Genus Rathayibacter Research Articles

New lactate- and pyruvate-containing polysaccharide and rhamnomannan with xylose residues from the cell wall of Rathayibacter oskolensis VKM Ac-2121T

The cell wall of endophytic strain Rathayibacter oskolensis VKM Ac-2121T (family Microbacteriaceae, class Actinomycetes) was found to contain neutral and acidic glycopolymers. The neutral polymer is a block-type rhamnomannan partially should be substitutied by xylose residues, [→2)-α-[β-D-Xylp-(1 → 3)]-D-Manp-(1 → 3)-α-D-Rhap-(1→]∼30 [→2)-α-D-Manp-(1 → 3)-α-D-Rhap-(1→]∼45. The acidic polymer has branched chain, bearing lactate and pyruvate residues, →4)-α-D-[S-Lac-(2–3)-α-L-Rhap-(1 → 3)]-D-Manp-(1 → 3)-α-D-[4,6-R-Pyr]-D-Galp-(1 → 3)-β-D-Glcp-(1 →. The structures of both glycopolymers were not described in the Gram-positive bacteria to date. The glycopolymers were studied by chemical and NMR spectroscopic methods. The results of this study provide new data on diversity of bacterial glycopolymers and may prove useful in the taxonomy of the genus Rathayibacter and for understanding the molecular mechanisms of interaction between plants and plant endophytes.

Rathayibacter tanaceti sp. nov., a Novel Actinobacterium from Tanacetum vulgare Infested by Foliar Nematode Aphelenchoides sp.

Two novel yellow-pigmented, rod-shaped and non-motile coryneform actinobacteria, strains VKM Ac-2596T and VKM Ac-2761, were isolated from a plant Tanacetum vulgare (Asteraceae) infested by foliar nematode Aphelenchoides sp. The strains exhibited the highest 16S rRNA gene sequence similarities to Rathayibacter agropyri CA4T (99.71%), Rathayibacter rathayi DSM 7485T (99.65%) and Rathayibacter iranicus VKM Ac-1602T (99.65%). The pairwise average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between VKM Ac-2596T and VKM Ac-2671 towards the type strains of Rathayibacter species did not exceed 85.24% and 29.40%, respectively, that are well below the thresholds for species delineation. The target strains had key chemotaxonomic properties typical of the genus Rathayibacter, namely, the DAB-based peptidoglycan, rhamnose and mannose as the predominant sugars and a rhamnomannan in the cell, the major menaquinone MK-10 and fatty acids of iso-anteiso type, with a large proportion of anteiso-15:0. The strains showed clear differences from the recognized Rathayibacter species in several phenotypic characteristics, including the difference in the composition of cell wall glycopolymers. Based on the results obtained in this study and the data published previously, we provide a description of a new species, Rathayibacter tanaceti sp. nov., with DL-642T (= VKM Ac-2596T = LMG 33114T) as the type strain.

Rathayibacter rubneri sp. nov. isolated from Allium cepa var. Rijnsburger, an onion landrace.

The novel, aerobic, Gram-stain-positive, rod-shaped bacterial strain, ZW T2_19T, was isolated from an onion sample (Allium cepa var. Rijnsburger). Analyses of the 16S rRNA gene sequence revealed that ZW T2_19T represented a member of the genus Rathayibacter but may represent a novel species of this genus. Analyses of the whole draft genome sequences, i.e. digital DNA-DNA hybridisation (dDDH) and average nucleotide identity (ANI) of ZW T2_19T and all type strains of species of the genus Rathayibacter confirmed that ZW T2_19T represents a novel species of the genus Rathayibacter. The genome size of ZW T2_19T is 4.01 Mbp and the DNA G+C content is 71.8 mol%. Glucose, mannose, rhamnose and ribose were detected as whole-cell sugars of ZW T2_19T. The major respiratory quinone of ZW T2_19T is menaquinone MK-10, at 78.9 %. The detected peptidoglycan type in ZW T2_19T is a variant of type B2γ with {Gly} [l-diaminobutyric acid (l-DAB)/l-homoserine (l-Hse)] d-Glu-l-DAB. Polar lipids in ZW T2_19T consisted of one diphosphatidylglycerol, one phosphatidylglycerol, seven glycolipids, one phospholipid and one lipid. The fatty acid profile of ZW T2_19T predominantly consisted of anteiso-C15 : 0 (53 %), iso-C16 : 0 (21 %) and anteiso-C17 : 0 (18 %). In addition, API 20NE, API 50CH, API Coryne, API ZYM, antibiotic susceptibility, haemolysis and growth at different temperatures and with different supplements was investigated. On the basis of the results obtained using this polyphasic approach, including molecular, phenotypic and biochemical analyses, we propose the novel species Rathayibacter rubneri with the type and only strain ZW T2_19T (= DSM 114294T = LMG 32700T).

