Improvement of duplex-specific nuclease salt tolerance by fusing DNA-binding domain of DNase from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix.

Salt tolerance is an important property of duplex-specific nuclease (DSN). DSN with high salt tolerance can be more widely used in genetic engineering, especially in the production of nucleic acid drugs. To improve the salt tolerance of DSN, we selected five DNA-binding domains from extremophilic organisms, which have been shown the ability to improve salt tolerance of DNA polymerases and nucleases. The experimental results demonstrated that the fusion protein TK-DSN produced by fusing a N-terminal DNA-binding domain, which comprised two HhH (helix-hairpin-helix) motifs domain from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix, has a significantly improved salt tolerance. TK-DSN can tolerate the concentration of NaCl up to 800 mM; in addition, the ability of digesting DNA was also enhanced during in vitro transcription and RNA purification. This strategy provides the method for the personalized customization of biological tool enzymes for different applications.

CRISPR/Cas12a-Mediated Genome Editing in Thioalkalivibrio versutus.

Haloalkaliphilic Thioalkalivibrio versutus, a dominant species for sulfide removal, has attracted increasing attention. However, research on T. versutus is limited by the lack of genetic manipulation tools. In this work, we developed a CRISPR/AsCas12a-mediated system in T. versutus for an efficient and implementable genome editing workflow. Compared to the CRISPR/Cas9-mediated system, the CRISPR/AsCas12a system exhibited enhanced editing efficiency. Additionally, as Cas12a is capable of processing the crRNA maturation independently, the CRISPR/AsCas12a system allowed multiplex gene editing and large-fragment DNA knockout by expressing more than one crRNA under the control of one promoter. Using the CRISPR/AsCas12a system, five key genes of the elemental sulfur oxidation pathway were knocked out. Simultaneous deletion of the rhd and tusA genes disrupted the ability of T. versutus to metabolize elemental sulfur, resulting in a 24.7% increase in elemental sulfur generation and a 15.2% reduction in sulfate production. This genome engineering strategy significantly improved our understanding of sulfur metabolism in Thioalkalivibrio spp.

Unlocking Survival Mechanisms for Metal and Oxidative Stress in the Extremely Acidophilic, Halotolerant Acidihalobacter Genus.

Microorganisms used for the biohydrometallurgical extraction of metals from minerals must be able to survive high levels of metal and oxidative stress found in bioleaching environments. The Acidihalobacter genus consists of four species of halotolerant, iron-sulfur-oxidizing acidophiles that are unique in their ability to tolerate chloride and acid stress while simultaneously bioleaching minerals. This paper uses bioinformatic tools to predict the genes and mechanisms used by Acidihalobacter members in their defense against a wide range of metals and oxidative stress. Analysis revealed the presence of multiple conserved mechanisms of metal tolerance. Ac. yilgarnensis F5T, the only member of this genus that oxidizes the mineral chalcopyrite, contained a 39.9 Kb gene cluster consisting of 40 genes encoding mobile elements and an array of proteins with direct functions in copper resistance. The analysis also revealed multiple strategies that the Acidihalobacter members can use to tolerate high levels of oxidative stress. Three of the Acidihalobacter genomes were found to contain genes encoding catalases, which are not common to acidophilic microorganisms. Of particular interest was a rubrerythrin genomic cluster containing genes that have a polyphyletic origin of stress-related functions.

Covalent immobilization of a halophilic, alkalithermostable lipase LipR2 on Florisil® nanoparticles for production of alkyl levulinates.

