Martha E. Trujillo

Nodulation of Lupinus albus by strains of Ochrobactrum lupini sp. nov.

ABSTRACT The nodulation of legumes has for more than a century been considered an exclusive capacity of a group of microorganisms commonly known as rhizobia and belonging to the α-Proteobacteria. However, in the last 3 years four nonrhizobial species, belonging to α and β subclasses of the Proteobacteria, have been described as legume-nodulating bacteria. In the present study, two fast-growing strains, LUP21 and LUP23, were isolated from nodules of Lupinus honoratus. The phylogenetic analysis based on the 16S and 23S rRNA gene sequences showed that the isolates belong to the genus Ochrobactrum. The strains were able to reinfect Lupinus plants. A plasmid profile analysis showed the presence of three plasmids. The nodD and nifH genes were located on these plasmids, and their sequences were obtained. These sequences showed a close resemblance to the nodD and nifH genes of rhizobial species, suggesting that the nodD and nifH genes carried by strain LUP21T were acquired by horizontal gene transfer. A polyphasic study including phenotypic, chemotaxonomic, and molecular features of the strains isolated in this study showed that they belong to a new species of the genus Ochrobactrum for which we propose the name Ochrobactrum lupini sp. nov. Strain LUP21T (LMG 20667T) is the type strain.

Biodiversity of Bradyrhizobia Nodulating Lupinus spp.

The genetic structure of Bradyrhizobium isolates recovered from three Lupinus species (Lupinus campestris, Lupinus montanus, and Lupinus exaltatus) grown in Mexico was examined. Among 41 Bradyrhizobium isolates, 18 electrophoretic types (ETs) were distinguished by multilocus enzyme electrophoresis of five metabolic enzymes. The mean genetic diversity, 0.64, indicated that there was great genetic diversity in the population sampled. Most isolates (63%) fell into two closely related clusters (clusters I and II) and were the types most frequently isolated from the root nodules of L. montanus and L. campestris. ET cluster III isolates were frequent nodule occupants of L. exaltatus. The isolates also were assigned to three main groups by using Curie point pyrolysis mass spectrometry. In general, the multilocus enzyme electrophoretic data and pyrolysis mass spectrometric data agreed. We determined the 16S rRNA sequences of representative Lupinus isolates and of Bradyrhizobium japonicum USDA 6T and found that the lupine isolates were highly related to the B. japonicum type strain, although not all B. japonicum type strains (subcultures maintained in different bacterial collections) had identical small-subunit rRNA.

The genus Micromonospora is widespread in legume root nodules: the example of Lupinus angustifolius

Our current knowledge of plant-microbe interactions indicate that populations inhabiting a host plant are not restricted to a single microbial species but comprise several genera and species. No one knows if communities inside plants interact, and it has been speculated that beneficial effects are the result of their combined activities. During an ecological study of nitrogen-fixing bacterial communities from Lupinus angustifolius collected in Spain, significant numbers of orange-pigmented actinomycete colonies were isolated from surface-sterilized root nodules. The isolates were analysed by BOX-PCR fingerprinting revealing an unexpectedly high genetic variation. Selected strains were chosen for 16S rRNA gene sequencing and phylogenetic analyses confirmed that all strains isolated belonged to the genus Micromonospora and that some of them may represent new species. To determine the possibility that the isolates fixed atmospheric nitrogen, chosen strains were grown in nitrogen-free media, obtaining in some cases, significant growth when compared with the controls. These strains were further screened for the presence of the nifH gene encoding dinitrogenase reductase, a key enzyme in nitrogen fixation. The partial nifH-like gene sequences obtained showed a 99% similarity with the sequence of the nifH gene from Frankia alni ACN14a, an actinobacterium that induces nodulation and fixes nitrogen in symbiosis with Alnus. In addition, in situ hybridization was performed to determine if these microorganisms inhabit the inside of the nodules. This study strongly suggests that Micromonospora populations are natural inhabitants of nitrogen-fixing root nodules.

Micromonospora pisi sp. nov., isolated from root nodules of Pisum sativum.

