of the gene encoding this activity, called agcA, showed it to be about 60% identical to T-DNA ags genes. Relatedness decreased abruptly in the 5' and 3' untranslated regions of the genes. ags is preceded by a promoter that functions only in the plant. Expression analysis showed that agcA also is preceded by its own promoter, which is active in the bacterium. Translation of agcA yielded a protein of about 45 kDa, consistent with the size predicted from the DNA sequence. Antibodies raised against the agcA product cross-reacted with the anabolic enzyme. These results indicate that the agropine system arose by a duplication of a progenitor gene, one copy of which became associated with the T-DNA and the other copy of which remained associated with the bacterium.

Genetically engineered plants producing opines alter their biological environment.

Little is known about the consequences of releasing genetically engineered plants (GEP) into the environment. Using opine-producing GEP, we show that transgenic plants alter their biological environment, more precisely the root-associated bacterial populations. The alterations were both transgene-specific and target population-specific. Therefore, assessment studies on the introduction of a given transgene into a GEP will be valid on the given transgene. Evidence of any transgene-associated biological effect will depend on the determination of the pertinent target populations, the identification of which is a key step of such studies.

Somatic excision of the Ac transposable element in transgenic Arabidopsis thaliana after 5-azacytidine treatment.

We have introduced the maize Ac transposable element in Arabidopsis thaliana and found that after three selfing generations, the element is immobile and extensively methylated. Moreover, the nopaline synthase (nos) gene present on the same transferred T-DNA, was active early after transformation and regeneration, but inactive in most of the S1 progeny. We used 5-azacytidine (5AzaC) to determine whether a reduction in the methylation would affect both Ac transposition and expression of the nos gene. After treatment with 5AzaC doses from 0.3 mM to 1.0 mM, approximately 25% of the plants produced detectable amounts of nopaline, indicating that the nos gene was reactivated. Using the polymerase chain reaction (PCR) to detect the empty donor site left by Ac transposition, we demonstrated that 5AzaC also activates Ac excision in the transgenic plants. Approximately 13% of the 5AzaC treated plants (doses from 0.1 mM to 1.0 mM) were shown to have empty donor sites due to Ac excision. None of the plants cultivated in the absence of 5AzaC showed evidence for Ac transposition or reactivation of the nos gene. Further analysis using Southern blot indicate that some demethylation occurred in the genome of individual plants. These results may represent demethylation in few cells during development which may be sufficient to reactivate in these cells the expression of the nos and Ac transposase transgenes, the latter promoting Ac transposition in somatic cells.

The use of digoxigenin-labelled probes to detect DNA sequences specific for plant pathogenic bacteria.

Southern blot hybridization is a valuable method in the assessment of the pathogenicity of bacterial strains or isolates. It is also a powerful tool for the demonstration of the presence of foreign DNA sequences in the genome of genetically-engineered plant cells. In this respect, cold, digoxigenin-labelled DNA probes can be used in place of classical radioactive probes, whether hybridizations are performed on bacterial genomic or plasmidic DNA, or on plant genomic DNA. The versatility of this cold labelling makes it suitable for the detection of unique bacterial genomic or plasmid sequences, even though these are located on large plasmids. The sensitivity of this cold probe technique also permits the detection of subpicogram quantities of DNA in plant genomic preparations. Their long term storage stability allows them to be frequently re-used over long periods of time, making this technique quite cost efficient.

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.