Proposed minimal standards for description of new taxa of the class Halobacteria.

Halophilic archaea of the class Halobacteria are the most salt-requiring prokaryotes within the domain Archaea. In 1997, minimal standards for the description of new taxa in the order Halobacteriales were proposed. From then on, the taxonomy of the class Halobacteria provides an excellent example of how changing concepts on prokaryote taxonomy and the development of new methods were implemented. The last decades have witnessed a rapid expansion of the number of described taxa within the class Halobacteria coinciding with the era of genome sequencing development. The current members of the International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Halobacteria propose these revisions to the recommended minimal standards and encourage the use of advanced technologies in the taxonomic description of members of the Halobacteria. Most previously required and some recommended minimal standards for the description of new taxa in the class Halobacteria were retained in the present revision, but changes have been proposed in line with the new methodologies. In addition to the 16S rRNA gene, the rpoB' gene is an important molecular marker for the identification of members of the Halobacteria. Phylogenomic analysis based on concatenated conserved, single-copy marker genes is required to infer the taxonomic status of new taxa. The overall genome relatedness indexes have proven to be determinative in the classification of the taxa within the class Halobacteria. Average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values should be calculated for rigorous comparison among close relatives.

Draft Genome Sequence of a Novel Species of Halococcus (Strain IIIV-5B), an Endophytic Archaeon Isolated from the Leaf Tissue of Avicennia germinans.

Strain IIIV-5B was isolated from the leaf tissue of the black mangrove, Avicennia germinans This microorganism belongs to the genus Halococcus Here, we present the draft genome sequence of strain IIIV-5B, a novel species of this genus. The 3,869,808-bp genome has a G+C content of 63.9% and around 3,812 coding sequences.

Insights through Genetics of Halophilic Microorganisms and Their Viruses.

Halophilic microorganisms are found in all domains of life and thrive in hypersaline (high salt content) environments. These unusual microbes have been a subject of study for many years due to their interesting properties and physiology. Study of the genetics of halophilic microorganisms (from gene expression and regulation to genomics) has provided understanding into mechanisms of how life can occur at high salinity levels. Here we highlight recent studies that advance knowledge of biological function through study of the genetics of halophilic microorganisms and their viruses.

Draft Genome Sequences of 16 Halophilic Prokaryotes Isolated from Diverse Environments.

Halophile-specific enzymes have wide-ranging industrial and commercial applications. Despite their importance, there is a paucity of available halophile whole-genome sequences. Here, we report the draft genome sequences of 16 diverse salt-tolerant strains of bacteria and archaea isolated from a variety of high-salt environments.

Temporal Analysis of the Microbial Community from the Crystallizer Ponds in Cabo Rojo, Puerto Rico, Using Metagenomics.

The Cabo Rojo solar salterns are a hypersaline environment located in a tropical climate, where conditions remain stable throughout the year. These conditions can favor the establishment of steady microbial communities. Little is known about the microbial composition that thrives in hypersaline environments in the tropics. The main goal of this study was to assess the microbial diversity present in the crystallizer ponds of Cabo Rojo, in terms of structure and metabolic processes across time using metagenomic techniques. Three samplings (December 2014, March and July 2016) were carried out, where water samples (50 L each) were filtered through a Millipore pressurized filtering system. DNA was subsequently extracted using physical-chemical methods and sequenced using paired end Illumina technologies. The sequencing effort produced three paired end libraries with a total of 111,816,040 reads, that were subsequently assembled into three metagenomes. Out of the phyla detected, the microbial diversity was dominated in all three samples by Euryarchaeota, followed by Bacteroidetes and Proteobacteria. However, sample MFF1 (for Muestreo Final Fraternidad) exhibited a higher diversity, with 12 prokaryotic phyla detected at 34% NaCl (w/v), when compared to samples MFF2 and MFF3, which only exhibited three phyla. Precipitation events might be one of the contributing factors to the change in the microbial community composition through time. Diversity at genus level revealed a more stable community structure, with an overwhelming dominance of the square archaeon Haloquadratum in the three metagenomes. Furthermore, functional annotation was carried out in order to detect genes related to metabolic processes, such as carbon, nitrogen, and sulfur cycles. The presence of gene sequences related to nitrogen fixation, ammonia oxidation, sulfate reduction, sulfur oxidation, and phosphate solubilization were detected. Through binning methods, four putative novel genomes were obtained, including a possible novel genus belonging to the Bacteroidetes and possible new species for the genera Natronomonas, Halomicrobium, and Haloquadratum. Using a metagenomic approach, a 3-year study has been performed in a Caribbean hypersaline environment. When compared to other salterns around the world, the Cabo Rojo salterns harbor a similar community composition, which is stable through time. Moreover, an analysis of gene composition highlights the importance of the microbial community in the biogeochemical cycles at hypersaline environments.

