Halophilic bacteria in a Lake Michigan drainage basin as potential biological indicators of chloride-impacted freshwaters.

There are few biological indicators for freshwater systems subjected to high chloride levels. Freshwater systems receive many forms of chloride such as road salts (e.g., NaCl, CaCl2, MgCl2), fertilizers (e.g., KCl), and year-round water softener pollution. The goal our study was to investigate Halomonadaceae populations as prospective biological indicators of chloride-impacted freshwaters. The bacterial family Halomonadaceae are halophiles that generally require the presence of salt to survive, which make them an attractive candidate in determining chloride impaired areas. Field sediment surveys assessed how salt tolerant and halophilic bacteria abundance corresponded to chloride and conductivity measurements. Colony forming unit (CFU) counts on modified M9 6% NaCl plates (w/v) at urbanized sites compared to the rural sites had highest counts during winter and spring when chloride concentrations were also highest. Select isolates identified as Halomonadaceae through 16S rRNA sequencing were kept as active cultures to determine the NaCl concentration and temperature preference that resulted in the isolates optimal growth. Isolates tested under 5 °C (cold) grew optimally in 2 % NaCl (w/v), whereas under 18 °C (warm), isolates showed optimal growth at 6 % NaCl. The majority of isolates had maximum growth in the warmer temperature, however, select isolates grew better in the cold temperature. Culture-independent methods were used and identified Halomonadaceae were widespread and permeant members of the microbial community in a Lake Michigan drainage basin. Quantitative polymerase chain reaction (qPCR) targeting Halomonadaceae genera demonstrated that abundance varied by site, but overall were present throughout the year. However, community sequencing revealed there were a large relative proportion of specific Halomonadaceae populations present in winter versus summer. Methods targeting salt tolerant bacteria and specific members of Halomonadaceae appears to be a promising approach to assess chloride-impacted areas to better understand the long-term ecological impacts as we continue to salinize freshwater resources.

Isolation and characterization of a halophilic Modicisalibacter sp. strain Wilcox from produced water.

We report the isolation a halophilic bacterium that degrades both aromatic and aliphatic hydrocarbons as the sole sources of carbon at high salinity from produced water. Phylogenetic analysis of 16S rRNA-gene sequences shows the isolate is a close relative of Modicisalibacter tunisiensis isolated from an oil-field water in Tunisia. We designate our isolate as Modicisalibacter sp. strain Wilcox. Genome analysis of strain Wilcox revealed the presence of a repertoire of genes involved in the metabolism of aliphatic and aromatic hydrocarbons. Laboratory culture studies corroborated the predicted hydrocarbon degradation potential. The strain degraded benzene, toluene, ethylbenzene, and xylenes at salinities ranging from 0.016 to 4.0 M NaCl, with optimal degradation at 1 M NaCl. Also, the strain degraded phenol, benzoate, biphenyl and phenylacetate as the sole sources of carbon at 2.5 M NaCl. Among aliphatic compounds, the strain degraded n-decane and n-hexadecane as the sole sources of carbon at 2.5 M NaCl. Genome analysis also predicted the presence of many heavy metal resistance genes including genes for metal efflux pumps, transport proteins, and enzymatic detoxification. Overall, due to its ability to degrade many hydrocarbons and withstand high salt and heavy metals, strain Wilcox may prove useful for remediation of produced waters.

Isolation, identification, and whole genome sequence analysis of the alginate-degrading bacterium Cobetia sp. cqz5-12.

