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A strictly aerobic, Gram-stain-negative, rod-shaped, and motile bacterium, designated strain KMM 296, isolated from the coelomic fluid of the mussel Crenomytilus grayanus , was investigated in detail due to its ability to produce a highly active alkaline phosphatase CmAP of the structural family PhoA. A previous taxonomic study allocated the strain to the species Cobetia marina , a member of the family Halomonadaceae of the class Gammaproteobacteria . However, 16S rRNA gene sequencing showed KMM 296's relatedness to Cobetia amphilecti NRIC 0815 T . The isolate grew with 0.5-19% NaCl at 4-42 °C and hydrolyzed Tweens 20 and 40 and L-tyrosine. The DNA G+C content was 62.5 mol%. The prevalent fatty acids were C 18:1 ω7c, C 12:0 3-OH, C 18:1 ω7c, C 12:0 , and C 17:0 cyclo. The polar lipid profile was characterized by the presence of phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, and also an unidentified aminolipid, phospholipid, and a few unidentified lipids. The major respiratory quinone was Q-8. According to phylogenomic and chemotaxonomic evidence, and the nearest neighbors, the strain KMM 296 represents a member of the species C. amphilecti . The genome-based analysis of C. amphilecti NRIC 0815 T and C. litoralis NRIC 0814 T showed their belonging to a single species. In addition, the high similarity between the C. pacifica NRIC 0813 T and C. marina LMG 2217 T genomes suggests their affiliation to one species. Based on the rules of priority, C. litoralis should be reclassified as a later heterotypic synonym of C. amphilecti , and C. pacifica is a later heterotypic synonym of C. marina . The emended descriptions of the species C. amphilecti and C. marina are also proposed.

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, CaCl 2 , MgCl 2 ), 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Horizontally transferred genes (HTGs) play a key role in animal symbiosis, and some horizontally transferred genes or proteins are highly expressed in specialized host cells (bacteriocytes). However, it is not clear how HTGs are regulated, but microRNAs (miRNAs) are prime candidates given their previously demonstrated roles in symbiosis and impacts on the expression of host genes. A horizontally acquired PanBC that is highly expressed in whitefly bacteriocytes can cooperate with an obligate symbiont Portiera for pantothenate production, facilitating whitefly performance and Portiera titre. Here, we found that a whitefly miRNA, novel-m0780-5p, was up-regulated and its target panBC was down-regulated in Portiera-eliminated whiteflies. This miRNA was located in the cytoplasmic region of whitefly bacteriocytes. Injection of novel-m0780-5p agomir reduced the expression of PanBC in whitefly bacteriocytes, while injection of novel-m0780-5p antagomir enhanced PanBC expression. Agomir injection also reduced the pantothenate level, Portiera titre and whitefly performance. Supplementation with pantothenate restored Portiera titre and the fitness of agomir-injected whiteflies. Thus, we demonstrate that a whitefly miRNA regulates panBC-mediated host-symbiont collaboration required for pantothenate synthesis, benefiting the whitefly-Portiera symbiosis. Both panBC and novel-m0780-5p are present in the genomes of six Bemisia tabaci species. The expression of a novel miRNA in multiple B. tabaci species suggests that the miRNA evolved after panBC acquisition, and allowed this gene to be more tightly regulated. Our discovery provides the first account of a HTG being regulated by a miRNA from the host genome, and suggests key roles for interactions between miRNAs and HTGs in the functioning of symbiosis., (© 2022 John Wiley & Sons Ltd.)