A novel halophilic, alkalithermostable lipase LipR2 from Alkalispirillum sp. NM-ROO2 was cloned and expressed. LipR2 was covalently immobilized on Florisil® functionalized with glutaraldehyde. Protein binding efficiency of functionalized Florisil® was 94.7%. Immobilized LipR2 retained 97.5% of specific activity of the free enzyme. Free LipR2 has maximal activity at 52 °C, pH 9.3 and 1.9 M NaCl and is resistant to surfactants and organic solvents. Immobilization enhanced LipR2's extreme characteristics, and increased thermostability of LipR2 with the half-life at 50 °C increasing three-fold. Immobilized LipR2 was used as a biocatalyst for esterification of levulinic acid with n-butanol. Under optimal conditions, a 45.9% ester yield was obtained after 12 h. Immobilized LipR2 catalyzed production of ethyl levulinate and 1-dodecyl levulinate with 48.8% and 26.2% ester yields, respectively. When used in repetitive batch esterification, LipR2 retained 69%, 57% and 18.5% of initial activity on esterification of levulinic acid with ethanol, n-butanol and 1-dodecanol, respectively.

Adaptability as the key to success for the ubiquitous marine nitrite oxidizer Nitrococcus.

Nitrite-oxidizing bacteria (NOB) have conventionally been regarded as a highly specialized functional group responsible for the production of nitrate in the environment. However, recent culture-based studies suggest that they have the capacity to lead alternative lifestyles, but direct environmental evidence for the contribution of marine nitrite oxidizers to other processes has been lacking to date. We report on the alternative biogeochemical functions, worldwide distribution, and sometimes high abundance of the marine NOB Nitrococcus. These largely overlooked bacteria are capable of not only oxidizing nitrite but also reducing nitrate and producing nitrous oxide, an ozone-depleting agent and greenhouse gas. Furthermore, Nitrococcus can aerobically oxidize sulfide, thereby also engaging in the sulfur cycle. In the currently fast-changing global oceans, these findings highlight the potential functional switches these ubiquitous bacteria can perform in various biogeochemical cycles, each with distinct or even contrasting consequences.

Complete genome sequence of Acidihalobacter prosperus strain F5, an extremely acidophilic, iron- and sulfur-oxidizing halophile with potential industrial applicability in saline water bioleaching of chalcopyrite.

Successful process development for the bioleaching of mineral ores, particularly the refractory copper sulfide ore chalcopyrite, remains a challenge in regions where freshwater is scarce and source water contains high concentrations of chloride ion. In this study, a pure isolate of Acidihalobacter prosperus strain F5 was characterized for its ability to leach base metals from sulfide ores (pyrite, chalcopyrite and pentlandite) at increasing chloride ion concentrations. F5 successfully released base metals from ores including pyrite and pentlandite at up to 30gL-1 chloride ion and chalcopyrite up to 18gL-1 chloride ion. In order to understand the genetic mechanisms of tolerance to high acid, saline and heavy metal stress the genome of F5 was sequenced and analysed. As well as being the first strain of Ac. prosperus to be isolated from Australia it is also the first complete genome of the Ac. prosperus species to be sequenced. The F5 genome contains genes involved in the biosynthesis of compatible solutes and genes encoding monovalent cation/proton antiporters and heavy metal transporters which could explain its abilities to tolerate high salinity, acidity and heavy metal stress. Genome analysis also confirmed the presence of genes involved in copper tolerance. The study demonstrates the potential biotechnological applicability of Ac. prosperus strain F5 for saline water bioleaching of mineral ores.

Natronospira proteinivora gen. nov., sp. nov, an extremely salt-tolerant, alkaliphilic gammaproteobacterium from hypersaline soda lakes.

Brine samples from Kulunda Steppe soda lakes (Altai, Russia) were inoculated into a hypersaline alkaline mineral medium with ß-keratin (chicken feather) as a substrate. The micro-organisms dominating the enrichment culture were isolated by limiting serial dilution on the same medium with casein as a substrate. The cells of strain BSker1T were motile, curved rods. The strain was an obligately aerobic heterotroph utilizing proteins and peptides as growth substrates. The isolate was an obligate alkaliphile with a pH range for growth from pH 8.5 to 10.25 (optimum at pH 9.5), and it was extremely salt tolerant, growing with between 1 and 4.5 M total Na+ (optimally at 2-2.5 M). BSker1T had a unique composition of polar lipid fatty acids, dominated by two C17 species. The membrane polar lipids included multiple unidentified phospholipids and two aminolipids. According to phylogenetic analysis of the 16S rRNA gene sequence, the isolate forms a novel branch within the family Ectothiorhodospiraceae (class Gammaproteobacteria) with the highest sequence similarity to the members of this family being 91 %. On the basis of distinct phenotypic and genotypic properties, strain BSker1T (=JCM 31341T=UNIQEM U1008T) is proposed to be classified as a representative of a novel genus and species, Natronospira proteinivora gen. nov., sp. nov.