Alginate-degrading bacteria or alginate lyases can be used to oligomerize alginate. In this study, an alginate-degrading bacterium with high alginolytic activity was successfully screened by using Sargassum fusiforme sludge. When the strain was grown on a plate containing sodium alginate, the transparent ring diameter (D) was 2.2 cm and the ratio (D/d) of transparent ring diameter to colony diameter (d) was 8.8. After 36 h in culture at a temperature of 28 °C shaken at 150 r/min, the enzymatic activity of the fermentation supernatant reached 160 U/mL, and the enzymatic activity of the bacterial precipitate harvested was 2,645 U/mL. The strain was named Cobetia sp. cqz5-12. Its genome is circular in shape, 4,209,007 bp in size, with a 62.36% GC content. It contains 3,498 predicted coding genes, 72 tRNA genes, and 21 rRNA genes. The functional annotations for the coding genes demonstrated that there were 181 coding genes in the genome related to carbohydrate transport and metabolism and 699 coding genes with unknown functions. Three putative coding genes, alg2107, alg2108 and alg2112, related to alginate degradation were identified by analyzing the carbohydrate active enzyme (CAZy) database. Moreover, proteins Alg2107 and Alg2112 were successfully expressed and exhibited alginate lyase activity.

Description of Salinicola corii sp. nov., a Halotolerant Bacterium Isolated from Wetsalted Hides.

A novel Gram-stain-negative, moderately halotolerant, rod-shaped bacterium, designated strain L3T, was isolated from a wetsalted hide in Chengdu, China. The organism grew optimally at 30 °C, at pH 8 and with 5-10% (w/v) NaCl. The major cellular fatty acids were C16:0, C16:1ω7c, C18:1ω7c and C19:0 cyclo ω8c; the predominant respiratory quinone was Q-9; the phospholipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and three unidentified phospholipids. Phylogenetic trees based on the 16S rRNA, gyrB and rpoD genes' sequences, obtained using three different algorithms, clearly revealed the isolate belonged to the genus Salinicola, and was found to be closely related to Salinicola acroporae JCM 30412T, Salinicola socius CGMCC 1.12383T and Salinicola lusitanus CR50T. The draft genome was approximately 4.5 Mb in size with 4486 predicted coding sequences, and the G+C content was 62.6 mol%. The maximum values of ANI and dDDH between strain L3T and the three above-mentioned type species were 89.2% and 63.8%, respectively. Differential phenotypic properties, together with the genome analysis, support the view that strain L3T represents a novel species, Salinicola corii sp. nov., with the type strain L3T (=CGMCC 1.17272T=KCTC 72572T).

Aidingimonas lacisalsi sp. nov., isolated from a salt lake in China.

A novel bacterium, XHU 5135T, belonging to the genus Aidingimonas, was isolated from a salt lake sample collected in Xinjiang Province, north-west PR China. The isolate was Gram-stain-negative, rod-shaped and non-motile. The strain was catalase-positive and oxidase-negative. Growth occurred at NaCl concentrations of 5-25 % (optimum, 10-13 %), at 13-41 °C (35-37 °C) and at pH 6.0-10.0 (pH 7.0-8.0). The predominant ubiquinone was Q-9. The major fatty acids were C19 : 0 cyclo ω8c and C16 : 0. The G+C content of the genomic DNA was 58.1 mol%. The affiliation of strain XHU 5135T with the genus Aidingimonas was confirmed by 16S rRNA gene sequence comparisons. The closest type strain was Aidingimonas halophile YIM 90637T, which showed a 16S rRNA gene sequence similarity of 97.5 %. The ANI value between XHU 5135T and the closest type strain was 80.01 %. The estimated digital DNA-DNA hybridization estimate value between strain XHU 5135T and the closest type strain was 22.80 %. Phenotypically, the characteristics of XHU 5135T were shown to differ from the most closely related species, A. halophila. On the basis of the data from this polyphasic study, strain XHU 5135T represents a novel species of the genus Aidingimonas, for which the name Aidingimonas lacisalsi sp. nov. is proposed. The type strain is strain XHU 5135T (=CCTCC AB 2016344T=KCTC 42945T=DSM 104700T).

The endosphere of the salt marsh plant Halimione portulacoides is a diversity hotspot for the genus Salinicola: description of five novel species Salinicola halimionae sp. nov., Salinicola aestuarinus sp. nov., Salinicola endophyticus sp. nov., Salinicola halophyticus sp. nov. and Salinicola lusitanus sp. nov.