Nutritional symbionts are restricted to specialized host cells called bacteriocytes in various insect orders. These symbionts can provide essential nutrients to the host. However, the cellular mechanisms underlying the regulation of these insect-symbiont metabolic associations remain largely unclear. The whitefly Bemisia tabaci MEAM1 hosts " Candidatus Portiera aleyrodidarum" (here, " Ca . Portiera") and " Candidatus Hamiltonella defensa" (here, " Ca . Hamiltonella") bacteria in the same bacteriocyte. In this study, the induction of autophagy by chemical treatment and gene silencing decreased symbiont titers and essential amino acid (EAA) and B vitamin contents. In contrast, the repression of autophagy in bacteriocytes via Atg8 silencing increased symbiont titers, and amino acid and B vitamin contents. Furthermore, dietary supplementation with non-EAAs or B vitamins alleviated autophagy in whitefly bacteriocytes, elevated TOR (target of rapamycin) expression, and increased symbiont titers. TOR silencing restored symbiont titers in whiteflies after dietary supplementation with B vitamins. These data suggest that " Ca. Portiera" and " Ca . Hamiltonella" evade autophagy of the whitefly bacteriocytes by activating the TOR pathway via providing essential nutrients. Taken together, we demonstrate that autophagy plays a critical role in regulating the metabolic interactions between the whitefly and two intracellular symbionts. Therefore, this study reveals that autophagy is an important cellular basis for bacteriocyte evolution and symbiosis persistence in whiteflies. The whitefly symbiosis unravels the interactions between cellular and metabolic functions of bacteriocytes. IMPORTANCE Nutritional symbionts, which are restricted to specialized host cells called bacteriocytes, can provide essential nutrients for many hosts. However, the cellular mechanisms of regulation of animal-symbiont metabolic associations have been largely unexplored. Here, using the whitefly-" Ca . Portiera"/" Ca . Hamiltonella" endosymbiosis, we demonstrate autophagy regulates the symbiont titers and thereby alters the essential amino acid and B vitamin contents. For persistence in the whitefly bacteriocytes, " Ca . Portiera" and " Ca . Hamiltonella" alleviate autophagy by activating the TOR (target of rapamycin) pathway through providing essential nutrients. Therefore, we demonstrate that autophagy plays a critical role in regulating the metabolic interactions between the whitefly and two intracellular symbionts. This study also provides insight into the cellular basis of bacteriocyte evolution and symbiosis persistence in the whitefly. The mechanisms underlying the role of autophagy in whitefly symbiosis could be widespread in many insect nutritional symbioses. These findings provide a new avenue for whitefly control via regulating autophagy in the future.

Background: Several members of the bacterial Halomonadacea family are natural producers of polyhydroxyalkanoates (PHA), which are promising materials for use as biodegradable bioplastics. Type-strain species of Cobetia are designated PHA positive, and recent studies have demonstrated relatively high PHA production for a few strains within this genus. Industrially relevant PHA producers may therefore be present among uncharacterized or less explored members. In this study, we characterized PHA production in two marine Cobetia strains. We further analyzed their genomes to elucidate pha genes and metabolic pathways which may facilitate future optimization of PHA production in these strains., Results: Cobetia sp. MC34 and Cobetia marina DSM 4741 T were mesophilic, halotolerant, and produced PHA from four pure substrates. Sodium acetate with- and without co-supplementation of sodium valerate resulted in high PHA production titers, with production of up to 2.5 g poly(3-hydroxybutyrate) (PHB)/L and 2.1 g poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/L in Cobetia sp. MC34, while C. marina DSM 4741 T produced 2.4 g PHB/L and 3.7 g PHBV/L. Cobetia marina DSM 4741 T also showed production of 2.5 g PHB/L from glycerol. The genome of Cobetia sp. MC34 was sequenced and phylogenetic analyses revealed closest relationship to Cobetia amphilecti. PHA biosynthesis genes were located at separate loci similar to the arrangement in other Halomonadacea. Further genome analyses revealed some differences in acetate- and propanoate metabolism genes between the two strains. Interestingly, only a single PHA polymerase gene (phaC 2 ) was found in Cobetia sp. MC34, in contrast to two copies (phaC 1 and phaC 2 ) in C. marina DSM 4741 T . In silico analyses based on phaC genes show that the PhaC 2 variant is conserved in Cobetia and contains an extended C-terminus with a high isoelectric point and putative DNA-binding domains., Conclusions: Cobetia sp. MC34 and C. marina DSM 4741 T are natural producers of PHB and PHBV from industrially relevant pure substrates including acetate. However, further scale up, optimization of growth conditions, or use of metabolic engineering is required to obtain industrially relevant PHA production titers. The putative role of the Cobetia PhaC 2 variant in DNA-binding and the potential implications remains to be addressed by in vitro- or in vivo methods., (© 2021. The Author(s).)

