MALDI-TOF mass spectrometry is a fast and reliable platform for identification and ecological studies of species from family Rhizobiaceae

Family Rhizobiaceae includes fast growing bacteria currently arranged into three genera, Rhizobium, Ensifer and Shinella, that contain pathogenic, symbiotic and saprophytic species. The identification of these species is not possible on the basis of physiological or biochemical traits and should be based on sequencing of several genes. Therefore alternative methods are necessary for rapid and reliable identification of members from family Rhizobiaceae. In this work we evaluated the suitability of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for this purpose. Firstly, we evaluated the capability of this methodology to differentiate among species of family Rhizobiaceae including those closely related and then we extended the database of MALDI Biotyper 2.0 including the type strains of 56 species from genera Rhizobium, Ensifer and Shinella. Secondly, we evaluated the identification potential of this methodology by using several strains isolated from different sources previously identified on the basis of their rrs, recA and atpD gene sequences. The 100% of these strains were correctly identified showing that MALDI-TOF MS is an excellent tool for identification of fast growing rhizobia applicable to large populations of isolates in ecological and taxonomic studies.     

Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria.

Diverse interactions between hosts and microbes are initiated by the detection of host-released chemical signals. Detection of these signals leads to altered patterns of gene expression that culminate in specific and adaptive changes in bacterial physiology that are required for these associations. This concept was first demonstrated for the members of the family Rhizobiaceae and was later found to apply to many other plant-associated bacteria as well as to microbes that colonize human and animal hosts. The family Rhizobiaceae includes various genera of rhizobia as well as species of Agrobacterium. Rhizobia are symbionts of legumes, which fix nitrogen within root nodules, while Agrobacterium tumefaciens is a pathogen that causes crown gall tumors on a wide variety of plants. The plant-released signals that are recognized by these bacteria are low-molecular-weight, diffusible molecules and are detected by the bacteria through specific receptor proteins. Similar phenomena are observed with other plant pathogens, including Pseudomonas syringae, Ralstonia solanacearum, and Erwinia spp., although here the signals and signal receptors are not as well defined. In some cases, nutritional conditions such as iron limitation or the lack of nitrogen sources seem to provide a significant cue. While much has been learned about the process of host detection over the past 20 years, our knowledge is far from being complete. The complex nature of the plant-microbe interactions makes it extremely challenging to gain a comprehensive picture of host detection in natural environments, and thus many signals and signal recognition systems remain to be described.     

Lipopolysaccharide structures from Agrobacterium and Rhizobiaceae species

This review reports and discusses the structural and the biological data available for the lipopolysaccharides from the Gram-negative bacterium Agrobacterium together with those of other related Rhizobiaceae species.     

Cyclic beta-glucans of members of the family Rhizobiaceae.

Cyclic beta-glucans are low-molecular-weight cell surface carbohydrates that are found almost exclusively in bacteria of the Rhizobiaceae family. These glucans are major cellular constituents, and under certain culture conditions their levels may reach up to 20% of the total cellular dry weight. In Agrobacterium and Rhizobium species, these molecules contain between 17 and 40 glucose residues linked solely by beta-(1,2) glycosidic bonds. In Bradyrhizobium species, the cyclic beta-glucans are smaller (10 to 13 glucose residues) and contain glucose linked by both beta-(1,6) and beta-(1,3) glycosidic bonds. In some rhizobial strains, the cyclic beta-glucans are unsubstituted, whereas in other rhizobia these molecules may become highly substituted with moieties such as sn-1-phosphoglycerol. To date, two genetic loci specifically associated with cyclic beta-glucan biosynthesis have been identified in Rhizobium (ndvA and ndvB) and Agrobacterium (chvA and chvB) species. Mutants with mutations at these loci have been shown to be impaired in their ability to grow in hypoosmotic media, have numerous alterations in their cell surface properties, and are also impaired in their ability to infect plants. The present review will examine the structure and occurrence of the cyclic beta-glucans in a variety of species of the Rhizobiaceae. The possible functions of these unique molecules in the free-living bacteria as well as during plant infection will be discussed.     

Genotypic and phenotypic similarity between bacteria of Brucella genus and Rhizobiaceae family as a basis for reconsideration of Brucella taxonomy

Accumulated data on assessment of genome of bacteria from Brucella genus and Rhizobiaceae family (results of sequencing, DNA-rRNA hybridization, 16S rRNA gene sequencing etc.) as well as their phenotypic characteristics (first of all, composition of cell fatty acids) were summarized. Data point to phylogenetic proximity of these bacteria and possibility to unite them in one Rhizobiaceae family together with the closest relatives of Brucella--first of all, with bacteria from Ochrobactrum genus). This seems to be more objective than recreation of Brucellaceae family (Rhizobiales order) with genera Brucella, Ochrobactrum and, possibly, others.     

Reiterated DNA sequences in Rhizobium and Agrobacterium spp.