Cloning and Molecular Characterization of an Alpha-Glucosidase (MalH) from the Halophilic Archaeon Haloquadratum walsbyi.

We report the heterologous expression and molecular characterization of the first extremely halophilic alpha-glucosidase (EC 3.2.1.20) from the archaeon Haloquadratum walsbyi. A 2349 bp region (Hqrw_2071) from the Hqr. walsbyi C23 annotated genome was PCR-amplified and the resulting amplicon ligated into plasmid pET28b(+), expressed in E. coli Rosetta cells, and the resulting protein purified by Ni-NTA affinity chromatography. The recombinant protein showed an estimated molecular mass of 87 kDa, consistent with the expected value of the annotated protein, and an optimal activity for the hydrolysis of α-PNPG was detected at 40 °C, and at pH 6.0. Enzyme activity values were the highest in the presence of 3 M NaCl or 3-4 M KCl. However, specific activity values were two-fold higher in the presence of 3-4 M KCl when compared to NaCl suggesting a cytoplasmic localization. Phylogenetic analyses, with respect to other alpha-glucosidases from members of the class Halobacteria, showed that the Hqr. walsbyi MalH was most similar (up to 41%) to alpha-glucosidases and alpha-xylosidases of Halorubrum. Moreover, computational analyses for the detection of functional domains, active and catalytic sites, as well as 3D structural predictions revealed a close relationship with an E. coli YicI-like alpha-xylosidase of the GH31 family. However, the purified enzyme did not show alpha-xylosidase activity. This narrower substrate range indicates a discrepancy with annotations from different databases and the possibility of specific substrate adaptations of halophilic glucosidases due to high salinity. To our knowledge, this is the first report on the characterization of an alpha-glucosidase from the halophilic Archaea, which could serve as a new model to gain insights into carbon metabolism in this understudied microbial group.

Draft genome sequence of strain MC1A, a UV-resistant bacterium isolated from dry soil in Puerto Rico.

We report here the draft genome sequence of a novel UV-resistant bacterium isolated from dry soil on the south coast of Puerto Rico. Based on polyphasic taxonomy, strain MC1A represents a new species and the name Solirubrum puertoriconensis is proposed. Assembly was performed using NGEN Assembler into eight contigs (N50 = 1,292,788), the largest of which included 1,549,887 bp. The draft genome consists of 4,810,875 bp and has a GC content of 58.7%. Several genes related to DNA repair and UV resistance were found. The Whole Genome Shotgun project is available at DDBJ/EMBL/GenBank under the accession LNAL00000000.

Draft genome sequence of Halorubrum tropicale strain V5, a novel halophilic archaeon isolated from the solar salterns of Cabo Rojo, Puerto Rico.

The genus Halorubrum is a member of the family Halobacteriaceae which currently has the highest number of described species (31) of all the haloarchaea. Here we report the draft genome sequence of strain V5, a new species within this genus that was isolated from the solar salterns of Cabo Rojo, Puerto Rico. Assembly was performed and rendered the genome into 17 contigs (N50 = 515,834 bp), the largest of which contains 1,031,026 bp. The genome consists of 3.57 MB in length with G + C content of 67.6%. In general, the genome includes 4 rRNAs, 52 tRNAs, and 3246 protein-coding sequences. The NCBI accession number for this genome is LIST00000000 and the strain deposit number is CECT9000.

Draft genome of Haloarcula rubripromontorii strain SL3, a novel halophilic archaeon isolated from the solar salterns of Cabo Rojo, Puerto Rico.

The genus Haloarcula belongs to the family Halobacteriaceae which currently has 10 valid species. Here we report the draft genome sequence of strain SL3, a new species within this genus, isolated from the Solar Salterns of Cabo Rojo, Puerto Rico. Genome assembly performed using NGEN Assembler resulted in 18 contigs (N50 = 601,911 bp), the largest of which contains 1,023,775 bp. The genome consists of 3.97 MB and has a GC content of 61.97%. Like all species of Haloarcula, the genome encodes heterogeneous copies of the small subunit ribosomal RNA. In addition, the genome includes 6 rRNAs, 48 tRNAs, and 3797 protein coding sequences. Several carbohydrate-active enzymes genes were found, as well as enzymes involved in the dihydroxyacetone processing pathway which are not found in other Haloarcula species. The NCBI accession number for this genome is LIUF00000000 and the strain deposit number is CECT9001.