Spiribacter aquaticus sp. nov., a novel member of the genus Spiribacter isolated from a saltern.

A moderately halophilic bacterium, designated strain SP30T, was isolated from a solar saltern located in Santa Pola, Alicante, on the East coast of Spain. It was a Gram-stain-negative, strictly aerobic bacterium, able to grow in 7.5-25 % (w/v) NaCl and optimally in 12.5 % (w/v) NaCl. Phylogenetic analyses, based on 16S rRNA gene sequences, showed that the novel isolate is a member of the genus Spiribacter, with the most closely related species being Spiribacter roseus SSL50T (99.9 % sequence similarity) and Spiribacter curvatus UAH-SP71T (99.4 % sequence similarity). The 16S rRNA gene sequence similarity with the type species Spiribacter salinus M19-40T was 96.6 %. The DNA-DNA relatedness value between strain SP30T and S. roseus SSL50T and S. curvatus UAH-SP71T was 40 and 55 %, respectively; these values are lower than the 70 % threshold accepted for species delineation. The major fatty acids were C16:0, C18 : 1ω7c, C19 : 0 cyclo ω8c and C12 : 0. Similarly to other species of the genus Spiribacter, strain SP30Twas observed as curved rods and spiral cells. Metabolic versatility was reduced to the utilization of a few organic compounds as the sole carbon and energy sources, as with other members of Spiribacter. However, it differed in terms of colony pigmentation (brownish-yellow instead of pink) and in having a higher growth rate. Based on these data and on the phenotypic, genotypic and chemotaxonomic characterization, we propose the classification of strain SP30T as a novel species within the genus Spiribacter, with the name Spiribacter aquaticus sp. nov. The type strain is SP30T (=CECT 9238T=LMG 30005T).

Improvement of desulfurizing activity of haloalkaliphilic Thialkalivibrio versutus SOB306 with the expression of Vitreoscilla hemoglobin gene.

OBJECTIVE: To construct efficient transformation and expression system and further improve desulfurizing activity of cells through expression of Vitreoscilla hemoglobin (VHb) in haloalkaliphilic Thialkalivibrio versutus SOB306. RESULTS: We transferred plasmids pKT230 and pBBR-smr into T. versutus SOB306 via a conjugation method. We identified four promoters from among several predicted promoters by scoring for streptomycin resistance, and finally selected tac and p3 based on the efficiency of expression of red fluorescent protein (RFP). Expression of RFP when regulated by tac was more than three times that of p3 in SOB306. Further, we expressed VHb under the control of tac promoter in SOB306. Expression of VHb was verified using CO-difference spectra. The results showed that VHb expression can boost sulfur metabolism, as evidenced by an increase of about 11.7 ± 1.8% in the average rate of thiosulfate removal in the presence of VHb. CONCLUSION: A conjugation transfer and an expression system for Thialkalivibrio, has been developed for the first time and used for expression of VHb to improve desulfurizing activity.

Spiribacter roseus sp. nov., a moderately halophilic species of the genus Spiribacter from salterns.