Seven endophytic strains were isolated from the halophyte Halimione portulacoides, collected from Ria de Aveiro, Portugal. To determine their exact taxonomic position, comparative analyses were performed with these strains and closely related type strains of Salinicola species. Genome sequencing and comparison indicated that five of the seven isolated strains comprised distinct and novel species (average nucleotide identity <0.95; in silico DNA-DNA hybridization <70 %; G+C difference >1 %). Multilocus sequence analysis was performed using gyrB, rpoD and 16S rRNA gene sequences from the novel and type strains to determine their phylogenetic positions. The novel strains are facultative anaerobes, mesophilic, facultative alkaliphic and halophilic, test positive for catalase and oxidase activities, for hydrolysis of Tween 20 and phosphate, for production of indole-3-acetic acid, but do not produce H2S. Ubiquinone UQ-9 is present in major amounts in all strains. The major fatty acids include C16 : 0 and the summed feature containing C18 : 1ω7c and/or C18 : 1ω6c. The DNA G+C content ranges from 60.6 to 65.8 mol%. Five strains were confirmed as new species belonging to the genus Salinicola, for which the names Salinicolahalimionae sp. nov. (type strain CPA60T=CECT 9338T=LMG 30107T), Salinicolaaestuarinus sp. nov. (type strain CPA62T=CECT 9339T=LMG 30108T), Salinicolaendophyticus sp. nov. (type strain CPA92T=CECT 9340T=LMG 30109T), Salinicolahalophyticus sp. nov. (type strain CR45T=CECT 9341T=LMG 30105T) and Salinicola lusitanus sp. nov. (type strain CR50T=CECT 9342T=LMG 30106T) are proposed.

Infection dynamics of endosymbionts reveal three novel localization patterns of Rickettsia during the development of whitefly Bemisia tabaci.

The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is a severe agricultural pest that harbors at least seven endosymbionts. Many important aspects of the symbiosis mechanism between these bacterial endosymbionts and their hosts are poorly understood, such as endosymbiont proliferation dynamics, spatial distribution and titer regulation during host development. In this study, infection by bacterial endosymbionts in the whitefly B. tabaci Middle East-Asia Minor-1 (MEAM1, formerly B biotype) South China population, their infection titers in various stages of whitefly host development and their spatial localization were investigated. Results revealed that the MEAM1 B. tabaci harbors the primary symbiont Portiera and secondary symbionts Rickettsia and Hamiltonella. The titers of these three endosymbionts increased with the development of their B. tabaci host. Significant proliferation of Portiera and Hamiltonella mainly occurred during the second to fourth instar nymphal stages, while Rickettsia proliferated mainly during adult eclosion. Fluorescence in situ hybridization analysis of B. tabaci adults revealed three novel infection patterns of Rickettsia: assemblage in the bacteriocytes that scattered through the entire abdomen of the female host, localization in wax glands and localization in the colleterial gland. These novel infection patterns may help to uncover the function of Rickettsia in its insect hosts.

Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov., plant growth promoting endophytes isolated from the halophyte plant Arthrocnemum macrostachyum.

Two endophytic bacteria (EAod3T and EAod7T) were isolated from the aerial part of plants of Arthrocnemum macrostachyum growing in the Odiel marshes (Huelva, Spain). Phylogenetic analysis based on 16S rRNA gene sequences indicated their affiliation to the genus Kushneria. 16S rRNA gene sequences of strains EAod3T and EAod7T showed the highest similarity to Kushneria marisflavi DSM 15357T (99.0 and 97.6 %, respectively). Digital DNA-DNA hybridization studies between the draft genomes of strain EAod3T and K. marisflavi DSM 15357T corresponded to 28.5 % confirming the novel lineage of strain EAod3T in the genus Kushneria. Cells of both strains were Gram-staining-negative, aerobic and motile rods able to grow at 4-37 °C, at pH 5.0-8.0 and tolerate 0.5-25 % NaCl (w/v). They presented ubiquinone Q9 and C16 : 0, C16 : 1ω7c/C16 : 1ω6c and C18 : 1ω7c as the major fatty acids. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Based on the phenotypic and phylogenetic results, strains EAod3T (=CECT 9073T=LMG 29856T) and EAod7T (=CECT 9075T=LMG 29858T) are proposed as new representatives of the genus Kushneria, and the proposed names are Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov., respectively. The whole genome sequence of strain EAod3T has a total length of 3.8 Mbp and a G+C content of 59.3 mol%.