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.

Marine bacteria of the genus Cobetia, which are promising sources of unique enzymes and secondary metabolites, were found to be complicatedly identified both by phenotypic indicators due to their ecophysiology diversity and 16S rRNA sequences because of their high homology. Therefore, searching for the additional methods for the species identification of Cobetia isolates is significant. The species-specific coding sequences for the enzymes of each functional category and different structural families were applied as additional molecular markers. The 13 closely related Cobetia isolates, collected in the Pacific Ocean from various habitats, were differentiated by the species-specific PCR patterns. An alkaline phosphatase PhoA seems to be a highly specific marker for C. amphilecti. However, the issue of C. amphilecti and C. litoralis , as well as C. marina and C. pacifica, belonging to the same or different species remains open.

Whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) are a superfamily of small phloem-feeding insects. They rely on their primary endosymbionts "Candidatus Portiera aleyrodidarum" to produce essential amino acids not present in their diet. Portiera has been codiverging with whiteflies since their origin and therefore reflects its host's evolutionary history. Like in most primary endosymbionts, the genome of Portiera stays stable across the Aleyrodidae superfamily after millions of years of codivergence. However, Portiera of the whitefly Bemisia tabaci has lost the ancestral genome order, reflecting a rare event in the endosymbiont evolution: the appearance of genome instability. To gain a better understanding of Portiera genome evolution, identify the time point in which genome instability appeared and contribute to the reconstruction of whitefly phylogeny, we developed a new phylogenetic framework. It targeted five Portiera genes and determined the presence of the DNA polymerase proofreading subunit (dnaQ) gene, previously associated with genome instability, and two alternative gene rearrangements. Our results indicated that Portiera gene sequences provide a robust tool for studying intergenera phylogenetic relationships in whiteflies. Using these new framework, we found that whitefly species from the Singhiella, Aleurolobus, and Bemisia genera form a monophyletic tribe, the Aleurolobini, and that their Portiera exhibit genome instability. This instability likely arose once in the common ancestor of the Aleurolobini tribe (at least 70 Ma), drawing a link between the appearance of genome instability in Portiera and the switch from multibacteriocyte to a single-bacteriocyte mode of inheritance in this tribe., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

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.

A gram-stain-negative, aerobic, non-spore-forming, rod-shaped, non-motile bacterium strain R4HLG17 T was isolated from Tamarix ramosissima roots growing in Kumtag desert. The strain grew at salinities of 0-16% (w/v) NaCl (optimum 5-6%), pH 5-9 (optimum 7) and at 16-45 °C. Based on 16S rRNA gene sequence similarity, strain R4HLG17 T belonged to the family Halomonadaceae and was most closely related to Halomonas lutea DSM 23508 T (95.1%), followed by Halotalea alkalilenta AW-7 T (94.8%), Salinicola acroporae S4-41 T (94.8%), Salinicola halophilus CG4.1 T (94.6%), and Larsenimonas salina M1-18 T (94.4%). Multilocus sequence analysis (MLSA) based on the partial sequences of 16S rRNA, atpA, gyrB, rpoD, and secA genes indicated that the strain R4HLG17 T formed an independent and monophyletic branch related to other genera of Halomonadaceae, supporting its placement as a new genus in this family. The draft genome of strain R4HLG17 T was 3.6 Mb with a total G + C content of 55.1%. The average nucleotide identity to Halomonas lutea DSM 23508 T was 83.5%. Q-9 was detected as the major respiratory quinone and summed feature 8 (C 18:1 ω7c/C 18:1 ω6c), summed feature 3 (C 16:1 ω7c/C 16:1 ω6c), and C 16:0 as predominant cellular fatty acids. On the basis of chemotaxonomic, phylogenetic, and phenotypic evidence, strain R4HLG17 T is concluded to represent a novel species of a new genus within Halomonadaceae, for which the name Phytohalomonas tamaricis gen. nov., sp. nov., is proposed. The type strain is R4HLG17 T (=ACCC 19929 T =KCTC 52415 T ).

