xenoGI 3: using the DTLOR model to reconstruct the evolution of gene families in clades of microbes.

To understand genome evolution in a group of microbes, we need to know the timing of events such as duplications, deletions and horizontal transfers. A common approach is to perform a gene-tree / species-tree reconciliation. While a number of software packages perform this type of analysis, none are geared toward a complete reconstruction for all families in an entire clade. Here we describe an update to the xenoGI software package which allows users to perform such an analysis using the newly developed DTLOR (duplication-transfer-loss-origin-rearrangement) reconciliation model starting from genome sequences as input.

Automating microbial taxonomy workflows with PHANTASM: phylogenomic analyses for the taxonomy and systematics of microbes.

The nucleotide sequences of 16S ribosomal RNA (rRNA) genes have been used to inform the taxonomic placement of prokaryotes for several decades. Whole-genome approaches can better resolve evolutionary relationships of organisms, but these analyses often require computational proficiencies that are uncommon among microbiologists. PHANTASM is a new tool capable of automating these workflows. This tool was designed to work for a wide range of prokaryotes and is the first example of an automated reconciliation of NCBI's Taxonomy database with that of the List of Prokaryotic names with Standing in Nomenclature (LPSN). In this study, we describe the workflow of PHANTASM and provide several examples of results generated by it. The source code is freely-available on GitHub. In order to facilitate the ease-of-access for researchers, PHANTASM is also available as a Docker image. While other tools exist to facilitate starting points for these analyses, PHANTASM provides users with a greater degree of control and produces outputs that can be used to make publication-quality figures.

Phylogenomic analyses of the Staphylococcaceae family suggest the reclassification of five species within the genus Staphylococcus as heterotypic synonyms, the promotion of five subspecies to novel species, the taxonomic reassignment of five Staphylococcus species to Mammaliicoccus gen. nov., and the formal assignment of Nosocomiicoccus to the family Staphylococcaceae.

Phylogenetic analyses based on 16S rRNA gene sequences of members of the family Staphylococcaceae showed the presence of para- and polyphyletic genera. This finding prompted a thorough investigation into the taxonomy of the Staphylococcaceae family by analysing their core genome phylogeny complemented with genome-based indices such as digital DNA-DNA hybridization, average nucleotide identity and average amino acid identity. The resulting data suggested the following proposals: Auricoccus indicus was reduced in taxonomic rank as a later heterotypic synonym of Abyssicoccus albus; Staphylococcus petrasii subsp. jettensis as a later heterotypic synonym of Staphylococcus petrasii subsp. petrasii; the unification of Staphylococcus aureus subsp. anaerobius and Staphylococcus aureus subsp. aureus as Staphylococcus aureus; the unification of Staphylococcus carnosus subsp. utilis and Staphylococcus carnosus subsp. carnosus as Staphylococcus carnosus; the unification of Staphylococcus saprophyticus subsp. bovis and Staphylococcus saprophyticus subsp. saprophyticus as Staphylococcus saprophyticus; Staphylococcus succinis subsp. casei as the novel species Staphylococcus casei; Staphylococcus schleiferi subsp. coagulans as the novel species Staphylococcus coagulans; Staphylococcus petrasii subsp. croceilyticus as the novel species Staphylococcus croceilyticus; Staphylococcus petrasii subsp. pragensis as the novel species Staphylococcus pragensis; Staphylococcus cohnii subsp. urealyticus as the novel species Staphylococcus urealyticus; the reassignment of Staphylococcus sciuri, Staphylococcus fleurettii, Staphylococcus lentus, Staphylococcus stepanovicii and Staphylococcus vitulinus to the novel genus Mammaliicoccus with Mammaliicoccus sciuri as the type species; and the formal assignment of Nosocomiicoccus as a member of the family Staphylococcaceae.

Sphingomonas palmae sp. nov. and Sphingomonas gellani sp. nov., endophytically associated phyllosphere bacteria isolated from economically important crop plants.