Kushneria konosiri sp. nov., isolated from the Korean salt-fermented seafood Daemi-jeot.

A halophilic bacterial strain, X49T, was isolated from the Korean traditional salt-fermented seafood Daemi-jeot. X49T was an obligately aerobic, Gram-stain-negative, motile, oval or rod-shaped (0.5-1.0×1.2-3.2 µm) bacterium. After 2 days of growth, colonies on Marine agar medium were orange and circular with entire margins. X49T growth was detected at 10-37 °C and pH 4.5-8.5 in the presence of 0-26 % (w/v) NaCl. The 16S rRNA gene sequence of strain X49T was most similar to that of the type strain of Kushneria marisflavi SW32T and shared a sequence similarity of 94.7-98.6 % with type strains of species of the genus Kushneria. The predominant fatty acids were C16 : 0, C18 : 1ω7c and C12 : 0 3OH. The major isoprenoid quinone was Q9 (93 %), and minor quinones were Q8 (4 %) and Q10 (3 %). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylserine, two unidentified aminolipids, two unidentified phospholipids and two unidentified lipids. The genomic DNA G+C content was 59.1 mol%. The level of the ANI value between strain X49T and K. marisflavi SW32T, the most closely related species of the genus Kushneria, was 89.32 %. Based on the low ANI value, strain X49T and its reference strains represent genotypically distinct species. Based on this polyphasic taxonomic analysis, strain X49T represents a novel species of the genus Kushneria. The name Kushneria konosiri sp. nov. is proposed and the type strain is X49T (=KACC 14623T=JCM 16805T).

Salinicola tamaricis sp. nov., a heavy-metal-tolerant, endophytic bacterium isolated from the halophyte Tamarix chinensis Lour.

A Gram-stain-negative, rod-shaped bacterium, strain F01T, was isolated from leaves of Tamarix chinensis Lour. The isolate grew optimally at 30 °C, at pH 7.0 and with 5.0 % (w/v) NaCl, and showed a high tolerance to manganese, lead, nickel, ferrous ions and copper ions. The major fatty acids were C18 : 1ω7c and C16 : 0, and the predominant respiratory quinone was Q-9. Polar lipids were dominated by diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, unidentified aminoglycolipids and phospholipids. The DNA G+C content was 65.8 %. Based on multilocus phylogenetic analysis, strain F01T belonged to the genus Salinicola, with highest 16S rRNA gene sequence similarity to Salinicola peritrichatus CGMCC 1.12381T (97.7 %). The level of DNA-DNA hybridization between strain F01T and closely related Salinicola strains was well below 70 %. According to the phenotypic, genetic and chemotaxonomic data, strain F01T is considered to represent a novel species in the genus Salinicola, for which the name Salinicola tamaricis sp. nov. is proposed. The type strain is F01T (=CCTCC AB 2015304T=KCTC 42855T).

Compositional differences among female-associated and embryo-associated microbiota of the viviparous Pacific Beetle cockroach, Diploptera punctata.