Repeated DNA sequences are a general characteristic of eucaryotic genomes. Although several examples of DNA reiteration have been found in procaryotic organisms, only in the case of the archaebacteria Halobacterium halobium and Halobacterium volcanii [C. Sapienza and W. F. Doolittle, Nature (London) 295:384-389, 1982], has DNA reiteration been reported as a common genomic feature. The genomes of two Rhizobium phaseoli strains, one Rhizobium meliloti strain, and one Agrobacterium tumefaciens strain were analyzed for the presence of repetitive DNA. Rhizobium and Agrobacterium spp. are closely related soil bacteria that interact with plants and that belong to the taxonomical family Rhizobiaceae. Rhizobium species establish a nitrogen-fixing symbiosis in the roots of legumes, whereas Agrobacterium species is a pathogen in different plants. The four strains revealed a large number of repeated DNA sequences. The family size was usually small, from 2 to 5 elements, but some presented more than 10 elements. Rhizobium and Agrobacterium spp. contain large plasmids in addition to the chromosomes. Analysis of the two Rhizobium strains indicated that DNA reiteration is not confined to the chromosome or to some plasmids but is a property of the whole genome.     

Purification and partial characterization of the Rhizobium leguminosarum biovar viciae Ca2+-dependent adhesin, which mediates the first step in attachment of cells of the family Rhizobiaceae to plant root hair tips.

The Ca2+-dependent adhesin which mediates the first step in attachment of bacteria of the family Rhizobiaceae to plant root hair tips was isolated from the surface of Rhizobium leguminosarum biovar viciae cells; its ability to inhibit attachment of R. leguminosarum to pea root hair tips was used as a bioassay. Isolated adhesin was found to be able to inhibit attachment of both carbon-limited and manganese-limited R. leguminosarum cells. A multicolumn purification procedure was developed which resulted in pure adhesin, as judged from silver staining of isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electropherograms. The crucial step in purification was the elution of rhizobial proteins by a CaCl2 gradient from a hydroxyapatite matrix. The specific activity increased 1,250 times during purification. The isoelectric point of the adhesin was determined to be 5.1, and the molecular mass was 14 kilodaltons (kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By using gel filtration in the presence and absence of Ca2+, the molecular mass of the adhesin was determined to be 15 and 6 kDa, respectively. The adhesin appeared to be a calcium-binding protein. The purified adhesin inhibited attachment of various other rhizobia to pea root hair tips. Also, cell surface preparations of several other rhizobial strains, including Agrobacterium, Bradyrhizobium, and Phyllobacterium spp., showed adhesin activity, suggesting that a common plant receptor is used for attachment of Rhizobiaceae cells and that the adhesin is common among Rhizobiaceae. No attachment-inhibiting activity was detected in cell surface preparations from various other bacterial strains tested. Cell surface preparations from Sym or Ti plasmid-cured Rhizobium and Agrobacterium strains, respectively, also showed adhesin activity, indicating that Sym or Ti plasmid-borne genes are not required for the synthesis and biogenesis of the adhesin. The adhesin was also found to be involved in the attachment of rhizobia to the root hairs of various other legumes and nonlegume plants, including monocotyledonous ones. Since the adhesin appears to be specific for Rhizobiaceae and is Ca2+ dependent, we propose to designate it rhicadhesin. A more detailed model for rhizobial attachment to plant root hairs is discussed.     

Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp

Some properties of glutamine synthetase I (GSI) and GSII are described for a fast-growing Rhizobium sp. (Rhizobium trifolii T1), a slow-growing Rhizobium sp. (Rhizobium japonicum USDA 83), and Agrobacterium tumefaciens C58. GSII of the fast-growing Rhizobium sp. and GSII of the Agrobacterium sp. were considerably more heat labile than GSII of the slow-growing Rhizobium sp. As previously shown in R. japonicum 61A76, GSI became adenylylated rapidly in all species tested in response to ammonium. GSII activity disappeared within one generation of growth in two of the strains, but the disappearance of GSII activity required two generations in another. Isoactivity points for transferase assay, which were derived from the pH curves of adenylylated GSI and deadenylylated GSI, were approximately pH 7.8 for both R. trifolii and A. tumefaciens. No isoactivity point was found for R. japonicum under the standard assay conditions used. When the feedback inhibitor glycine was used to inhibit differentially the adenylylated GSI and deadenylylated GSI of R. japonicum, an isoactivity point was observed at pH 7.3. Thus, the transferase activity of GSI could be determined independent of the state of adenylation. A survey of 23 strains of bacteria representing 11 genera indicated that only Rhizobium spp. and Agrobacterium spp. contained GSII. Thus, this enzyme appears to be unique for the Rhizobiaceae.     

Identification of two glutamine synthetases in Agrobacterium.

Two distinct glutamine synthetases have been identified in Agrobacterium and in the fast-growing rhizobia. A limited survey indicates that GSII may be found only in the Rhizobiaceae family.