The plant growth promoting model bacterium FZB42T was proposed as the type strain of B. amyloliquefaciens subsp. plantarum (Borriss et al., Int J Syst Evol Microbiol. 2011, vol. 61, 1786-801), but has been recently recognized as being synonymous to Bacillus velezensis due to phylogenomic analysis (Dunlap et al. Int J Syst Evol Microbiol. 2016, vol. 66, 1212-1217). However, until now, majority of publications consider plant-associated close relatives of FZB42 still as ´B. amyloliquefaciens´. Here, we reinvestigated the taxonomic status of FZB42 and related strains in its context to the free-living soil bacterium DSM7T, the type strain of B. amyloliquefaciens. We identified 66 bacterial genomes from the NCBI data bank with high similarity to DSM7T. Dendrograms based on complete rpoB nucleotide sequences and on core genome sequences, respectively, clustered into a clade consisting of three tightly linked branches: (1) B. amyloliquefaciens, (2) B. siamensis, and (3) a conspecific group containing the type strains of B. velezensis, B. methylotrophicus, and B. amyloliquefaciens subsp. plantarum. The three monophyletic clades shared a common mutation rate of 0.01 substitutions per nucleotide position, but were distantly related to B. subtilis (0.1 substitutions per nucleotide position). The tight relatedness of the three clusters was corroborated by TETRA, dDDH, ANI and AAI analysis of the core genomes, but dDDH and ANI values were found slightly below species level thresholds when B.amyloliquefaciens DSM7T genome sequence was used as query sequence. Due to these results, we propose that the B. amyloliquefaciens clade should be considered as a taxonomic unit above of species level, designated here as ´operational group B. amyloliquefaciens´ consisting of the soil borne B. amyloliquefaciens, and plant associated B. siamensis and B. velezensis, whose members are closely related and allow identifying changes on the genomic level due to developing the plant-associated life-style.

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Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis Form an "Operational Group B. amyloliquefaciens" within the B. subtilis Species Complex.

Understanding the evolutionary background of a bacterial isolate has applications for a wide range of research. However generating an accurate species phylogeny remains challenging. Reliance on 16S rDNA for species identification currently remains popular. Unfortunately, this widespread method suffers from low resolution at the species level due to high sequence conservation. Currently, there is now a wealth of genomic data that can be used to yield more accurate species designations via modern phylogenetic methods and multiple genetic loci. However, these often require extensive expertise and time. The Automated Multi-Locus Species Tree (autoMLST) was thus developed to provide a rapid ‘one-click’ pipeline to simplify this workflow at: https://automlst.ziemertlab.com. This server utilizes Multi-Locus Sequence Analysis (MLSA) to produce high-resolution species trees; this does not preform multi-locus sequence typing (MLST), a related classification method. The resulting phylogenetic tree also includes helpful annotations, such as species clade designations and secondary metabolite counts to aid natural product prospecting. Distinct from currently available web-interfaces, autoMLST can automate selection of reference genomes and out-group organisms based on one or more query genomes. This enables a wide range of researchers to perform rigorous phylogenetic analyses more rapidly compared to manual MLSA workflows.

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AutoMLST: an automated web server for generating multi-locus species trees highlighting natural product potential.

Bacillus velezensis FZB42, the model strain for Gram-positive plant-growth-promoting and biocontrol rhizobacteria, has been isolated in 1998 and sequenced in 2007. In order to celebrate these anniversaries, we summarize here the recent knowledge about FZB42. In last 20 years, more than 140 articles devoted to FZB42 have been published. At first, research was mainly focused on antimicrobial compounds, apparently responsible for biocontrol effects against plant pathogens, recent research is increasingly directed to expression of genes involved in bacteria-plant interaction, regulatory small RNAs (sRNAs), and on modification of enzymes involved in synthesis of antimicrobial compounds by processes such as acetylation and malonylation. Till now, 13 gene clusters involved in non-ribosomal and ribosomal synthesis of secondary metabolites with putative antimicrobial action have been identified within the genome of FZB42. These gene clusters cover around 10% of the whole genome. Antimicrobial compounds suppress not only growth of plant pathogenic bacteria and fungi, but could also stimulate induced systemic resistance (ISR) in plants. It has been found that besides secondary metabolites also volatile organic compounds are involved in the biocontrol effect exerted by FZB42 under biotic (plant pathogens) and abiotic stress conditions. In order to facilitate easy access to the genomic data, we have established an integrating data bank 'AmyloWiki' containing accumulated information about the genes present in FZB42, available mutant strains, and other aspects of FZB42 research, which is structured similar as the famous SubtiWiki data bank.

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Bacillus velezensis FZB42 in 2018: The Gram-Positive Model Strain for Plant Growth Promotion and Biocontrol.