Rhamnomannans and Teichuronic Acid from the Cell Wall of Rathayibacter tritici VKM Ac-1603T.

The structures of three cell wall glycopolymers of the phytopathogen Rathayibacter tritici VKM Ac-1603T (family Microbacteriaceae, order Micrococcales, class Actinobacteria) were established by chemical methods and NMR spectroscopy. Polymer 1 is a branched rhamnomannan with the repeating unit →3)-α-[β-D-Xylp-(1→2)]-D-Manp-(1→2)-α-D-Rhap-(1→3)-α-D-Manp-(1→2)-α-D-Rhap-(1→; polymer 2 is a linear rhamnomannan with the repeating unit →2)-α-D-Manp-(1→2)-α-D-Rhap-(1→3)-α-D-Manp-(1→2)-α-D-Rhap-(1→; polymer 3 is a branched teichuronic acid containing monosaccharide residues GlcA, Gal, Man, and Glc at a 1 : 1 : 1 : 5 ratio (see the text for the structures). It has been demonstrated that representatives of four Rathayibacter species studied to date (R. tritici VKM Ac-1603T, R. iranicus VKM Ac-1602 T, R. toxicus VKM Ac-1600 and "Rathayibacter tanaceti" VKM Ac-2596) contain differing patterns of phosphate-free glycopolymers. At the same time, the above Rathayibacter strains have a common property - the presence of rhamnomannans with D-rhamnose. These rhamnomannans may be linear or branched and differing in the positions of glycosidic bonds and side substituents. The presence in the cell wall of rhamnomannans with D-rhamnose may serve as useful chemotaxonomic marker of the genus Rathayibacter.

Rhamnose-Containing Cell Wall Glycopolymers from Rathayibacter toxicus VKM Ac-1600 and "Rathayibacter tanaceti" VKM Ac-2596.

Structures of the cell wall glycopolymers from two representatives of the genus Rathayibacter were investigated using chemical, NMR spectroscopy, and optical methods. The R. toxicus VKM Ac-1600 strain contains two neutral glycopolymers - a linear rhamnomannan →2)-α-D-Rhap-(1→3)-α-D-Manp-(1→ and a branched polysaccharide containing in the repeating unit the residues of D-Manp, D-Glcp, and L-Rhap in the ratios of 2 : 4 : 1, respectively (the structure is presented in the text). The "Rathayibacter tanaceti" VKM Ac-2596 contains a rhamnomannan that is different from the above-described one by localization of glycosidic bonds on the residues of α-Rhap and α-Manp, i.e. →3)-α-D-Rhap (1→2)-α-D-Manp-(1→. The structures of all identified glycopolymers are described for the first time in actinobacteria. The data obtained make it possible to characterize representatives of the studied actinobacteria more fully and can be used to differentiate Rathayibacter species at the phenotype level.

Rathayibacter agropyri (non O'Gara 1916) comb. nov., nom. rev., isolated from western wheatgrass (Pascopyrum smithii).

