Species belonging to the genus of Pantoea are commonly isolated from plants, humans and the natural environment. The species of the genus are phenotypically closely related, making rapid identification of Pantoea strains to the species level difficult. Multilocus sequence analysis (MLSA) was evaluated as a means for rapid classification and identification of Pantoea strains. Four housekeeping genes, gyrB, rpoB, atpD and infB, were sequenced for strains assigned to the genus. Included in the study were (1) reference strains from the seven currently recognized species of Pantoea, (2) strains belonging to Brenner DNA groups II, IV and V, previously isolated from clinical samples and difficult to identify because of high phenotypic similarity to P. agglomerans or P. ananatis and (3) isolates from diseased Eucalyptus, maize and onion, assigned to the genus on the basis of phenotypic tests. Phylogenetic trees were constructed from the sequences of the four housekeeping genes. The ''core'' Pantoea species formed a cluster separate from the ''Japanese'' species which formed a tight cluster that included the genus Tatumella when the tree was based on concatenated sequences of the four genes. The MLSA data further suggested the existence of ten potential novel species, phylogenetically related to the currently recognized Pantoea species and the possible inclusion of Pectobacterium cypripedii in the genus Pantoea. When compared with DNA-DNA hybridization data, a good congruence was observed between both methods, with gyrB sequence data being the most consistent. In conclusion, MLSA of partial nucleotide sequences of the genes gyrB, rpoB, atpD and infB can be used for classification, identification and phylogenetic analyses of Pantoea strains. r 2008 Elsevier GmbH. All rights reserved. The GenBank/EMBL accession numbers for the sequences presented in this study are: EF988753-EF988838, EU145260-EU145275, EU344757-EU344760, FJ187830-FJ187834 (gyrB gene); EF988925-EF989010, EU145292-EU145307, EU344765-EU344768, FJ187840-FJ187844 (rpoB gene); EF988667-EF988752, EU145244-EU145259, EU344753-EU344756, FJ187825-FJ187829 (atpD gene); EF988839-EF988924, EU145276-EU145291, EU344761-EU344764, FJ187835-FJ187839 (infB gene) and EF688006-EF688012, EU216734-EU216737, EU344769-EU344770 (16S rRNA).Ã Corresponding author. Tel.: +2712 420 3934; fax: +2712 420 3960.E-mail address: teresa.coutinho@fabi.up.ac.za (T. Coutinho).Please cite this article as: C. Brady, et al., Phylogeny and identification of Pantoea species associated with plants, humans and the natural environment based on multilocus sequence analysis

The taxonomy of Enterobacter has a complicated history, with several species transferred to and from this genus. Classification of strains is difficult owing to its polyphyletic nature, based on 16S rRNA gene sequences. It has been previously acknowledged that Enterobacter contains species which should be transferred to other genera. In an attempt to resolve the taxonomy of Enterobacter, MLSA based on partial sequencing of protein-encoding genes (gyrB, rpoB, infB and atpD) was performed on the type strains and reference strains of Enterobacter, Cronobacter and Serratia species, as well as members of the closely related genera Citrobacter, Klebsiella, Kluyvera, Leclercia, Mangrovibacter, Raoultella and Yokenella. Phylogenetic analyses of the concatenated nucleotide sequences revealed that Enterobacter can be divided into five strongly supported MLSA groups, suggesting that the species should be reclassified into five different genera. Further support for this was provided by a concatenated amino acid tree, phenotypic characteristics and fatty acid profiles, enabling differentiation of the MLSA groups. Three novel genera are proposed: Lelliottia gen. nov., Pluralibacter gen. nov. and Kosakonia gen. nov. and the following new combinations: Lelliottia nimipressuralis comb. nov., Lelliottia amnigena comb. nov., Pluralibacter gergoviae comb. nov., Pluralibacter pyrinus comb. nov., Kosakonia cowanii comb. nov., Kosakonia radicincitans comb. nov., Kosakonia oryzae comb. nov., Kosakonia arachidis comb. nov., Cronobacter helveticus comb. nov. and Cronobacter pulveris comb. nov. Additionally, the novel epithet Cronobacter zurichensis nom. nov. is proposed for the reclassification of Enterobacter turicensis into the genus Cronobacter, as Cronobacter turicensis (Iversen et al., 2008) is already in use.

Pantoea ananatis infects both monocotyledonous and dicotyledonous plants. The symptoms are diverse depending on the host infected, and include leaf blotches and spots, die-back, and stalk, fruit and bulb rot. BIOLOGICAL CONTROL AGENT: Pantoea ananatis has both antifungal and antibacterial properties. These characteristics have the potential of being exploited by biological control specialists.

