Phylogenomics of Haloarchaea: The Controversy of the Genera Natrinema-Haloterrigena

The haloarchaeal genera Natrinema and Haloterrigena were described almost simultaneously by two different research groups and some strains studied separately were described as different species of these genera. Furthermore, the description of additional species were assigned to either Natrinema or Haloterrigena, mainly on the basis of the phylogenetic comparative analysis of single genes (16S rRNA gene and more recently rpoB’ gene), but these species were not adequately separated or assigned to the corresponding genus. Some studies suggested that the species of these two genera should be unified into a single genus, while other studies indicated that the genera should remain but some of the species should be reassigned. In this study, we have sequenced or collected the genomes of the type strains of species of Natrinema and Haloterrigena and we have carried out a comparative genomic analysis in order to clarify the controversy related to these two genera. The phylogenomic analysis based on the comparison of 525 translated single-copy orthologous genes and the Overall Genome Relatedness Indexes (i.e., AAI, POCP, ANI, and dDDH) clearly indicate that the species Haloterrigena hispanica, Haloterrigena limicola, Haloterrigena longa, Haloterrigena mahii, Haloterrigena saccharevitans, Haloterrigena thermotolerans, and Halopiger salifodinae should be transferred to the genus Natrinema, as Natrinema hispanicum, Natrinema limicola, Natrinema longum, Natrinema mahii, Natrinema saccharevitans, Natrinema thermotolerans, and Natrinema salifodinae, respectively. On the contrary, the species Haloterrigena turkmenica, Haloterrigena salifodinae, and Haloterrigena salina will remain as the only representative species of the genus Haloterrigena. Besides, the species Haloterrigena daqingensis should be reclassified as a member of the genus Natronorubrum, as Natronorubrum daqingense. At the species level, Haloterrigena jeotgali and Natrinema ejinorense should be considered as a later heterotypic synonyms of the species Haloterrigena (Natrinema) thermotolerans and Haloterrigena (Natrinema) longa, respectively. Synteny analysis and phenotypic features also supported those proposals.España Junta de Andalucía grants US-1263771 [US/JUNTA/FEDER, UE]España FEDER/Ministry of Science and Innovation-State Research Agency projects CGL2017-83385-P and PID2020-118136GB-I0

Helicobacter ibis sp. nov., isolated from faecal droppings of black-faced ibis (Theristicus melanopis)

As part of a larger study on Epsilonproteobacteria carried by wild birds in the city of Valdivia (southern Chile), two curved rod-shaped Gram-stain-negative strains (A82T and WB-40) were recovered from faecal samples and subjected to a taxonomic study. Results of a genus-specific PCR showed that these isolates belonged to the genus Helicobacter. Further identification by 16S rRNA and hsp60 (60 kDa heat-shock protein) gene sequence analysis revealed that they formed a separate phylogenetic clade, different from other known Helicobacter species with ‘Helicobacter burdigaliensis’ CNRCH 2005/566HT and Helicobacter valdiviensis WBE14T being the most closely related species. This was confirmed by core-genome phylogeny as well as digital DNA–DNA hybridization and average nucleotide identity analyses between the genomes of strains A82T and WB-40 and all other Helicobacter species. The draft genome sequences of A82T and WB-40, obtained by Illumina NextSeq 2000 sequencing, consisted of 1.6 Mb with a G+C content of 31.9–32.0 mol%. The results obtained from the phylogenetic and genomic characteri-zation, together with their different morphological and biochemical features, revealed that these two strains represent a novel species, for which we propose the name Helicobacter ibis sp. nov. with A82T (=LMG 32718T=CCCT 22.04T) as the type strain.Comisión Nacional de Investigación Científica y Tecnológica (CONICYT). Chile 1200125Ministerio de Ciencia e Innovación PID2020-118136GB-I00Junta de Andalucía P20_01066, BIO-21

Salinivibrio kushneri sp. nov., a moderately halophilic bacterium isolated from salterns