A novel actinomycete, designated strain GUI 15(T), isolated from the root nodules of a Pisum sativum plant was characterized taxonomically by using a polyphasic approach. The 16S rRNA gene sequence of strain GUI 15(T) showed highest similarity to Micromonospora pattaloongensis TJ2-2(T) (98.7 %) and Polymorphospora rubra TT 97-42(T) (98.5 %). Phylogenetic analysis based on the gyrase B gene also supported the close relationship of these three strains, but indicated that strain GUI 15(T) should be assigned to the genus Micromonospora. Chemotaxonomic results confirmed the position of the isolate in the genus Micromonospora, but revealed differences at the species level. The novel strain could be distinguished from recognized Micromonospora species by using a combination of physiological and biochemical tests. Based on these observations, strain GUI 15(T) is considered to represent a novel species of the genus Micromonospora, for which the name Micromonospora pisi sp. nov. is proposed. The type strain is GUI 15(T) (=DSM 45175(T)=LMG 24546(T)).

Biodiversity of populations of phosphate solubilizing rhizobia that nodulates chickpea in different Spanish soils

Within rhizobia, two species nodulating chickpea, Mesorhizobium ciceri and Mesorhizobium mediterraneum, are known as good phosphate solubilizers. For this reason, we have analysed the ability to solubilize phosphate of a wide number of strains isolated from Cicer arietinum growing in several soils in Spain. The aim of this work was to analyse microbial populations nodulating chickpea, that are able to solubilize phosphates, using molecular techniques. In the present work we analyzed 19 strains isolated from effective nodules of C. arietinum growing in three soils from the North of Spain. Nineteen strains showed ability to solubilize phosphate in YED-P medium. These strains were separated into 4 groups according to the results obtained by 879F-RAPD fingerprinting. The 16S rDNA sequencing of a representative strain from each group allowed the identification of strains as belonging to the genus Mesorhizobium. Strains from groups I and II showed a 99.4% and 99.2% similarity with M. mediterraneum UPM-CA142T, respectively. The strains from group III were related to M. tianshanense USDA 3592T at a 99.4% similarity level. Finally, the strain from group IV was related to M. ciceri USDA 3383T with a 99.3% similarity. The LMW RNA profiles confirmed these results. Strains from groups I and II showed an identical LMW RNA profile to that of M. mediterraneum UPM-CA142T; the profile of strains from group III was identical to that of M.␣tianshanense USDA 3592T and the profile of strains from group IV was identical to that of M. ciceri USDA 3383T. Different 879F-RAPD patterns were obtained for strains of the group I, group II and the M.␣mediterraneum type strain (UPM-CA142T). The 879-RAPD patterns obtained for group III also differed from the pattern shown by M.tianshanense USDA 3592T. Finally, the patterns between group IV and M. ciceri USDA 3383T were also different. These results suggest that groups I and II may be subspecies of M. mediterraneum, group III a subspecies of M. tianshanense and group IV a subspecies of M. ciceri. Nevertheless, more studies are needed to establish the taxonomic status of strains isolated in this study.

Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes

Advancement of DNA sequencing technology allows the routine use of genome sequences in the various fields of microbiology. The information held in genome sequences proved to provide objective and reliable means in the taxonomy of prokaryotes. Here, we describe the minimal standards for the quality of genome sequences and how they can be applied for taxonomic purposes.

Introducing a digital protologue: a timely move towards a database-driven systematics of archaea and bacteria

