Heavy metal tolerant Metalliresistens boonkerdii gen. nov., sp. nov., a new genus in the family Bradyrhizobiaceae isolated from soil in Thailand

Abstract

Bacterial strains from inoculated soybean field soil in Thailand were directly isolated using Bradyrhizobium japonicum selective medium (BJSM), on the basis of Zn²⁺ and Co²⁺ resistance of B. japonicum and B. elkanii. The isolates were classified into symbiotic and non-symbiotic groups by inoculation assays and Southern hybridization of nod and nif genes. In this study, a nearly full-length 16S rRNA gene sequence showed that the non-symbiotic isolates were more closely related to members of Rhodopseudomonas and to a number of uncultured bacterial clones than to members of Bradyrhizobium. Therefore, a polyphasic study was performed to determine the taxonomic positions of four representatives of the non-symbiotic isolates. Multilocus phylogenetic analysis of individual genes and a combination of the 16S rRNA and three housekeeping genes (atpD, recA and glnII) supported the placement of the non-symbiotic isolates in a different genus. The ability of heavy metal resistance in conjunction with phenotypic analyses, including cellular fatty acid content and biochemical characteristics, showed that the non-symbiotic isolates were differentiated from the other related genera in the family Bradyrhizobiaceae. Therefore, the non-symbiotic isolates represented a novel genus and species, for which the name Metalliresistens boonkerdii gen. nov., sp. nov. is proposed. The type strain is NS23 (= NBRC 106595^T = BCC 40155^T).

Introduction

Rhizobia are nitrogen-fixing bacteria that form root nodules on legume plants. All currently known rhizobia are in the phylum Proteobacteria. Most of the rhizobia species are in the class Alphaproteobacteria, which include Rhizobium, Mesorhizobium, Ensifer, Bradyrhizobium, Azorhizobium, Phyllobacterium, Ochrobactrum, Devosia, and Methylobacterium [8], [10], [15], [17]. There are also nodule-forming Betaproteobacteria species in the family Burkholderiaceae [2], [7], [35]. Methods have been developed to allow the direct isolation of rhizobia from field soil, and occurrences of non-symbiotic rhizobia have been observed in many studies. For example, non-symbiotic strains of R. leguminosarum, R. etli and M. loti have been reported as a significant component of rhizobial populations in the environment [27], [46], [48], [49], [53]. These non-symbiotic bacteria failed to nodulate legume plants due to the lack of symbiotic genes. It has been hypothesized that the non-symbiotic bacteria seem to be saprophytic with the capacity of becoming symbiotic upon acquisition of symbiotic genes [39], [54].

The family Bradyrhizobiaceae consists of nine genera, which are Bradyrhizobium, Afipia, Agromonas, Blastobacter, Nitrobacter, Oligotropha, Rhodopseudomonas, Rhodoblastus, and Bosea. Sequence analysis of the 16S rDNA region has revealed a very close relationship between these bacteria [42], [47], [57], [63], leading to caution in the taxonomy and systematics of this group [46], [59]. In the case of the genus Bradyrhizobium, for example, B. elkanii is clearly a separate species from Bradyrhizobium japonicum and B. liaoningense, and members of B. japonicum are phylogenetically closer to the other non-symbiotic genera (Rhodopseudomonas, Afipia and Nitrobacter) than to B. elkanii, based on 16S rDNA similarity [46], [64]. Rhodopseudomonas palustris is a phototrophic purple non-sulfur bacterium and, based on 16S rRNA sequence analysis, it is phylogenetically close to nodule-forming non-phototrophic B. japonicum [65], [67], as well as stem-nodulating phototrophic Bradyrhizobium sp. strains BTAi1 and ORS278 [12], [16], [19], [67], [68]. The fact that other genera (Afipia, Agromonas, Blastobacter, Nitrobacter and Rhodopseudomonas) are phylogenetically closer to B. japonicum than to B. elkanii leaves the current classification of Bradyrhizobium looking rather unsatisfactory. 16S rRNA gene sequencing serves as a gold standard for bacterial species determination [52]; however, it has been argued that closely related species cannot always be distinguished because of the high level of sequence conservation [32]. Trees based on a single gene do not always reflect organismal phylogeny because of possible horizontal gene transfer or recombination and variable mutation rates. The use of information from the comparison and combination of multiple genes can give a global and reliable overview of interorganismal relationships [31]. Therefore, analysis of a combination of several housekeeping genes, such as atpD, recA, dnaK, glnII, gyrB and rpoB, have been used to elucidate taxonomic relationships among Bradyrhizobium species and other rhizobia [22], [31], [36], [40], [44], [60]. In addition, phenotypic and chemotaxonomic studies should be performed and extended in a comprehensive way.

In our previous study [38], 130 Bradyrhizobium isolates were obtained from heavily inoculated soybean field soil and uninoculated soybean free soil in Thailand. Bradyrhizobium species were expected to be directly isolated from soil by using B. japonicum selective medium (BJSM) [56]. The BJSM medium is based on the resistance of B. japonicum and B. elkanii strains to more than 40 μg mL⁻¹ of the metal ions Zn²⁺ and Co²⁺. The isolated strains were classified into symbiotic and non-symbiotic groups by inoculation assay on soybean (Glycine max), mungbean (Vigna radiata) and cowpea (V. unguiculata). The non-symbiotic isolates were found to be a significant component in soil from both fields. The non-symbiotic isolates failed to hybridize to nod and nif gene probes, indicating that they lacked the symbiotic genes. From partial 16S rRNA gene sequence analysis, a number of non-symbiotic isolates appeared to belong to the family Bradyrhizobiaceae. However, the non-symbiotic isolates could not be clearly affiliated, since they had 96–97% identity to different genera in the Bradyrhizobiaceae family, including Bradyrhizobium, Rhodopseudomonas and Afipia. Therefore, the objective of this present study was to characterize the non-symbiotic isolates in more detail by using a polyphasic approach, and to describe the novel isolates in the proposed new genus.