Ten Gram-strain-negative, facultatively anaerobic, moderately halophilic bacterial strains, designated AL184T, IB560, IB563, IC202, IC317, MA421, ML277, ML318, ML328A and ML331, were isolated from water ponds of five salterns located in Spain. The cells were motile, curved rods and oxidase and catalase positive. All of them grew optimally at 37 °C, at pH 7.2–7.4 and in the presence of 7.5% (w/v) NaCl. Based on phylogenetic analyses of the 16S rRNA, the isolates were most closely related to Salinivibrio sharmensis BAGT (99.6–98.2% 16S rRNA gene sequence similarity) and Salinivibrio costicola subsp. costicola ATCC 35508T (99.0–98.1%). According to the MLSA analyses based on four (gyrB, recA, rpoA and rpoD) and eight (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA) concatenated gene sequences, the most closely relatives were S. siamensis JCM 14472T (96.8–95.4% and 94.9–94.7%, respectively) and S. sharmensis DSM 18182T (94.0–92.6% and 92.9–92.7%, respectively). In silico DNA–DNA hybridization (GGDC) and average nucleotide identity (ANI) showed values of 23.3–44.8% and 80.2–91.8%, respectively with the related species demonstrating that the ten isolates constituted a single novel species of the genus Salinivibrio. Its pangenome and core genome consist of 6041 and 1230 genes, respectively. The phylogeny based on the concatenated orthologous core genes revealed that the ten strains form a coherent phylogroup well separated from the rest of the species of the genus Salinivibrio. The major cellular fatty acids of strain AL184T were C16:0 and C18:1. The DNA G + C content range was 51.9–52.5 mol% (Tm) and 50.2–50.9 mol% (genome). Based on the phylogenetic-phylogenomic, phenotypic and chemotaxonomic data, the ten isolates represent a novel species of the genus Salinivibrio, for which the name Salinivibrio kushneri sp. nov. is proposed. The type strain is AL184T (= CECT 9177T = LMG 29817T).Ministerio de Economía y Competitividad CGL2013-46941-P, CGL2017-83385-

Salimicrobium salexigens sp. nov., a moderately halophilic bacterium from salted hides

Two Gram-positive, moderately halophilic bacteria, designated strains 29CMIT and 53CMI, were isolated from salted hides. Both strains were non-motile, strictly aerobic cocci, growing in the presence of 3-25 % (w/v) NaCl (optimal growth at 7.5-12.5 % [w/v] NaCl), between pH 5.0 and 10.0 (optimal growth at pH 7.5) and at temperatures between 15 and 40 ºC (optimal growth at 37 ºC). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed a similarity of 98.7 % and were closely related to species of the genus Salimicrobium, within the phylum Firmicutes. Strains 29CMIT and 53CMI exhibited 16S rRNA gene sequence similarity values of 97.9 % to 97.6 % with Salimicrobium album DSM 20748T, Salimicrobium halophilum DSM 4771T, Salimicrobium flavidum ISL-25T and Salimicrobium luteum BY-5T. The DNA G+C content was 50.7 mol% and 51.5 mol% for strains 29CMIT and 53CMI, respectively. The DNA-DNA hybridization between both strains was 98 %, whereas the values between strain 29CMIT and the species Salimicrobium album CCM 3517T, Salimicrobium luteum BY-5T, Salimicrobium flavidum ISL-25T and Salimicrobium halophilum CCM 4074T were 45 %, 28 %,

Assessment of MultiLocus Sequence Analysis As a Valuable Tool for the Classification of the Genus Salinivibrio