Burkholderia sensu lato is a large and complex group, containing pathogenic, phytopathogenic, symbiotic and non-symbiotic strains from a very wide range of environmental (soil, water, plants, fungi) and clinical (animal, human) habitats. Its taxonomy has been evaluated several times through the analysis of 16S rRNA sequences, concantenated 4–7 housekeeping gene sequences, and lately by genome sequences. Currently, the division of this group into Burkholderia, Caballeronia, Paraburkholderia, and Robbsia is strongly supported by genome analysis. These new genera broadly correspond to the various habitats/lifestyles of Burkholderia s.l., e.g., all the plant beneficial and environmental (PBE) strains are included in Paraburkholderia (which also includes all the N2-fixing legume symbionts) and Caballeronia, while most of the human and animal pathogens are retained in Burkholderia sensu stricto. However, none of these genera can accommodate two important groups of species. One of these includes the closely related Paraburkholderia rhizoxinica and Paraburkholderia endofungorum, which are both symbionts of the fungal phytopathogen Rhizopus microsporus. The second group comprises the Mimosa-nodulating bacterium Paraburkholderia symbiotica, the phytopathogen Paraburkholderia caryophylli, and the soil bacteria Burkholderia dabaoshanensis and Paraburkholderia soli. In order to clarify their positions within Burkholderia sensu lato, a phylogenomic approach based on a maximum likelihood analysis of conserved genes from more than 100 Burkholderia sensu lato species was carried out. Additionally, the average nucleotide identity (ANI) and amino acid identity (AAI) were calculated. The data strongly supported the existence of two distinct and unique clades, which in fact sustain the description of two novel genera Mycetohabitans gen. nov. and Trinickia gen. nov. The newly proposed combinations are Mycetohabitans endofungorum comb. nov., Mycetohabitans rhizoxinica comb. nov., Trinickia caryophylli comb. nov., Trinickia dabaoshanensis comb. nov., Trinickia soli comb. nov., and Trinickia symbiotica comb. nov. Given that the division between the genera that comprise Burkholderia s.l. in terms of their lifestyles is often complex, differential characteristics of the genomes of these new combinations were investigated. In addition, two important lifestyle-determining traits—diazotrophy and/or symbiotic nodulation, and pathogenesis—were analyzed in depth i.e., the phylogenetic positions of nitrogen fixation and nodulation genes in Trinickia via-à-vis other Burkholderiaceae were determined, and the possibility of pathogenesis in Mycetohabitans and Trinickia was tested by performing infection experiments on plants and the nematode Caenorhabditis elegans. It is concluded that (1) T. symbiotica nif and nod genes fit within the wider Mimosa-nodulating Burkholderiaceae but appear in separate clades and that T. caryophylli nif genes are basal to the free-living Burkholderia s.l. strains, while with regard to pathogenesis (2) none of the Mycetoh...

Bacterial isolates from oak trees in Spain and Britain, showing symptoms of bark canker and Acute Oak Decline (AOD), respectively, were examined by a polyphasic approach. Both 16S rRNA gene sequencing and multilocus sequence analysis (MLSA), based on partial sequences of gyrB, rpoB, infB and atpD genes, revealed that the isolates were separated into two genetic groups according to their origin. Their closest phylogenetic relative was Brenneria quercina, the causal agent of drippy nut disease of oak, which clustered distant to the other species of the genus Brenneria. MLSA data for species of the genera Brenneria, Pectobacterium, Dickeya, Erwinia, Pantoea and Samsonia confirmed the polyphyletic nature of the genus Brenneria and indicated synonymy of Dickeya dadantii and Dickeya dieffenbachiae. DNA-DNA hybridization experiments confirmed this synonymy and also revealed DNA-DNA relatedness values of 58-73 % between the new oak isolates and B. quercina. Phenotypic and/or chemotaxonomic methods allowed B. quercina and the two genetic groups of new oak isolates to be discriminated from other recognized species of the genus Brenneria and from members of the closely related genera Dickeya, Pectobacterium and Samsonia. Based on the data obtained, the following taxonomic proposals are made: (1)

BackgroundPantoea ananatis is found in a wide range of natural environments, including water, soil, as part of the epi- and endophytic flora of various plant hosts, and in the insect gut. Some strains have proven effective as biological control agents and plant-growth promoters, while other strains have been implicated in diseases of a broad range of plant hosts and humans. By analysing the pan-genome of eight sequenced P. ananatis strains isolated from different sources we identified factors potentially underlying its ability to colonize and interact with hosts in both the plant and animal Kingdoms.ResultsThe pan-genome of the eight compared P. ananatis strains consisted of a core genome comprised of 3,876 protein coding sequences (CDSs) and a sizeable accessory genome consisting of 1,690 CDSs. We estimate that ~106 unique CDSs would be added to the pan-genome with each additional P. ananatis genome sequenced in the future. The accessory fraction is derived mainly from integrated prophages and codes mostly for proteins of unknown function. Comparison of the translated CDSs on the P. ananatis pan-genome with the proteins encoded on all sequenced bacterial genomes currently available revealed that P. ananatis carries a number of CDSs with orthologs restricted to bacteria associated with distinct hosts, namely plant-, animal- and insect-associated bacteria. These CDSs encode proteins with putative roles in transport and metabolism of carbohydrate and amino acid substrates, adherence to host tissues, protection against plant and animal defense mechanisms and the biosynthesis of potential pathogenicity determinants including insecticidal peptides, phytotoxins and type VI secretion system effectors.ConclusionsP. ananatis has an ‘open’ pan-genome typical of bacterial species that colonize several different environments. The pan-genome incorporates a large number of genes encoding proteins that may enable P. ananatis to colonize, persist in and potentially cause disease symptoms in a wide range of plant and animal hosts.Electronic supplementary materialThe online version of this article (doi: 10.1186/1471-2164-15-404) contains supplementary material, which is available to authorized users.