Isolation and molecular identification of landfill bacteria capable of growing on di-(2-ethylhexyl) phthalate and deteriorating PVC materials.

Waste materials containing Di-(2-ethylhexyl) phthalate (DEHP), a suspected endocrine disruptor and reasonably anticipated human carcinogen, are typically disposed of in landfills. Despite this, very few studies had been conducted to isolate and identify DEHP-degrading bacteria in landfill leachate. Therefore, this study was conducted to isolate and characterize bacteria in landfill leachate growing on DEHP as the sole carbon source and deteriorating PVC materials. Four strains LHM1, LHM2, LHM3 and LHM4, not previously reported as DEHP-degraders, were identified via 16S rRNA gene sequence. Gram-positive strains LHM1 and LHM2 had a greater than 97% similarity with Chryseomicrobium imtechense MW 10(T) and Lysinibacillus fusiformis NBRC 15717(T), respectively. Gram-negative strains LHM3 and LHM4 were related to Acinetobacter calcoaceticus DSM 30006(T) (90.7% similarity) and Stenotrophomonas pavanii ICB 89(T) (96.0% similarity), respectively. Phylogenetic analysis also corroborated these similarities of strains LHM1 and LHM2 to the corresponding bacteria species. Strains LHM2 and LHM4 grew faster than strains LHM1 and LHM3 in the enrichment where DEHP was the sole carbon source. When augmented to the reactors with PVC shower curtains containing DEHP, strains LHM1 and LHM2 developed greater optical densities in the solution phase and thicker biofilm on the surfaces of the shower curtains.

PVC biodeterioration and DEHP leaching by DEHP-degrading bacteria.

Newly isolated, not previously reported, di-(2-ethylhexyl) phthalate (DEHP)-degraders were augmented to assess their role in polyvinyl chloride (PVC) shower curtain deterioration and DEHP leaching. The biofilms that developed on the surfaces of the bioaugmented shower curtains with Gram-positive strains LHM1 and LHM2 were thicker than those of the biostimulated and Gram-negative strain LHM3-augmented shower curtains. The first derivative thermogravimetric (DTG) peaks of the bioaugmented shower curtains with the Gram-positive bacteria were observed at ~287°C, whereas the control and Gram-negative strain LHM3-augmented shower curtains were detected at ~283°C. This slight delay in the first DTG peak temperature is indicative of lower plasticizer concentrations in the shower curtains that were bioaugmented with Gram positive bacteria. Despite bioaugmentation with DEHP-degraders, aqueous solutions of the bioaugmentation reactors were not DEHP-free due probably to the presence of co-solutes that must have supported microbial growth. Generally, the bioaugmented reactors with the Gram-positive strains LHM1 and LHM2 had greater aqueous DEHP concentrations in the first-half (<3 wk) of the biodeterioration experiment than the biostimulated and strain LHM3-augmented reactors. Therefore, strains LHM1 and LHM2 may play an important role in DEHP leaching to the environment and PVC biodeterioration.

Description of Kushneria aurantia gen. nov., sp. nov., a novel member of the family Halomonadaceae, and a proposal for reclassification of Halomonas marisflavi as Kushneria marisflavi comb. nov., of Halomonas indalinina as Kushneria indalinina comb. nov. and of Halomonas avicenniae as Kushneria avicenniae comb. nov.