In this study, two endophytic bacterial strains designated JS21-1T and S6-262T isolated from leaves of Elaeis guineensis and stem tissues of Jatropha curcas respectively, were subjected for polyphasic taxonomic approach. On R2A medium, colonies of strains JS21-1T and S6-262T are orange and yellow, respectively. Phylogenetic analyses using 16S rRNA gene sequencing and whole-genome sequences placed the strains in distinct clades but within the genus Sphingomonas. The DNA G + C content of JS21-1T and S6-262T were 67.31 and 66.95%, respectively. Furthermore, the average nucleotide identity and digital DNA-DNA hybridization values of strains JS21-1T and S6-262T with phylogenetically related Sphingomonas species were lower than 95% and 70% respectively. The chemotaxonomic studies indicated that the major cellular fatty acids of the strain JS21-1T were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), C16:0, and C14:0 2OH; strain S6-262T possessed summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH) and summed feature 8 (C18:1 ω6c and/or C18:1 ω7c). The major quinone was Q10, and the unique polyamine observed was homospermidine. The polar lipid profile comprised of mixture of sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and certain uncharacterised phospholipids and lipids. Based on this polyphasic evidence, strains JS21-1T and S6-262T represent two novel species of the genus Sphingomonas, for which the names Sphingomonas palmae sp. nov. and Sphingomonas gellani sp. nov. are proposed, respectively. The type strain of Sphingomonas palmae sp. nov. is JS21-1T (= DSM 27348T = KACC 17591T) and the type strain of Sphingomonas gellani sp. nov. is S6-262T (= DSM 27346T =  KACC 17594T).

Dimethylsulfoniopropionate Sulfur and Methyl Carbon Assimilation in Ruegeria Species.

Dimethylsulfoniopropionate (DMSP) is abundant in marine environments and an important source of reduced carbon and sulfur for marine bacteria. While both Ruegeria pomeroyi and Ruegeria lacuscaerulensis possessed genes encoding the DMSP demethylation and cleavage pathways, their responses to DMSP differed. A glucose-fed, chemostat culture of R. pomeroyi consumed 99% of the DMSP even when fed a high concentration of 5 mM. At the same time, cultures released 19% and 7.1% of the DMSP as dimethylsulfide (DMS) and methanethiol, respectively. Under the same conditions, R. lacuscaerulensis consumed only 28% of the DMSP and formed one-third of the amount of gases. To examine the pathways of sulfur and methyl C assimilation, glucose-fed chemostats of both species were fed 100 µM mixtures of unlabeled and doubly labeled [dimethyl-13C, 34S]DMSP. Both species derived nearly all of their sulfur from DMSP despite high sulfate availability. In addition, only 33% and 50% of the methionine was biosynthesized from the direct capture of methanethiol in R. pomeroyi and R. lacuscaerulensis, respectively. The remaining methionine was biosynthesized by the random assembly of free sulfide and methyl-tetrahydrofolate derived from DMSP. Thus, although the two species possessed similar genes encoding DMSP metabolism, their growth responses were very different.IMPORTANCE Dimethylsulfoniopropionate (DMSP) is abundant in marine environments and an important source of reduced carbon and sulfur for marine bacteria. DMSP is the precursor for the majority of atmospheric dimethylsulfide (DMS), a climatically active gas that connects the marine and terrestrial sulfur cycles. Although research into the assimilation of DMSP has been conducted for over 20 years, the fate of DMSP in microbial biomass is not well understood. In particular, the biosynthesis of methionine from DMSP has been a focal point, and it has been widely believed that most methionine was synthesized via the direct capture of methanethiol. Using an isotopic labeling strategy, we have demonstrated that the direct capture of methanethiol is not the primary pathway used for methionine biosynthesis in two Ruegeria species, a genus comprised primarily of globally abundant marine bacteria. Furthermore, although the catabolism of DMSP by these species varied greatly, the anabolic pathways were highly conserved.

Chitinasiproducens palmae gen. nov., sp. nov., a new member of the family Burkholderiaceae isolated from leaf tissues of oil palm (Elaeis guineensis Jacq.).