Four pink-pigmented, non-motile, Gram-staining-negative and moderately halophilic curved rods, designated strains SSL50T, SSL25, SSL97 and SSL4, were isolated from a saltern located in Isla Cristina, Huelva, south-west Spain. Phylogenetic analyses based on 16S rRNA gene sequences showed that they were members of the genus Spiribacter, most closely related to Spiribacter curvatus UAH-SP71T (99.3-99.5 % sequence similarity) and Spiribacter salinus M19-40T (96.5-96.7 %). Other related strains were Alkalilimnicola ehrlichii MLHE-1T (95.1-95.3 %), Arhodomonas recens RS91T (95.1-95.2 %) and Arhodomonas aquaeolei ATCC 49307T (95.0-95.1 %), all members of the family Ectothiorhodospiraceae. The major fatty acids were C18 : 1ω6c and/or C18 : 1ω7c, C16 : 0 and C12 : 0. The DNA G+C range was 64.0-66.3 mol%. The DNA-DNA hybridization values between strains SSL50T, SSL25, SSL97, SSL4 and S. piribacter. curvatus UAH-SP71T were 37-49 %. The average nucleotide identity (ANIb) values between the genome of strain SSL50T and those of the two other representatives of the genus Spiribacter, S. curvatus UAH-SP71T and S. salinus M19-40T, were 82.4 % and 79.1 %, respectively, supporting the proposal of a novel species of the genus Spiribacter. On the basis of the polyphasic analysis, the four new isolates are considered to represent a novel species of the genus Spiribacter, for which the name Spiribacter roseus sp. nov. is proposed. The type strain is SSL50T (=CECT 9117T=IBRC-M 11076T).

Biocathodes reducing oxygen at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes.

Biocathodes polarized at high potential are promising for enhancing Microbial Fuel Cell performances but the microbes and genes involved remain poorly documented. Here, two sets of five oxygen-reducing biocathodes were formed at two potentials (-0.4V and +0.1V vs. saturated calomel electrode) and analyzed combining electrochemical and metagenomic approaches. Slower start-up but higher current densities were observed at high potential and a distinctive peak increasing over time was recorded on cyclic voltamogramms, suggesting the growth of oxygen reducing microbes. 16S pyrotag sequencing showed the enrichment of two operational taxonomic units (OTUs) affiliated to Ectothiorodospiraceae on high potential electrodes with the best performances. Shotgun metagenome sequencing and a newly developed method for the identification of Taxon Specific Gene Annotations (TSGA) revealed Ectothiorhodospiraceae specific genes possibly involved in electron transfer and in autotrophic growth. These results give interesting insights into the genetic features underlying the selection of efficient oxygen reducing microbes on biocathodes.

Complete genome sequence of Thialkalivibrio versutus D301 isolated from Soda Lake in northern China, a typical strain with great ability to oxidize sulfide.

Thioalkalivibrio versutus D301 isolated from Soda Lake is a haloalkaliphilic and obligated chemolithoautotrophic Gram-negative bacterium. The strain has a good adaption to hyperhaline and highly alkaline environment and a powerful sulfur-oxidizing ability. Here, we present the complete genome sequence of T. versutus D301, providing insights into the genomic basis of its effects and facilitating its application in microbial desulfurization.

Halopeptonella vilamensis gen. nov, sp. nov., a halophilic strictly aerobic bacterium of the family Ectothiorhodospiraceae.

A Gram-negative, halophilic, heterotrophic, rod-shaped, non-spore-forming bacterium (SV525T) was isolated from the sediment of a hypersaline lake located at 4600 m above sea level (Laguna Vilama, Argentina). Strain SV525T was strictly aerobic and formed pink-to-magenta colonies. Growth occurred at 10­35 °C (optimum 25­30 °C), at pH levels 6.0­8.5 (optimum 7.0) and at NaCl concentrations of 7.5­25 % (w/v) with an optimum at 10­15 % (w/v). The strain required sodium and magnesium but not potassium ions for growth. Grows with tryptone, or Bacto Peptone as sole carbon and energy source and requires yeast extract for growth. It produced catalase and oxidase. The predominant ubiquinone was Q-8 and the major fatty acids comprised C18:1 ω7c, C16:0 and C18:0. The DNA G+C content was 60.4 mol% and its polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and a phosphoglycolipid. Phylogenetic analysis based on 16S rRNA gene indicated that strain SV525T belongs to the family Ectothiorhodospiraceae within the class Gammaproteobacteria. On the basis of phylogenetic and phenotypic data, SV525T represents a novel genus and species, for which the name Halopeptonella vilamensis gen. nov., sp. nov. is proposed. The type strain is SV525T (=DSM 21056T =JCM 16388T =NCIMB 14596T).