The genus Salinivibrio includes obligatory halophilic bacteria and is commonly isolated from hypersaline habitats and salted food products. They grow optimally between 7.5 and 10% salts and are facultative anaerobes. Currently, this genus comprises four species, one of them, S. costicola, with three subspecies. In this study we isolated and characterized an additional 70 strains from solar salterns located in different locations. Comparative 16S rRNA gene sequence analysis identified these strains as belonging to the genus Salinivibrio but could not differentiate strains into species-like groups. To achieve finer phylogenetic resolution, we carried out a MultiLocus Sequence Analysis (MLSA) of the new isolates and the type strains of the species of Salinivibrio based on the individual as well as concatenated sequences of four housekeeping genes: gyrB, recA, rpoA, and rpoD. The strains formed four clearly differentiated species-like clusters called phylogroups. All of the known type and subspecies strains were associated with one of these clusters except S. sharmensis. One phylogroup had no previously described species coupled to it. Further DNA–DNA hybridization (DDH) experiments with selected representative strains from these phylogroups permitted us to validate the MLSA study, correlating the species level defined by the DDH (70%) with a 97% cut-off for the concatenated MLSA gene sequences. Based on these criteria, the novel strains forming phylogroup 1 could constitute a new species while strains constructing the other three phylogroups are members of previously recognized Salinivibrio species. S. costicola subsp. vallismortis co-occurs with S. proteolyticus in phylogroup 4, and separately from other S. costicola strains, indicating its need for reclassification. On the other hand, genome fingerprinting analysis showed that the environmental strains do not form clonal populations and did not cluster according to their site of cultivation. In future studies regarding the classification and identification of new Salinivibrio strains we recommend the following strategy: (i) initial partial sequencing of the 16S rRNA gene for genus-level identification; (ii) sequencing and concatenation of the four before mentioned housekeeping genes for species-level discrimination; (iii) DDH experiments, only required when the concatenated MLSA similarity values among a new isolate and other Salinivibrio strains are above the 97% cut-off.España, MINECO project CGL2013- 46941-

Comparative Genomics and Phylogenomic Analysis of the Genus Salinivibrio

In the genomic era phylogenetic relationship among prokaryotes can be inferred from the core orthologous genes (OGs) or proteins in order to elucidate their evolutionary history and current taxonomy should benefits of that. The genus Salinivibrio belongs to the family Vibrionaceae and currently includes only five halophilic species, in spite the fact that new strains are very frequently isolated from hypersaline environments. Species belonging to this genus have undergone several reclassifications and, moreover, there are many strains of Salinivibrio with available genomes which have not been affiliated to the existing species or have been wrongly designated. Therefore, a phylogenetic study using the available genomic information is necessary to clarify the relationships of existing strains within this genus and to review their taxonomic affiliation. For that purpose, we have also sequenced the first complete genome of a Salinivibrio species, Salinivibrio kushneri AL184T, which was employed as a reference to order the contigs of the draft genomes of the type strains of the current species of this genus, as well as to perform a comparative analysis with all the other available Salinivibrio sp. genomes. The genome of S. kushneri AL184T was assembled in two circular chromosomes (with sizes of 2.84 Mb and 0.60 Mb, respectively), as typically occurs in members of the family Vibrionaceae, with nine complete ribosomal operons, which might explain the fast growing rate of salinivibrios cultured under laboratory conditions. Synteny analysis among the type strains of the genus revealed a high level of genomic conservation in both chromosomes, which allow us to hypothesize a slow speciation process or homogenization events taking place in this group of microorganisms to be tested experimentally in the future. Phylogenomic and orthologous average nucleotide identity (OrthoANI)/average amino acid identity (AAI) analyses also evidenced the elevated level of genetic relatedness within members of this genus and allowed to group all the Salinivibrio strains with available genomes in seven separated species. Genome-scale attribute study of the salinivibrios identified traits related to polar flagellum, facultatively anaerobic growth and osmotic response, in accordance to the phenotypic features described for species of this genus.Spanish Ministry of Economy and Competitiveness Project CGL2017- 83385-PEspaña Junta de Andalucía BIO-213Spanish, University of Seville VIPPIT-US-201

Draft Genome Sequence of the Moderately Halophilic Bacterium Pseudoalteromonas ruthenica Strain CP76