A novel extracellular alkaline phosphatase/phosphodiesterase from the structural protein family PhoD that encoded by the genome sequence of the marine bacterium Cobetia amphilecti KMM 296 (CamPhoD) has been expressed in Escherichia coli cells. The calculated molecular weight, the number of amino acids, and the isoelectric point (pI) of the mature protein's subunit are equal to 54832.98 Da, 492, and 5.08, respectively. The salt-tolerant, bimetal-dependent enzyme CamPhoD has a molecular weight of approximately 110 kDa in its native state. CamPhoD is activated by Co 2+ , Mg 2+ , Ca 2+ , or Fe 3+ at a concentration of 2 mM and exhibits maximum activity in the presence of both Co 2+ and Fe 3+ ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn 2+ , Cu 2+ , and Mn 2+ , as well as chelating agents EDTA and EGTA, do not have an appreciable effect on the CamPhoD activity. The temperature optimum for the CamPhoD activity is 45 °C. The enzyme catalyzes the cleavage of phosphate mono- and diester bonds in nucleotides, releasing inorganic phosphorus from p-nitrophenyl phosphate (pNPP) and guanosine 5'-triphosphate (GTP), as determined by the Chen method, with rate approximately 150- and 250-fold higher than those of bis-pNPP and 5'-pNP-TMP, respectively. The Michaelis-Menten constant (K m ), V max , and efficiency (k cat /K m ) of CamPhoD were 4.2 mM, 0.203 mM/min, and 7988.6 S -1 /mM; and 6.71 mM, 0.023 mM/min, and 1133.0 S -1 /mM for pNPP and bis-pNPP as the chromogenic substrates, respectively. Among the 3D structures currently available, in this study we found only the low identical structure of the Bacillus subtilis enzyme as a homologous template for modeling CamPhoD, with a new architecture of the phosphatase active site containing Fe 3+ and two Ca 2+ ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family., Competing Interests: The authors declare no conflict of interest.

Two endophytic bacteria (EAod3 T and EAod7 T ) 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 EAod3 T and EAod7 T 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 EAod3 T and K. marisflavi DSM 15357 T corresponded to 28.5 % confirming the novel lineage of strain EAod3 T 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 EAod3 T (=CEC T 9073 T =LMG 29856 T ) and EAod7 T (=CECT 9075 T =LMG 29858 T ) 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 EAod3 T has a total length of 3.8 Mbp and a G+C content of 59.3 mol%.

Background: Diaphorina citri is the vector of 'Candidatus Liberibacter asiaticus', the most widespread pathogen associated huanglongbing, the most serious disease of citrus. To enhance our understanding of the distribution and origin of the psyllid, we investigated the genetic diversity and population structures of 24 populations in Asia and one from Florida based on the mtCOI gene. Simultaneously, genetic diversity and population structures of the primary endosymbiont (P-endosymbiont) 'Candidatus Carsonella ruddii' and secondary endosymbiont (S-endosymbiont) 'Candidatus Profftella armatura' of D. citri were determined with the housekeeping genes., Result: AMOVA analysis indicated that populations of D. citri and its endosymbionts in east and south-east Asia were genetically distinct from populations in Pakistan and Florida. Furthermore, P-endosymbiont populations displayed a strong geographical structure across east and south-east Asia, while low genetic diversity indicated the absence of genetic structure among the populations of D. citri and its S-endosymbiont across these regions., Conclusion: The 'Ca. C. ruddii' is more diverse and structured than the D. citri and the 'Ca. P. armatura' across east and south-east Asia. Multiple introductions of the psyllid have occurred in China. Management application for controlling the pest is proposed based on the genetic information of D. citri and its endosymbionts. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

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).