Spiribacter curvatus sp. nov., a moderately halophilic bacterium isolated from a saltern.

A novel pink-pigmented bacterial strain, UAH-SP71T, was isolated from a saltern located in Santa Pola, Alicante (Spain) and the complete genome sequence was analysed and compared with that of Spiribacter salinus M19-40T, suggesting that the two strains constituted two separate species, with a 77.3% ANI value. In this paper, strain UAH-SP71T was investigated in a taxonomic study using a polyphasic approach. Strain UAH-SP71T was a Gram-stain-negative, strictly aerobic, non-motile curved rod that grew in media containing 5-20% (w/v) NaCl (optimum 10% NaCl), at 5-40 °C (optimum 37 °C) and at pH 5-10 (optimum pH 8). Phylogenetic analysis based on the comparison of 16S rRNA gene sequences revealed thatstrain UAH-SP71T is a member of the genus Spiribacter, showing a sequence similarity of 96.5% with Spiribacter salinus M19-40T. Other related species are also members of the family Ectothiorhodospiraceae, including Arhodomonas recens RS91T (95.5% 16S rRNA gene sequence similarity), Arhodomonas aquaeolei ATCC 49307T (95.4 %) and Alkalilimnicola ehrlichii MLHE-1T (94.9 %). DNA-DNA hybridization between strain UAH-SP71T and Spiribacter salinus M19-40T was 39 %. The major cellular fatty acids of strain UAH-SP71T were C18 : 1ω6c and/or C18 : 1ω7c, C16 : 0, C16 : 1ω6c and/or C16 : 1ω7c, C10 : 0 3-OH and C12 : 0, a pattern similar to that of Spiribacter salinus M19-40T. Phylogenetic, phenotypic and genotypic differences between strain UAH-SP71T and Spiribacter salinus M19-40T indicate that strainUAH-SP71T represents a novel species of the genus Spiribacter, for which the name Spiribacter curvatus sp. nov. is proposed. The type strain is UAH-SP71T (5CECT8396T5DSM 28542T).

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp.

Laccases are oxidoreductases mostly studied in fungi, while bacterial laccases remain poorly studied despite their high genetic diversity and potential for biotechnological application. Our previous bioinformatic analysis identified alkaliphilic bacterial strains Thioalkalivibrio sp. as potential sources of robust bacterial laccases that would be stable at high pH. In the present work, a gene for a laccase-like enzyme from Thioalkalivibrio sp. ALRh was cloned and expressed as a 6× His-tagged protein in Escherichia coli. The purified enzyme was a pH-tolerant laccase stable in the pH range between 2.1 and 9.9 at 20 °C as shown by intrinsic fluorescence emission spectrometry. It had optimal activities at pH 5.0 and pH 9.5 with the laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol, respectively. In addition, it could oxidize several other monophenolic compounds and potassium hexacyanoferrate(II) but not tyrosine. It showed highest activity at 50 °C, making it suitable for prolonged incubations at this temperature. The present study shows that Thioalkalivibrio sp. encodes an active, alkaliphilic, and thermo-tolerant laccase and contributes to our understanding of the versatility of bacterial laccase-like multicopper oxidases in general.

Aquisalimonas lutea sp. nov., a moderately halophilic bacterium from a saltern.