A Gram-stain-negative, aerobic, rod-shaped, leaf-associated bacterium, designated JS23T, was isolated from surface-sterilized leaf tissue of an oil palm grown in Singapore and was investigated by polyphasic taxonomy. Phylogenetic analyses based on 16S rRNA gene sequences and 180 conserved genes in the genome of several members of Burkholderiaceae revealed that strain JS23T formed a distinct evolutionary lineage independent of other taxa within the family Burkholderiaceae. The predominant ubiquinone was Q-8. The primary polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and an unidentified aminophospholipid. The major fatty acids were C16 : 0, summed feature 3 (C16 : 1 ω7c /C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c /C18 : 1 ω6c). The size of the genome is 5.36 Mbp with a DNA G+C content of 66.2 mol%. Genomic relatedness measurements such as average nucleotide identity, genome-to-genome distance and digital DNA-DNA hybridization clearly distinguished strain JS23T from the closely related genera Burkholderia, Caballeronia, Mycetohabitans, Mycoavidus, Pandoraea, Paraburkholderia, Robbsia and Trinickia. Furthermore, average amino acid identity values and the percentages of conserved proteins, 56.0-68.4 and 28.2-45.5, respectively, were well below threshold values for genus delineation and supported the assignment of JS23T to a novel genus. On the basis of the phylogenetic, biochemical, chemotaxonomic and phylogenomic evidence, strain JS23T is proposed to represent a novel species of a new genus within the family Burkholderiaceae, for which the name Chitinasiproducens palmae gen. nov., sp. nov., is proposed with the type strain of JS23T (= DSM 27307T=KACC 17592T).

Reclassification of Sphingomonas aeria as a later heterotypic synonym of Sphingomonas carotinifaciens based on whole-genome sequence analysis.

The 16S rRNA gene sequences of Sphingomonas carotinifaciens L9-754T and Sphingomonas aeria B093034T possess 99.71 % sequence similarity. Further studies were undertaken to clarify the taxonomic assignments of these species. Whole-genome comparisons showed that S. aeria B093034Tand S. carotinifaciens L9-754T shared 96.9 % average nucleotide identity, 98.4 % average amino acid identity and 76.1 % digital DNA-DNA hybridization values. These values exceeded or approached the recommended species delineation threshold values. Furthermore, a phylogenetic tree based on 41 of the most conserved genes provided additional evidence that S. aeria B093034T and S. carotinifaciens L9-754T are very closely related. Based on this evidence we propose the reclassification of S. aeria Xue et al. 2018 as a later heterotypic synonym of S. carotinifaciens Madhaiyan et al. 2017.

An efficient method for synthesizing dimethylsulfonio-34 S-propionate hydrochloride from 34 S8.

Dimethylsulfoniopropionate (DMSP, (2-carboxyethyl)dimethylsulfonium) is a highly abundant compound in marine environments. As a precursor to the climatically active gas, dimethylsulfide (DMS), DMSP connects the marine and terrestrial sulfur cycles. However, the fate of DMSP in microbial biomass is not well understood as only a few studies have performed isotopic labeling experiments. A previously published method synthesized 34 S-labeled DMSP from 34 S8 , but the efficiency was only 26% and required five separate reactions, expensive reagents, and purification of the products of each reaction. In this study, a method of synthesizing 34 S-labeled DMSP from 34 S8 is described. Improvements include elemental steps, inexpensive reagents, purification of only one intermediate, and less time to complete. The efficiency of this method is 65% and results in pure DMSP with more than 98% isotope enrichment as determined by 1 H-nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS).

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.

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.

Genomic and phenotypic differentiation among Methanosarcina mazei populations from Columbia River sediment.

Methanogenic archaea are genotypically and phenotypically diverse organisms that are integral to carbon cycling in anaerobic environments. Owing to their genetic tractability and ability to be readily cultivated, Methanosarcina spp. have become a powerful model system for understanding methanogen biology at the cellular systems level. However, relatively little is known of how genotypic and phenotypic variation is partitioned in Methanosarcina populations inhabiting natural environments and the possible ecological and evolutionary implications of such variation. Here, we have identified how genomic and phenotypic diversity is partitioned within and between Methanosarcina mazei populations obtained from two different sediment environments in the Columbia River Estuary (Oregon, USA). Population genomic analysis of 56 M. mazei isolates averaging <1% nucleotide divergence revealed two distinct clades, which we refer to as 'mazei-T' and 'mazei-WC'. Genomic analyses showed that these clades differed in gene content and fixation of allelic variants, which point to potential differences in primary metabolism and also interactions with foreign genetic elements. This hypothesis of niche partitioning was supported by laboratory growth experiments that revealed significant differences in trimethylamine utilization. These findings improve our understanding of the ecologically relevant scales of genomic variation in natural systems and demonstrate interactions between genetic and ecological diversity in these easily cultivable and genetically tractable model methanogens.