A s new environments are explored and technological innovations improve tools for the characterization of microbial biodiversity, insights into bacterial and archaeal diversity are continually emerging 1,2 , including improved understanding of physiological capacity, ecology and evolution of organisms across the tree of life. These advances are based on both cultivation strategies 3,4 and cultivation-independent methods that directly access diversity using single-cell 5,6 or metagenomic sequencing 7-9 (Box 1). Though our ability to culture fastidious microorganisms is improving, success seems to vary depending on the environment. For example, the microbial diversity of host-associated systems such as the human microbiome 11,12 may be more amenable to cultivation compared to some environments such as soil. At present, it seems clear that most archaeal and bacterial diversity remains yet to be cultured 10,13. The reasons are many, but as demonstrated recently by the cultivation of a member of the Asgard archaea 14 , syntrophic interactions, slow growth and media optimization can present formidable challenges. Rules of prokaryotic nomenclature and current challenges Describing biodiversity and identifying organisms are the scientific goals of taxonomy. Taxonomy integrates classification and nomenclature to describe biological diversity. Classification circumscribes and ranks taxa, and nomenclature is the process of assigning names. The commonly used Linnaean nomenclatural system focuses on the recognition of species as the basic unit, which are included in taxa of successively higher ranks (genus, family, order, class and phylum). There is some flexibility on how to circumscribe microbial species using phylogenetic, genotypic and phenotypic data. Once a species is delineated, rules of nomenclature given in the International Code of Nomenclature of Prokaryotes (ICNP or 'the Code' 14 ; see Box 2) guide the creation and assignment of names. This is true of all codes of nomenclature that currently exist-prokaryotes, viruses, animals, algae, fungi and plants-in addition to separate codes for cultivated plants and plant associations. Roadmap for naming uncultivated Archaea and Bacteria The assembly of single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) has led to a surge in genome-based discoveries of members affiliated with Archaea and Bacteria, bringing with it a need to develop guidelines for nomenclature of uncultivated microorganisms. The International Code of Nomenclature of Prokaryotes (ICNP) only recognizes cultures as 'type material', thereby preventing the naming of uncultivated organisms. In this Consensus Statement, we propose two potential paths to solve this nomenclatural conundrum. One option is the adoption of previously proposed modifications to the ICNP to recognize DNA sequences as acceptable type material; the other option creates a nomenclatural code for uncultivated Archaea and Bacteria that could eventually be merged with the ICNP in the future. Regardless of the path taken, we b...

BackgroundThe Type VI secretion apparatus is assembled by a conserved set of proteins encoded within a distinct locus. The putative effector proteins Hcp and VgrG are also encoded within these loci. We have identified numerous distinct Type VI secretion system (T6SS) loci in the genomes of several ecologically diverse Pantoea and Erwinia species and detected the presence of putative effector islands associated with the hcp and vgrG genes.ResultsBetween two and four T6SS loci occur among the Pantoea and Erwinia species. While two of the loci (T6SS-1 and T6SS-2) are well conserved among the various strains, the third (T6SS-3) locus is not universally distributed. Additional orthologous loci are present in Pantoea sp. aB-valens and Erwinia billingiae Eb661. Comparative analysis of the T6SS-1 and T6SS-3 loci showed non-conserved islands associated with the vgrG and hcp, and vgrG genes, respectively. These regions had a G+C content far lower than the conserved portions of the loci. Many of the proteins encoded within the hcp and vgrG islands carry conserved domains, which suggests they may serve as effector proteins for the T6SS. A number of the proteins also show homology to the C-terminal extensions of evolved VgrG proteins.ConclusionsExtensive diversity was observed in the number and content of the T6SS loci among the Pantoea and Erwinia species. Genomic islands could be observed within some of T6SS loci, which are associated with the hcp and vgrG proteins and carry putative effector domain proteins. We propose new hypotheses concerning a role for these islands in the acquisition of T6SS effectors and the development of novel evolved VgrG and Hcp proteins.