A yellow-pigmented, motile, Gram-stain-negative, moderately halophilic and strictly aerobic bacterium, designated BA42AL-1(T), was isolated from water of a saltern of Santa Pola, Alicante, Spain. Strain BA42AL-1(T) grew in media containing 5-20% (w/v) salts (optimum 7.5% salts). It grew between pH 6.0 and 9.0 (optimally at pH 7.5) and at 15-45 °C (optimally at 37 °C). Phylogenetic analysis based on the comparison of 16S rRNA gene sequences revealed that strain BA42AL-1(T) is a member of the genus Aquisalimonas . The closest relatives to this strain were Aquisalimonas halophila YIM 95345(T) and Aquisalimonas asiatica CG12(T) with sequence similarities of 99.4% and 97.0%, respectively. DNA-DNA hybridization between the novel isolate and Aquisalimonas halophila YIM 95345(T) revealed a relatedness of 54%. The major fatty acids of strain BA42AL-1(T) were C(18 : 1)ω6c/C(18 : 1)ω7c, C(19 : 0) cyclo ω8c and C(16 : 0), and lower contents of C12 : 0 and C18 : 0. The polar lipid pattern of strain BA42AL-1(T) consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylcholine, two glycolipids, a lipid and four unknown phospholipids. The G+C content of the genomic DNA of this strain was 65.0 mol%. Based on the DNA-DNA hybridization, phenotypic, chemotaxonomic and phylogenetic data presented in this study, strain BA42AL-1(T) is proposed as a novel species of the genus Aquisalimonas , for which the name Aquisalimonas lutea sp. nov. is suggested. The type strain is BA42AL-1(T) ( = CCM 8472(T) = CECT 8326(T) = LMG 27614(T)).

Thiogranum longum gen. nov., sp. nov., an obligately chemolithoautotrophic, sulfur-oxidizing bacterium of the family Ectothiorhodospiraceae isolated from a deep-sea hydrothermal field, and an emended description of the genus Thiohalomonas.

A novel, obligately chemolithoautotrophic, sulfur-oxidizing bacterial strain, designated strain gps52(T), was isolated from a rock sample collected near the hydrothermal vents of the Suiyo Seamount in the Pacific Ocean. The cells possessed a Gram-stain-negative-type cell wall and contained menaquinone-8(H4) and menaquinone-9(H4) as respiratory quinones, and C16 : 1ω7c, C16 : 0 and C18 : 1ω7c as major cellular fatty acids. Neither storage compounds nor extensive internal membranes were observed in the cells. Strain gps52(T) grew using carbon dioxide fixation and oxidation of inorganic sulfur compounds with oxygen as electron acceptor. Optimal growth was observed at 32 °C, pH 6.5 and with 3 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain gps52(T) belongs to the family Ectothiorhodospiraceae and is different from any other known bacteria, with sequence similarities of less than 93 %. Based on phenotypic and phylogenetic findings, the isolate is considered to represent a novel genus and species in the family Ectothiorhodospiraceae, and the name Thiogranum longum gen. nov., sp. nov. is proposed. The type strain is gps52(T) ( = NBRC 101260(T) = DSM 19610(T)). An emended description of the genus Thiohalomonas is also proposed.

[Culturable psychrotolerant methanotrophic bacteria in landfill cover soil].

Methanotrophs closely related to psychrotolerant members of the genera Methylobacter and Methylocella were identified in cultures enriched at 10@C from landfill cover soil samples collected in the period from April to November. Mesophilic methanotrophs of the genera Methylobacter and Methylosinus were found in cultures enriched at 20 degrees C from the same cover soil samples. A thermotolerant methanotroph related to Methylocaldum gracile was identified in the culture enriched at 40 degrees C from a sample collected in May (the temperature of the cover soil was 11.5-12.5 degrees C). In addition to methanotrophs, methylobacteria of the genera Methylotenera and Methylovorus and members of the genera Verrucomicrobium, Pseudomonas, Pseudoxanthomonas, Dokdonella, Candidatus Protochlamydia, and Thiorhodospira were also identified in the enrichment cultures. A methanotroph closely related to the psychrotolerant species Methylobacter tundripaludum (98% sequence identity of 16S r-RNA genes with the type strain SV96(T)) was isolated in pure culture. The introduction of a mixture of the methanotrophic enrichments, grown at 15 degrees C, into the landfill cover soil resulted in a decrease in methane emission from the landfill surface in autumn (October, November). The inoculum used was demonstrated to contain methanotrophs closely related to Methylobacter tundripaludum SV96.