Aplanobacter agropyri was first described in 1915 by O'Gara and later transferred to the genus Corynebacterium by Burkholder in 1948 but it was not included in the Approved Lists of Bacterial Names in 1980 and, consequently, is not recognized as a validly published species. In the 1980s, bacteria resembling Corynebacterium agropyri were isolated from plant samples stored at the Washington State Mycological Herbarium and from a diseased wheatgrass plant collected in Cardwell, Montana, USA. In the framework of this study, eight additional isolates were recovered from the same herbarium plant samples in 2011. The isolates are slow-growing, yellow-pigmented, Gram-stain-positive and coryneform. The peptidoglycan is type B2γ containing diaminobutyric acid, alanine, glycine and glutamic acid, the cell-wall sugars are rhamnose and mannose, the major respiratory quinone is MK-10, and the major fatty acids are anteiso-15 : 0, anteiso 17 : 0 and iso-16 : 0, all of which are typical of the genus Rathayibacter. Phylogenetic analysis of 16S rRNA gene sequences placed the strains in the genus Rathayibacter and distinguished them from the six other described species of Rathayibacter. DNA-DNA hybridization confirmed that the strains were members of a novel species. Based on phenotypic, chemotaxonomic and phylogenetic characterization, it appears that strains CA-1 to CA-12 represent a novel species, and the name Rathayibacter agropyri (non O'Gara 1916) comb. nov., nom. rev. is proposed; the type strain is CA-4T (=DSM 104101T;=ATCC TSD-78T).

Rathayibacter oskolensis sp. nov., a novel actinobacterium from Androsace koso-poljanskii Ovcz. (Primulaceae) endemic to the Central Russian Upland.

A rod-shaped, non-endospore-forming and non-motile bacterium, strain DL-329T, was isolated from the above-ground part of a plant, Androsace koso-poljanskii Ovcz. (Primulaceae), at the the State Natural Reserve 'Belogorie', Russia. On the basis of 16S rRNA gene sequence comparisons, the strain clustered with members of the genus Rathayibacter, showing the highest sequence similarity to Rathayibacter tritici (98.89 %), Rathayibacter rathayi (98.82 %) and Rathayibacter festucae (98.82 %). The DNA hybridization experiments demonstrated that strain DL-329T represents a separate genomic species. The results of comparative studies of physiological and chemotaxonomic characteristics, including cell-wall sugar patterns, polar lipid profiles, and the matrix-assisted laser desorption/ionization time-of-flight mass spectra of bacterial cells, allowed clear differentiation of VKM Ac-2121T from the recognized Rathayibacter species at the phenotypic level. Based on the data obtained, a new species, Rathayibacter oskolensis sp. nov., is proposed, with DL-329T (=VKM Ac-2121T=LMG 22542T) as the type strain.

Rathayibacter toxicus, Other Rathayibacter Species Inducing Bacterial Head Blight of Grasses, and the Potential for Livestock Poisonings.

Rathayibacter toxicus, a Select Agent in the United States, is one of six recognized species in the genus Rathayibacter and the best known due to its association with annual ryegrass toxicity, which occurs only in parts of Australia. The Rathayibacter species are unusual among phytopathogenic bacteria in that they are transmitted by anguinid seed gall nematodes and produce extracellular polysaccharides in infected plants resulting in bacteriosis diseases with common names such as yellow slime and bacterial head blight. R. toxicus is distinguished from the other species by producing corynetoxins in infected plants; toxin production is associated with infection by a bacteriophage. These toxins cause grazing animals feeding on infected plants to develop convulsions and abnormal gate, which is referred to as "staggers," and often results in death of affected animals. R. toxicus is the only recognized Rathayibacter species to produce toxin, although reports of livestock deaths in the United States suggest a closely related toxigenic species may be present. A closely related but undescribed species, Rathayibacter sp. EV, originally isolated from Ehrharta villosa var. villosa in South Africa, is suspected of producing toxin. Many of the diseases caused by Rathayibacter species occur in arid areas and the extracellular polysaccharide they produce is believed to aid in their survival between crops. For example, R. "agropyri" was isolated from infected plant material after being stored for 50 years in a herbarium. Similarly, the anguinid vectors associated with these bacteria form seed galls in infected plants and are capable of surviving for very long periods of time under dry conditions. The addition of R. toxicus to the list of Select Agents has raised concern over its potential introduction and a realization that current diagnostic methods are inadequate to distinguish among Rathayibacter species. In addition, little is known about the Rathayibacter species and their seed gall nematode vectors present in the United States.