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., Mycetohabitansrhizoxinica comb. nov., Trinickia caryophylli comb. nov., Trinickiadabaoshanensis comb. nov., Trinickia soli comb. nov., and Trinickiasymbiotica 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-a-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. caryophyllinif genes are basal to the free-living Burkholderia s.l. strains, while with regard to pathogenesis (2) none of the Mycetohabitans and Trinickia strains tested are likely to be pathogenic, except for the known phytopathogen T. caryophylli.

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Whole Genome Analyses Suggests that Burkholderia sensu lato Contains Two Additional Novel Genera (Mycetohabitans gen. nov., and Trinickia gen. nov.): Implications for the Evolution of Diazotrophy and Nodulation in the Burkholderiaceae.

Roseobacters are a diverse and globally abundant group of Alphaproteobacteria within the Rhodobacteraceae family. Recent studies and the cophenetic correlations suggest that the 16S rRNA genes are poor phylogenetic markers within this group. In contrast, the cophenetic correlation coefficients of the core-gene average amino acid identity (cAAI) and RpoC protein sequences are high and likely more predictive of relationships. A maximum-likelihood phylogenetic tree calculated from 53 core genes demonstrated that some of the current genera were either polyphyletic or paraphyletic. The boundaries of bacterial genera were redefined based upon the cAAI, the percentage of conserved proteins, and phenotypic characteristics and resulted in the following taxonomic proposals. Loktanella vestfoldensis, Loktanella litorea, Loktanella maricola, Loktanella maritima, Loktanella rosea, Loktanella sediminilitoris, Loktanella tamlensis, and Roseobacter sp. CCS2 should be reclassified into the novel genus Yoonia. Loktanella hongkongensis, Loktanella aestuariicola, Loktanella cinnabarina, Loktanella pyoseonensis, Loktanellasoe soekkakensis and Loktanella variabilis should be reclassified in the novel genus Limimaricola. Loktanella koreensis and Loktanella sediminum should be reclassified in the novel genus Cognatiyoonia. Loktanella marina should be reclassified in the novel genus Flavimaricola. Aestuariivita atlantica should be reclassified in the novel genus Pseudaestuariivita. Thalassobius maritima should be reclassified in the novel genus Cognatishimia. Similarly, Ruegeria mobilis, Ruegeria scottomollicae, Ruegeria sp. TM1040 and Tropicibacter multivorans should be reclassified in the genus Epibacterium. Tropicibacter litoreus and Tropicibacter mediterraneus should be reclassified in the genus Ruegeria. Thalassobius abyssi and Thalassobius aestuarii should be reclassified in the genus Shimia. Citreicella aestuarii, Citreicella manganoxidans, Citreicella marina, Citreicella thiooxidans, Pelagibaca bermudensis and Thiobacimonas profunda should be reclassified in the genus Salipiger. Nautella italica should be reclassified in the genus Phaeobacter. Because these proposals to reclassify the type and all others species of Citreicella, Nautella, Pelagibaca and Thiobacimonas, these genera are not used in this taxonomy.

Phylogenomic analyses of a clade within the roseobacter group suggest taxonomic reassignments of species of the genera Aestuariivita, Citreicella, Loktanella, Nautella, Pelagibaca, Ruegeria, Thalassobius, Thiobacimonas and Tropicibacter, and the proposal of six novel genera.

The rapidly increasing availability of microbial genome sequences has led to a growing demand for bioinformatics software tools that support the functional analysis based on the comparison of closely related genomes. By utilizing comparative approaches on gene level it is possible to gain insights into the core genes which represent the set of shared features for a set of organisms under study. Vice versa singleton genes can be identified to elucidate the specific properties of an individual genome. Since initial publication, the EDGAR platform has become one of the most established software tools in the field of comparative genomics. Over the last years, the software has been continuously improved and a large number of new analysis features have been added. For the new version, EDGAR 2.0, the gene orthology estimation approach was newly designed and completely re-implemented. Among other new features, EDGAR 2.0 provides extended phylogenetic analysis features like AAI (Average Amino Acid Identity) and ANI (Average Nucleotide Identity) matrices, genome set size statistics and modernized visualizations like interactive synteny plots or Venn diagrams. Thereby, the software supports a quick and user-friendly survey of evolutionary relationships between microbial genomes and simplifies the process of obtaining new biological insights into their differential gene content. All features are offered to the scientific community via a web-based and therefore platform-independent user interface, which allows easy browsing of precomputed datasets. The web server is accessible at http://edgar.computational.bio.

https://serval.unil.ch/resource/serval:BIB_8105EEB44781.P001/REF.pdf

Julian Kreis

Papers

EDGAR 2.0: an enhanced software platform for comparative gene content analyses.

EDGAR: A Versatile Tool for Phylogenomics