Arhodomonas sp. strain Seminole and its genetic potential to degrade aromatic compounds under high-salinity conditions.

Arhodomonas sp. strain Seminole was isolated from a crude oil-impacted brine soil and shown to degrade benzene, toluene, phenol, 4-hydroxybenzoic acid (4-HBA), protocatechuic acid (PCA), and phenylacetic acid (PAA) as the sole sources of carbon at high salinity. Seminole is a member of the genus Arhodomonas in the class Gammaproteobacteria, sharing 96% 16S rRNA gene sequence similarity with Arhodomonas aquaeolei HA-1. Analysis of the genome predicted a number of catabolic genes for the metabolism of benzene, toluene, 4-HBA, and PAA. The predicted pathways were corroborated by identification of enzymes present in the cytosolic proteomes of cells grown on aromatic compounds using liquid chromatography-mass spectrometry. Genome analysis predicted a cluster of 19 genes necessary for the breakdown of benzene or toluene to acetyl coenzyme A (acetyl-CoA) and pyruvate. Of these, 12 enzymes were identified in the proteome of toluene-grown cells compared to lactate-grown cells. Genomic analysis predicted 11 genes required for 4-HBA degradation to form the tricarboxylic acid (TCA) cycle intermediates. Of these, proteomic analysis of 4-HBA-grown cells identified 6 key enzymes involved in the 4-HBA degradation pathway. Similarly, 15 genes needed for the degradation of PAA to the TCA cycle intermediates were predicted. Of these, 9 enzymes of the PAA degradation pathway were identified only in PAA-grown cells and not in lactate-grown cells. Overall, we were able to reconstruct catabolic steps for the breakdown of a variety of aromatic compounds in an extreme halophile, strain Seminole. Such knowledge is important for understanding the role of Arhodomonas spp. in the natural attenuation of hydrocarbon-impacted hypersaline environments.

From metagenomics to pure culture: isolation and characterization of the moderately halophilic bacterium Spiribacter salinus gen. nov., sp. nov.

Recent metagenomic studies on saltern ponds with intermediate salinities have determined that their microbial communities are dominated by both Euryarchaeota and halophilic bacteria, with a gammaproteobacterium closely related to the genera Alkalilimnicola and Arhodomonas being one of the most predominant microorganisms, making up to 15% of the total prokaryotic population. Here we used several strategies and culture media in order to isolate this organism in pure culture. We report the isolation and taxonomic characterization of this new, never before cultured microorganism, designated M19-40(T), isolated from a saltern located in Isla Cristina, Spain, using a medium with a mixture of 15% salts, yeast extract, and pyruvic acid as the carbon source. Morphologically small curved cells (young cultures) with a tendency to form long spiral cells in older cultures were observed in pure cultures. The organism is a Gram-negative, nonmotile bacterium that is strictly aerobic, non-endospore forming, heterotrophic, and moderately halophilic, and it is able to grow at 10 to 25% (wt/vol) NaCl, with optimal growth occurring at 15% (wt/vol) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that strain M19-40(T) has a low similarity with other previously described bacteria and shows the closest phylogenetic similarity with species of the genera Alkalilimnicola (94.9 to 94.5%), Alkalispirillum (94.3%), and Arhodomonas (93.9%) within the family Ectothiorhodospiraceae. The phenotypic, genotypic, and chemotaxonomic features of this new bacterium showed that it constitutes a new genus and species, for which the name Spiribacter salinus gen. nov., sp. nov., is proposed, with strain M19-40(T) (= CECT 8282(T) = IBRC-M 10768(T) = LMG 27464(T)) being the type strain.