Pseudoalteromonas ruthenica strain CP76, isolated from a saltern in Spain, is a moderately halophilic bacterium belonging to the Gammaproteobacteria. Here we report the draft genome sequence, which consists of a 4.0-Mb chromosome, of this strain, which is able to produce the extracellular enzyme haloprotease CPI

Genotypic and Lipid Analyses of Strains From the Archaeal Genus Halorubrum Reveal Insights Into Their Taxonomy, Divergence, and Population Structure

To gain a better understanding of how divergence occurs, and how taxonomy can benefit from studying natural populations, we isolated and examined 25 closely related Halorubrum strains obtained from different hypersaline communities and compared them to validly named species and other reference strains using five taxonomic study approaches: phylogenetic analysis using the 16S rRNA gene and multilocus sequencing analysis (MLSA), polar lipid profiles (PLP), average nucleotide identity (ANI) and DNA-DNA hybridization (DDH). 16S rRNA gene sequence could not differentiate the newly isolated strains from described species, while MLSA grouped strains into three major clusters. Two of those MLSA clusters distinguished candidates for new species. The third cluster with concatenated sequence identity equal to or greater than 97.5% was comprised of strains from Aran-Bidgol Lake (Iran) and solar salterns in Namibia and Spain, and two previously described species isolated from Mexico and Algeria. PLP and DDH analyses showed that Aran-Bidgol strains formed uniform populations, and that strains isolated from other geographic locations were heterogeneous and divergent, indicating that they may constitute different species. Therefore, applying only sequencing approaches and similarity cutoffs for circumscribing species may be too conservative, lumping concealed diversity into a single taxon. Further, our data support the interpretation that local populations experience unique evolutionary homogenization pressures, and once relieved of insular constraints (e.g., through migration) are free to diverge.España, MINECO Projects CGL2013-46941-P and CGL2017-83385-

Draft Genome Sequence of Saccharomonospora sp. Strain LRS4.154, a Moderately Halophilic Actinobacterium with the Biotechnologically Relevant Polyketide Synthase and Nonribosomal Peptide Synthetase Systems

The draft genome sequence of Saccharomonospora sp. strain LRS4.154, a moderately halophilic actinobacterium, has been determined. The genome has 4,860,108 bp, a G+C content of 71.0%, and 4,525 open reading frames (ORFs). The clusters of PKS and NRPS genes, responsible for the biosynthesis of a large number of biomolecules, were identified in the genome.España, Ministerio de Economía e Innovación grant number CGL2013-46941-

Bacilus locisalis sp. nov. , a new haloalkaliphilic species from hypersaline and alkaline lakes of China, Kenya and Tanzania

A polyphasic taxonomic study was performed on seven Bacillus-like bacteria isolated from three hypersaline and alkaline lakes located in China, Kenya and Tanzania. All strains were moderately halophilic and alkaliphilic, Gram positive, motile rods. The DNA G+C content from the seven isolates ranged from 42.2 to 43.4 mol% and their major fatty acid was anteiso-C15:0. Strain CG1T, selected as representative strain of the isolates, possesses meso-diaminopimelic acid in the cell wall peptidoglycan, MK-7 as the predominant menaquinone and diphosphatidyl glycerol, phosphatidylglycerol and phosphatidylethanolamine as the major polar lipids. Comparative 16S rRNA gene sequence analysis indicated that the isolates belonged to the genus Bacillus. The seven isolates shared 97.7-99.9% 16S rRNA gene sequence similarity, and formed a branch that was distinct from the type strains of the recognized species of the genus Bacillus. They were most closely related to Bacillus agaradhaerens DSM 8721T (92.6-93.8% 16S rRNA sequence similarity). DNA–DNA hybridization values between the seven isolates were 85-100%. According to the polyphasic characterization, the strains represent a novel species, for which the name Bacillus locisalis sp. nov. is proposed. The type strain is CG1T (CCM 7370T = CECT 7152T = CGMCC 1.6286T = DSM 18085T). Ke