Glycopolymers of the Cell Walls of Actinobacteria of the Genus Rathayibacter

Glycopolymers of the Cell Walls of Actinobacteria of the Genus Rathayibacter

Terminal Oxidases in Representatives of Different Genera of the Family Microbacteriaceae

The nature of terminal oxidases in representatives of four different genera of the family Microbacteriaceae was studied. It was found that the late-logarithmic and early-stationary cells of all of the investigated strains of the genera Plantibacter and Okibacterium contain the aa3-type cytochrome oxidase. Bacteria of the genera Rathayibacter and Agreia synthesize three oxidases, the bb3- and aa3-type cytochrome oxidases and nonheme cyanide-resistant oxidase, in proportions dependent on the cultivation conditions and the growth phase. Oxygen deficiency in the cultivation medium induces the synthesis of the bd-type oxidase in all of the microorganisms studied. The data obtained provide evidence that the type of terminal oxidases, along with the known chemotaxonomic characteristics, may serve to differentiate the genera of the family Microbacteriaceae at the phenotypic level.

Rathayibacter caricis sp. nov. and Rathayibacter festucae sp. nov., isolated from the phyllosphere of Carex sp. and the leaf gall induced by the nematode Anguina graminis on Festuca rubra L., respectively.

Two novel species, Rathayibacter caricis sp. nov. (type strain VKM Ac-1799T = UCM Ac-618T) and Rathayibacter festucae sp. nov. (type strain VKM Ac-1390T UCM Ac-619T), are proposed for two coryneform actinomycetes found in the phyllosphere of Carex sp. and in the leaf gall induced by the plant-parasitic nematode Anguina graminis on Festuca rubra L., respectively. The strains of the novel species are typical of the genus Rathayibacter in their chemotaxonomic characteristics and fall into the Rathayibacter 16S rDNA phylogenetic cluster. They belong to two separate genomic species and differ markedly from current validly described species of Rathayibacter at the phenotypic level. The most striking feature differentiating Rathayibacter caricis sp. nov. from other species of the genus is the presence of fucose in its cell wall and Rathayibacter festucae sp. nov. can be easily recognized among other yellow-pigmented rathayibacters because of its rose-orange-coloured colonies.

Diversity of grass-associated Microbacteriaceae isolated from the phyllosphere and litter layer after mulching the sward; polyphasic characterization of Subtercola pratensis sp. nov., Curtobacterium herbarum sp. nov. and Plantibacter flavus gen. nov., sp. nov.

A representative selection of coryneform bacteria, isolated from the phyllosphere of grasses and the litter layer after mulching the sward, was characterized by a polyphasic approach to clarify their taxonomic position in the family Microbacteriaceae, with particular reference to potentially plant-pathogenic bacteria. On the basis of 16S rDNA analysis, the isolates can be classified into six genotypes representing the genera Curtobacterium, Clavibacter, Subtercola and a subgroup, which was not affiliated to a known genus. One genotype, belonging to the genus Curtobacterium, had an identical 16S rDNA sequence to reference strains of the Curtobacterium flaccumfaciens pathovars. Another genotype, closely related to the potentially pathogenic Curtobacterium flaccumfaciens, could be distinguished from known species of the genus on the basis of phylogenetic and phenotypic characterization and is consequently proposed as a novel species, Curtobacterium herbarum sp. nov. (type strain P 420/07T DSM 14013T = LMG 19917T). Two genotypes assigned to Clavibacter showed a close relationship to Clavibacter michiganensis subsp. tessellarius, a pathogenic bacterium causing foliar lesions on wheat. A further genotype, which clustered clearly in the genus Subtercola by comparison of 16S rDNA sequences, showed a hitherto undescribed B-type of peptidoglycan containing the diagnostic diamino acids ornithine and 2,4-diaminobutyric acid, in the cell wall; this genotype is proposed as Subtercola pratensis sp. nov. (type strain P 229/10T = DSM 14246T = LMG 21000T). For one genotype, which formed a phylogenetically separate branch in the family of Microbacteriaceae showing chemotaxonomic similarities to the genus Rathayibacter, a novel genus, Plantibacter gen. nov., is proposed; the type species is Plantibacter flavus sp. nov. (type strain P 297/02T = DSM 14012T = LMG 19919T).