All cockroach species, except one, harbor the endosymbiont Blattabacterium, transmitted from females to embryos. Adult cockroaches acquire non-Blattabacterium bacteria as part of their gut microbiota over time, but our knowledge of the possible transmission of these non-Blattabacterium bacteria from females to embryos is rudimentary. We characterized the gut microbiota of gravid viviparous Diploptera punctata females and the non-Blattabacterium microbiota of associated developing embryos, as well as the gut microbiota of non-gravid females, and the microbiota of orphan embryos (females not included), following high-throughput sequencing of the 16S rRNA gene to assess bacterial transference. We determined significant differences in community composition between gravid females and associated embryos and overall greater similarity in community composition among embryos than adult females. Results suggest various routes of transference of bacteria from females or the environment to embryos. The bacterial families Halomonadaceae and Shewanellaceae were more abundant in embryos than in gravid females. The functional relevance of these families remains to be elucidated, but provisioning of amino acids deficient in the brood sac secretion is a possibility. Overall, our results highlight the need for further studies investigating the uptake and selective screening of microbes by D. punctata embryos, as well as their functions.

Phylogeographic analyses of bacterial endosymbionts in fig homotomids (Hemiptera: Psylloidea) reveal codiversification of both primary and secondary endosymbionts.

While obligate primary (P-) endosymbionts usually cospeciate with their insect hosts, less is known about codiversification of secondary (S-) endosymbionts that are generally considered facultative. Typically, insects of the superfamily Psylloidea harbour one P- (Carsonella) and at least one S-endosymbiont, thought to compensate for Carsonella genome reduction. Most codiversification studies have used phylogenies of psyllids and their endosymbionts across and within host families or genera, but few have explored patterns within species. We focussed on P- and S-endosymbionts of three Mycopsylla (Homotomidae) species to explore whether they have congruent phylogenies and within-species geographic structures. The P-endosymbiont Carsonella, a S-endosymbiont and Wolbachia all had 100% prevalence, while Arsenophonus was only found in one species at low prevalence. Congruent phylogenies of Mycopsylla and P-endosymbionts across populations and species support strict cospeciation. S-endosymbiont phylogenies were also congruent across host species but low genetic variation in the S-endosymbiont was not correlated with host phylogeography, possibly due to a shorter evolutionary association. Between species, Wolbachia and Mycopsylla phylogenies were incongruent, probably due to horizontal transmission events. Our study is the first to explore endosymbionts of Mycopsylla and further supports the codivergence of Psylloidea hosts and P-endosymbionts, with obligate host interactions for both P- and S-endosymbionts.

Pistricoccus aurantiacus gen. nov., sp. nov., a moderately halophilic bacterium isolated from a shark.

A novel Gram-stain negative, non-motile, moderately halophilic, facultatively anaerobic and spherical bacterium designated strain SS9T was isolated from the gill homogenate of a shark. Cells of SS9T were observed to be 0.8-1.2 µm in diameter. The strain was found to grow optimally at 33 °C, pH 7.0-8.0 and in the presence of 6.0 % (w/v) NaCl. On the basis of 16S rRNA gene phylogeny, strain SS9T can be affiliated with the family Halomonadaceae and is closely related to Chromohalobacter marismortui NBRC 103155T (95.6 % sequence similarity), Halomonas ilicicola SP8T (95.6 %) and Chromohalobacter salexigens DSM 3043T (95.5 %). Multilocus sequence analysis of strain SS9T using the housekeeping genes 16S rRNA, 23S rRNA, gyrB, rpoD and secA revealed the strain's distinct phylogenetic position, separate from other known genera of the family Halomonadaceae. Strain SS9T was found to contain ubiquinone-9 (Q-9) as the predominant ubiquinone and C18:1 ω7c, C16:0 and summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH) as the major fatty acids. The major polar lipids of strain SS9T were identified as phosphatidylglycerol and phosphatidylethanolamine. The DNA G + C content of strain SS9T was determined to be 60.4 mol%. It is evident from phylogenetic, genotypic, phenotypic and chemotaxonomic results that strain SS9T represents a novel species in a new genus, for which the name Pistricoccus aurantiacus gen. nov., sp. nov. is proposed. The type strain is SS9T (=KCTC 42586T = MCCC 1H00111T).

Larsenimonas suaedae sp. nov., a moderately halophilic, endophytic bacterium isolated from the halophyte Suaeda salsa.