It is self-evident that contemporary biological science has become a database-driven endeavour, ranging from databases serving broad communities (such as Genbank) to those that are more specialised. We have argued previously (Rosselló-Móra 2012; Sutcliffe et al. 2012) that it would greatly benefit Archaeal and Bacterial systematics to create an iterative taxonomic database of Archaeal and Bacterial species, which would provide a summative and evolving repository for information on species characteristics, such as those typically found in the protologues of descriptions of novel genera and species. Indeed, such a database would be a welcome complement to MycoBank, a comparable initiative in fungal taxonomy (Robert et al. 2013), and important databases of microbial nomenclature and identification such as LPSN, LTP and EzTaxon (Yarza et al. 2010; Parte 2014; Yoon et al. 2017). As Editors of Antonie van Leeuwenhoek and Systematic and Applied Microbiology, we are responsible for two journals that effectively publish the descriptions of significant numbers of Archaeal and Bacterial species. Given the importance of getting a database established, we have therefore developed a ‘digital protologue’ database (DPD) to accompany descriptions of novel Archaeal and Bacterial taxa published in these two journals. Having been tested by volunteer authors, we believe the DPD has good functionality and fitness for purpose as a repository for taxonomic data, and we expect to refine and improve its design and interactivity in response to feedback from users. Significant features include accession numbers to relevant sequence databases (which will become still more important as whole genome sequences increasingly accompany descriptions of novel microbial taxa) and the generation of unique TaxoNumbers for each entry (which can also be cited and used for microattribution purposes). Moreover, content is exportable in a tabulated format that can be used as Online Supplementary material to accompany published material. At present the database is focussed on descriptions of Archaeal and Bacterial species although options are available to create entries for the description of genera and Candidatus taxa. As the current version will no doubt evolve, we encourage all users to feed-back to the editors for improvements. R. Rosselló-Móra Marine Microbiology Group, Department of Ecology and Marine Resources, Institut Mediterrani d’Estudis Avançats (CSIC-UIB), E-07190 Esporles, Balearic Islands, Spain

Road map of the phylum Actinobacteria

This revised road map and the resulting taxonomic outline update the previous versions of Garrity and Holt (2001) and Garrity et al. (2005) with the description of additional taxa and new phylogenetic analyses. While the road map seeks to be complete for all taxa validly named prior to 1 January 2008, some taxa described after that date are included.

A call to arms for systematists: revitalising the purpose and practises underpinning the description of novel microbial taxa

Prokaryotic systematics is a fundamentally important discipline that provides a framework for the activities of all microbiologists. Here we propose that the field has become mired in a sea of perceived rules and regulations, many of which stipulate what is considered ‘sufficient’ for the phenotypic characterisation of novel prokaryotic taxa. Importantly, we argue also that the principles and practise of prokaryotic systematics have not yet fully embraced the revolution in biological understanding that has occurred through the availability of huge numbers of whole genome sequences. We therefore propose that a significant reappraisal of the procedures used to describe novel prokaryotic taxa is needed, including the likely introduction of new publication formats. Urgent action is needed to revitalise the practise of prokaryotic systematics in order to maintain this discipline as an attractive career choice for twenty first century life scientists.

Diversity of Micromonospora strains isolated from nitrogen fixing nodules and rhizosphere of Pisum sativum analyzed by multilocus sequence analysis

It was recently reported that Micromonospora inhabits the intracellular tissues of nitrogen fixing nodules of the wild legume Lupinus angustifolius. To determine if Micromonospora populations are also present in nitrogen fixing nodules of cultivated legumes such as Pisum sativum, we carried out the isolation of this actinobacterium from P. sativum plants collected in two man-managed fields in the region of Castilla and León (Spain). In this work, we describe the isolation of 93 Micromonospora strains recovered from nitrogen fixing nodules and the rhizosphere of P. sativum. The genomic diversity of the strains was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). Forty-six isolates and 34 reference strains were further analyzed using a multilocus sequence analysis scheme developed to address the phylogeny of the genus Micromonospora and to evaluate the species distribution in the two studied habitats. The MLSA results were evaluated by DNA-DNA hybridization to determine their usefulness for the delineation of Micromonospora at the species level. In most cases, DDH values below 70% were obtained with strains that shared a sequence similarity of 98.5% or less. Thus, MLSA studies clearly supported the established taxonomy of the genus Micromonospora and indicated that genomic species could be delineated as groups of strains that share > 98.5% sequence similarity based on the 5 genes selected. The species diversity of the strains isolated from both the rhizosphere and nodules was very high and in many cases the new strains could not be related to any of the currently described species.