Taxonomic significance of 2,4-diaminobutyric acid isomers in the cell wall peptidoglycan of actinomycetes and reclassification of Clavibacter toxicus as Rathayibacter toxicus comb. nov.

An HPLC procedure which separates D- and L-amino acid isomers was applied to an analysis of peptidoglycan of 2,4-diaminobutyric acid (DAB)-containing actinomycetes. The cell wall peptidoglycans of species of the genera Agromyces, Clavibacter and Rathayibacter contain DAB and have been differentiated principally by their menaquinone profile. These peptidoglycans are known to be identical in structure, all being of the B2 gamma type, possessing both D- and L-DAB. The type strains of all the subspecies of Clavibacter michiganesis have D- and L-DAB in almost equal proportions in their cell wall peptidoglycan as previously reported. In contrast, the type strains of Clavibacter toxicus and all valid species of the genera Agromyces and Rathayibacter contain the L-isomer of DAB almost exclusively. This characteristic is in good agreement with phylogenetic analyses based on 16S rDNA sequences and menaquinone profiles. On the basis of these data, the transfer of Clavibacter toxicus to the genus Rathayibacter as Rathayibacter toxicus comb. nov. is proposed. The isomer profile of DAB is shown to be a good taxonomic marker to differentiate these genera.

Rathayibacter gen. nov., Including the Species Rathayibacter rathayi comb. nov., Rathayibacter tritici comb. nov., Rathayibacter iranicus comb. nov., and Six Strains from Annual Grasses

A new genus, Rathayibacter, is proposed to accommodate three species of gram-positive, aerobic, coryneform bacteria previously placed in the genus Clavibacter (Rathayibacter rathayi comb. nov., Rathayibacter tritici comb. nov., and Rathayibacter iranicus comb. nov.), as well as six strains that were isolated from annual cereal grasses, may be responsible for ryegrass toxicity, and are very similar to the recently described organism Clavibacter toxicus sp. nov. (I. T. Riley and K. M. Ophel, Int. J. Syst. Bacteriol. 42:64-68, 1992). The properties of members of the genus Rathayibacter include coryneform morphology, peptidoglycan based on 2,4-diaminobutyric acid (type B2γ), predominant menaquinones of the MK-10 type, and phosphatidylglycerol and diphosphatidylglycerol as basic polar lipids. The DNA base compositions range from 63 to 72 mol% G+C. The members of the new genus form a phenetic cluster distinct from Clavibacter spp. at a level of 71% (simple matching coefficient) and exhibit 7 to 9% DNA-DNA reassociation with strains of Clavibacter spp. In contrast to Clavibacter spp., most Rathayibacter strains are associated with nematodes belonging to the genus Anguina. The Rathayibacter species differ from species belonging to related genera (e.g., Clavibacter and Agromyces species) in the following characteristics: Menaquinone and whole-cell sugar compositions, results of lysozyme-sodium dodecyl sulfate test (which indicates differences in cell wall composition), ability to utilize a number carbon sources, resistance to bacteriocins of some Clavibacter spp., and other characteristics. The Rathayibacter species can be differentiated from each other by following characteristics: Presence or absence of xylose and galactose in the cell walls, fatty acid composition, ability to assimilate various sources of carbon and nitrogen, hydrolytic activity, tolerance to 5% NaCI and 0.03% potassium tellurite, susceptibility to iranicin, and absence or presence of plasmids of certain sizes. The type species of the genus Rathayibacter is R. rathayi. The type strains of the species are R. rathayi VKM Ac-1601 (= ICMP 2574), R. iranicus VKM Ac-1602 (= ICMP 3496), and R. tritici VKM Ac-1603 (= ICMP 2626).