A moderately halophilic, Gram-stain-negative, non-endospore-forming endophytic bacterium designated strain ST307T was isolated from the euhalophyte Suaeda salsa in Dongying, China. Strain ST307T was aerobic, rod-shaped, motile and orange-yellow-pigmented. The organism grew at NaCl concentrations of 0.6-20 % (w/v) (optimum 5-6 %, w/v), at temperatures of 5-45 °C (optimum 35 °C) and at pH 5-9 (optimum pH 7-8). It accumulated poly-ß-hydroxybutyric acid and produced exopolysaccharides. The major fatty acids were C18 : 1ω7c/C18 : 1ω6c, C16 : 0 and C16 : 1ω7c/C16 : 1ω6c. The predominant lipoquinone was ubiquinone Q-9. The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, a glycoaminolipid and a phosphoglycoaminolipid. The DNA G+C content was 60.5 mol%. Phylogenetic analyses of 16S rRNA gene sequences and concatenated atpA, rpoD and secA gene sequences revealed that the strain represents a member of the genus Larsenimonas. The closest related type strain was Larsenimonas salina M1-18T. Mean DNA-DNA relatedness values between strain ST307T and the related species L. salina M1-18T, Chromohalobacter beijerinckii DSM 7218T, C. canadensis DSM 6769T, C. israelensis DSM 6768T, C. marismortui CGMCC 1.2321T, C. nigrandesensis DSM 14323T, C. salexigens DSM 3043T and C. sarecensis DSM 15547T were 15±2-45±1 %. On the basis of phenotypic, chemotaxonomic and molecular features, strain ST307T clearly represents a novel species of the genus Larsenimonas. The name Larsenimonassuaedae sp. nov. is proposed, with ST307T (=CGMCC 1.8902T=DSM 22428T) as the type strain.

Comparative 16S rRNA signatures and multilocus sequence analysis for the genus Salinicola and description of Salinicola acroporae sp. nov., isolated from coral Acropora digitifera.

A novel Gram-negative, aerobic, motile marine bacterium, strain S4-41(T), was isolated from mucus of the coral Acropora digitifera from the Andaman Sea. Heterotrophic growth was observed in 0-25 % NaCl, at 15-45 °C and pH 4.5-9. In phylogenetic trees, strain S4-41(T) was grouped within the genus Salinicola but formed a separate branch distant from a cluster composed of Salinicola salarius M27(T) and Salinicola socius SMB35(T). DNA-DNA relatedness between strain S4-41(T) and these reference strains were well below 70 %. Q-9 was the sole respiratory quinone. The DNA G+C content was determined to be 63.6 mol%. Based on a polyphasic analysis, strain S4-41(T) is concluded to represent a novel species in the genus Salinicola for which the name Salinicola acroporae sp. nov. is proposed. The type strain is S4-41(T) (=JCM 30412(T) = LMG 28587(T)). Comparative 16S rRNA analysis of the genera Salinicola, Kushneria, Chromohalobacter and Cobetia revealed the presence of genus specific sequence signatures. Multilocus sequence analysis based on concatenated sequences of rRNAs (16S and 23S) and four protein coding housekeeping genes (atpA, gyrB, secA, rpoD) was found to be unnecessary for phylogenetic studies of the genus Salinicola.

High-throughput screening for a moderately halophilic phenol-degrading strain and its salt tolerance response.

A high-throughput screening system for moderately halophilic phenol-degrading bacteria from various habitats was developed to replace the conventional strain screening owing to its high efficiency. Bacterial enrichments were cultivated in 48 deep well microplates instead of shake flasks or tubes. Measurement of phenol concentrations was performed in 96-well microplates instead of using the conventional spectrophotometric method or high-performance liquid chromatography (HPLC). The high-throughput screening system was used to cultivate forty-three bacterial enrichments and gained a halophilic bacterial community E3 with the best phenol-degrading capability. Halomonas sp. strain 4-5 was isolated from the E3 community. Strain 4-5 was able to degrade more than 94% of the phenol (500 mg · L(-1) starting concentration) over a range of 3%-10% NaCl. Additionally, the strain accumulated the compatible solute, ectoine, with increasing salt concentrations. PCR detection of the functional genes suggested that the largest subunit of multicomponent phenol hydroxylase (LmPH) and catechol 1,2-dioxygenase (C12O) were active in the phenol degradation process.