An aerobic, moderately halophilic, Gram-negative, motile, non-sporulating rod-shaped bacterium, designated strain A10(T), was isolated from the surface of leaves of the black mangrove Avicennia germinans and was subjected to a polyphasic taxonomic study. Strain A10(T) was able to grow at NaCl concentrations in the range 5-17.5 % (w/v) with optimum growth at 10 % (w/v) NaCl. Growth occurred at temperatures of 20-40 degrees C (optimal growth at 37 degrees C) and pH 5.5-8.5 (optimal growth at pH 7.0-8.0). The major respiratory quinone was ubiquinone 9. The major fatty acids were C(16 : 0), C(18 : 1)omega7c, C(19 : 0) cyclo omega8c and C(12 : 0) 3-OH. The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and unidentified phospholipids, glycolipids and an aminoglycolipid. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain A10(T) is closely related to Halomonas avicenniae MW2a(T) (95.7 % sequence similarity), Halomonas marisflavi SW32(T) (95.2 %) and Halomonas indalinina GC2.1(T) (95.0 %). Strain A10(T) formed a coherent phylogenetic branch with these three species, separated from other species of Halomonas and closely related genera (with 16S rRNA gene sequence similarities below 94.0 %). A complete 23S rRNA gene sequence comparison of strain A10(T) with closely related species confirmed the phylogenetic position of the novel isolate, forming a branch with the species Halomonas avicenniae, Halomonas indalinina and Halomonas marisflavi, separated from other species of the genera belonging to the family Halomonadaceae (showing sequence similarities below 91.7 %). DNA-DNA hybridization studies between strain A10(T) and Halomonas avicenniae MW2a(T), Halomonas marisflavi DSM 15357(T) and Halomonas indalinina CG2.1(T) were 21, 17 and 10 %, respectively. These levels of DNA-DNA relatedness were low enough to classify strain A10(T) as representing a genotypically distinct species. Overall, the phenotypic, genotypic, chemotaxonomic and phylogenetic results demonstrated that strain A10(T) represents a new genus and species. The name Kushneria aurantia gen. nov., sp. nov. is proposed, with strain A10(T) (=CCM 7415(T)=CECT 7220(T)) as the type strain. This is the type species of the new proposed genus, which belongs to the family Halomonadaceae. In addition, our data support the placement of the species Halomonas marisflavi, Halomonas indalinina and Halomonas avicenniae within this new genus, as Kushneria marisflavi comb. nov., Kushneria indalinina comb. nov. and Kushneria avicenniae comb. nov., respectively.

Halobacillus mangrovi sp. nov., a moderately halophilic bacterium isolated from the black mangrove Avicennia germinans.

A moderately halophilic, spore-forming, Gram-positive, short-rod-shaped bacterium, designated strain MS10(T), was isolated from the surface of leaves of the black mangrove Avicennia germinans and was subjected to a polyphasic taxonomic study. Strain MS10(T) was able to grow at NaCl concentrations in the range 5-20% (w/v) with optimum growth at 10% (w/v) NaCl. Growth occurred at temperatures of 10-50 degrees C (optimal growth at 33-35 degrees C) and pH 6.0-9.0 (optimal growth at pH 7.0). Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain MS10(T) fell within the branch encompassing members of the genus Halobacillus and was most closely related to Halobacillus dabanensis JCM 12772(T) (99.2% 16S rRNA gene sequence similarity). The DNA G+C content of strain MS10(T) was 45.7 mol%, the major respiratory isoprenoid quinone was MK-7 and the cell-wall peptidoglycan was of the L-Orn-D-Asp type, characteristics consistent with its affiliation to the genus Halobacillus. Strain MS10(T) showed a level of DNA-DNA hybridization with H. dabanensis JCM 12772(T) of 29% and levels below 70% were also obtained with respect to other recognized members of the genus Halobacillus. The major fatty acids of strain MS10(T) were iso-C(16:0), anteiso-C(15:0), iso-C(14:0) and iso-C(15:0). Overall, the phenotypic, genotypic and phylogenetic results presented in this study demonstrate that strain MS10(T) represents a novel species of the genus Halobacillus, for which the name Halobacillus mangrovi sp. nov. is proposed. The type strain is MS10(T) (=CECT 7206(T)=CCM 7397(T)).

Halomonas avicenniae sp. nov., isolated from the salty leaves of the black mangrove Avicennia germinans in Puerto Rico.

A Gram-negative, short rod to oval-shaped bacterium (strain MW2a(T)) was isolated from the surface of leaves of the black mangrove Avicennia germinans and subjected to a polyphasic taxonomic study. Strain MW2a(T) was moderately halophilic, growing at NaCl concentrations in the range 0-25 % (w/v) with optimum growth at 5 % (w/v) NaCl. Growth occurred at 12-40 degrees C (optimum, 30-35 degrees C) and at pH 5.0-9.0 (optimum, pH 7.0-8.0). Strain MW2a(T) was strictly aerobic. Phylogenetic analysis based on the 16S rRNA gene showed that the strain belongs to the genus Halomonas. The closest relative was Halomonas marisflavi, with 98.6 % 16S rRNA gene sequence similarity. The DNA G+C content of strain MW2a(T) was 61.5 mol%, which is in the range of values for Halomonas species. DNA-DNA hybridization with H. marisflavi showed a relatedness of 42 % and lower values were obtained with respect to other related Halomonas species. The major fatty acids were C(16 : 0), C(19 : 0) cyclo omega8c, C(18 : 1)omega7c and C(12 : 0) 3-OH. Overall, the phenotypic, genotypic and phylogenetic results presented in this study demonstrate that strain MW2a(T) represents a novel species within the genus Halomonas. The name Halomonas avicenniae sp. nov. is proposed, with strain MW2a(T) (=CECT 7193(T)=CCM 7396(T)) as the type strain.