Kushneria pakistanensis sp. nov., a novel moderately halophilic bacterium isolated from rhizosphere of a plant (Saccharum spontaneum) growing in salt mines of the Karak area in Pakistan.

The taxonomic position of a Gram-stain negative, moderately halophilic bacterium, designated NCCP-934(T), was investigated using polyphasic taxonomic approach. The strain NCCP-934(T) was isolated from rhizosphere of a plant (Saccharum spontaneum, family Poaceae) growing in salt mines area in the Karak district of Khyber Pakhtunkhwa Province, Pakistan. Cells of strain NCCP-934(T) are rod shaped and motile. The bacterium is strictly aerobic, can grow at a temperature range of 10-40 °C (optimum at 30-33 °C) and in a pH range of 6.0-10.5 (optimum pH 7.0-9.0). The strain can tolerate 1-30 % (w/v) NaCl (optimal growth occurs in the presence of approximately 3-9 % NaCl). The phylogenetic analysis based on the 16S rRNA gene sequence, showed that strain NCCP-934(T) belongs to the genus Kushneria with the highest sequence similarity to K. marisflavi SW32(T) (98.9 %), K. indalinina CG2.1(T) (98.7 %), K. avicenniae MW2a(T) (98.4 %) and less than 97 % similarity with other related species (94.7 % with the type species of the genus, K. aurantia A10(T)). DNA-DNA relatedness between strain NCCP-934(T) and the type strains of the closely related species was lower than 18 %. The chemotaxonomic data (major respiratory quinone, Q9; predominant fatty acids, C18:1 ω7c and C16:0 followed by C12:0 3-OH and Summed features 3 (C16:1 ω7c/iso-C15:0 2-OH); major polar lipids, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmonomethylethanol, phosphatidylserine, phosphatidylinositol and three polar lipid of unknown structure) supported the affiliation of strain NCCP-934(T) within the genus Kushneria. The DNA G+C content of strain NCCP-934(T) was 59.2 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain NCCP-934(T) can be distinguished from the closely related taxa and thus represents a novel species in the genus Kushneria, for which the name Kushneria pakistanensis sp. nov. is proposed, with the type strain NCCP-934(T) (=LMG 28525(T) = KCTC 42082(T) = JCM 18802(T)).

Larsenia salina gen. nov., sp. nov., a new member of the family Halomonadaceae based on multilocus sequence analysis.

Two Gram-staining-negative, moderately halophilic bacteria, strains M1-18(T) and L1-16, were isolated from a saltern located in Huelva (Spain). They were motile, strictly aerobic rods, growing in the presence of 3-25% (w/v) NaCl (optimal growth at 7.5-10% [w/v] NaCl), between pH 4.0 and 9.0 (optimal at pH 6.0-7.0) and at temperatures between 15 and 40°C (optimal at 37°C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed the higher similarity values with Chromohalobacter israelensis ATCC 43985(T) (95.2-94.8%) and Chromohalobacter salexigens DSM 3043(T) (95.0-94.9%), and similarity values lower than 94.6% with other species of the genera Chromohalobacter, Kushneria, Cobetia or Halomonas. Multilocus sequence analysis (MLSA) based on the partial sequences of atpA, rpoD and secA housekeeping genes indicated that the new isolates formed an independent and monophyletic branch that was related to the peripheral genera of the family Halomonadaceae, Halotalea, Carnimonas and Zymobacter, supporting their placement as a new genus of the Halomonadaceae. The DNA-DNA hybridization between both strains was 82%, whereas the values between strain M1-18(T) and the most closely related species of Chromohalobacter and Kushneria were equal or lower to 48%. The major cellular fatty acids were C18:1ω7c/C18:1ω6c, C16:0, and C16:1ω7c/C16:1ω6c, a profile that differentiate this new taxon from species of the related genera. We propose the placement of both strains as a novel genus and species, within the family Halomonadaceae, with the name Larsenia salina gen. nov., sp. nov. The type strain is M1-18(T) (=CCM 8464=CECT 8192(T)=IBRC-M 10767(T)=LMG 27461(T)).

No exception to the rule: Candidatus Portiera aleyrodidarum cell wall revisited.

Many insect endosymbionts described so far are gram-negative bacteria. Primary endosymbionts are obligatory bacteria usually harboured by insects inside vacuoles in specialized cells called bacteriocytes. This combination produces a typical three-membrane system with one membrane derived from the insect vacuole and the other two from the bacterial gram-negative cell envelope, composed by the cell wall (the outer membrane plus the periplasmic space) and the plasma membrane (the inner membrane). For the last 21 years, the primary endosymbiont of whiteflies 'Candidatus Portiera aleyrodidarum' was considered an exception to this rule. Previous works stated that only two membranes were present, the vacuolar membrane and one of the two bacterial membranes. The absence of the cell wall was related to the special vertical transmission of the endosymbionts in whiteflies. In this work, we present electron microscopic studies showing a complete cell envelope in 'Ca. Portiera aleyrodidarum' from the whitefly Bemisia tabaci. Additionally, comparison of the inferred metabolism from the gene content did not show any difference in cell envelope biogenesis compared with the closely related three-membrane endosymbionts 'Candidatus Carsonella ruddii' and 'Candidatus Evansia muelleri' Xc1. Our results rule out the proposal that 'Ca. Portiera aleyrodidarum' is an exception to the three-membrane system.

Induction of apoptosis in cancer cell lines by the Red Sea brine pool bacterial extracts.

BACKGROUND: Marine microorganisms are considered to be an important source of bioactive molecules against various diseases and have great potential to increase the number of lead molecules in clinical trials. Progress in novel microbial culturing techniques as well as greater accessibility to unique oceanic habitats has placed the marine environment as a new frontier in the field of natural product drug discovery. METHODS: A total of 24 microbial extracts from deep-sea brine pools in the Red Sea have been evaluated for their anticancer potential against three human cancer cell lines. Downstream analysis of these six most potent extracts was done using various biological assays, such as Caspase-3/7 activity, mitochondrial membrane potential (MMP), PARP-1 cleavage and expression of γH2Ax, Caspase-8 and -9 using western blotting. RESULTS: In general, most of the microbial extracts were found to be cytotoxic against one or more cancer cell lines with cell line specific activities. Out of the 13 most active microbial extracts, six extracts were able to induce significantly higher apoptosis (>70%) in cancer cells. Mechanism level studies revealed that extracts from Chromohalobacter salexigens (P3-86A and P3-86B(2)) followed the sequence of events of apoptotic pathway involving MMP disruption, caspase-3/7 activity, caspase-8 cleavage, PARP-1 cleavage and Phosphatidylserine (PS) exposure, whereas another Chromohalobacter salexigens extract (K30) induced caspase-9 mediated apoptosis. The extracts from Halomonas meridiana (P3-37B), Chromohalobacter israelensis (K18) and Idiomarina loihiensis (P3-37C) were unable to induce any change in MMP in HeLa cancer cells, and thus suggested mitochondria-independent apoptosis induction. However, further detection of a PARP-1 cleavage product, and the observed changes in caspase-8 and -9 suggested the involvement of caspase-mediated apoptotic pathways. CONCLUSION: Altogether, the study offers novel findings regarding the anticancer potential of several halophilic bacterial species inhabiting the Red Sea (at the depth of 1500-2500 m), which constitute valuable candidates for further isolation and characterization of bioactive molecules.