The Asian citrus psyllid, Diaphorina citri, is a major pest of citrus and vector of citrus greening (huanglongbing) in Asian. In our field-collected psyllid samples, we discovered that Fuzhou (China) and Faisalabad (Pakistan), populations harbored an obligate primary endosymbiont Candidatus Carsonella (gen. nov.) with a single species, Candidatus Carsonella ruddii (sp. nov.) and a secondary endosymbiont, Wolbachia surface proteins (WSP) which are intracellular endosymbionts residing in the bacteriomes. Responses of these symbionts to different temperatures were examined and their host survival assessed. Diagnostic PCR assays showed that the endosymbionts infection rates were not significantly reduced in both D. citri populations after 24 h exposure to cold or heat treatments. Although quantitative PCR assays showed significant reduction of WSP relative densities at 40°C for 24 h, a substantial decrease occurred as the exposure duration increased beyond 3 days. Under the same temperature regimes, Ca. C. ruddii density was initially less affected during the first exposure day, but rapidly reduced at 3-5 days compared to WSP. However, the mortality of the psyllids increased rapidly as exposure time to heat treatment increased. The responses of the two symbionts to unfavorable temperature regimes highlight the complex host-symbionts interactions between D. citri and its associated endosymbionts., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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).

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., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A novel Gram-stain negative, non-motile, moderately halophilic, facultatively anaerobic and spherical bacterium designated strain SS9 T was isolated from the gill homogenate of a shark. Cells of SS9 T 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 SS9 T can be affiliated with the family Halomonadaceae and is closely related to Chromohalobacter marismortui NBRC 103155 T (95.6 % sequence similarity), Halomonas ilicicola SP8 T (95.6 %) and Chromohalobacter salexigens DSM 3043 T (95.5 %). Multilocus sequence analysis of strain SS9 T 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 SS9 T was found to contain ubiquinone-9 (Q-9) as the predominant ubiquinone and C 18:1 ω7c, C 16:0 and summed feature 3 (C 16:1 ω7c and/or iso-C 15:0 2-OH) as the major fatty acids. The major polar lipids of strain SS9 T were identified as phosphatidylglycerol and phosphatidylethanolamine. The DNA G + C content of strain SS9 T was determined to be 60.4 mol%. It is evident from phylogenetic, genotypic, phenotypic and chemotaxonomic results that strain SS9 T represents a novel species in a new genus, for which the name Pistricoccus aurantiacus gen. nov., sp. nov. is proposed. The type strain is SS9 T (=KCTC 42586 T  = MCCC 1H00111 T ).

Reductive genome evolution is a universal phenomenon observed in endosymbiotic bacteria in insects. As the genome reduces its size and irreversibly losses coding genes, the functionalities of the cell system, including the energetics processes, are more restricted. Several energetic pathways can also be lost. How do these reduced metabolic networks sustain the energy needs of the system? Among the bacteria with reduced genomes Candidatus Portiera aleyrodidarum, obligate endosymbiont of whiteflies, represents an extreme case since lacks several key mechanisms for ATP generation. Thus, to analyze the cell energetics in this system, a genome-scale metabolic model of this endosymbiont was constructed, and its energy production capabilities dissected using stoichiometric analysis. Our results suggest that energy generation is coupled to the synthesis of essential amino acids and carotenoids, crucial metabolites in the symbiotic association. A deeper insight showed that ATP production via carotenoid synthesis is also connected with amino acid production. This unusual association of energy production with anabolism suggests that, although minimized, endosymbiont metabolic networks maintain a remarkable biosynthetic potential., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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-β-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.

A moderately halophilic, aerobic bacterium, strain BZ-SZ-XJ27T, belonging to the genus Halomonas, was isolated from a saline-alkaline lake in the Xinjiang Uyghur Autonomous Region of China. Phylogenetic analysis based on 16S rRNA gene sequences and a multilocus sequence analysis using the 16S rRNA, gyrB and rpoD genes demonstrated that strain BZ-SZ-XJ27T represents a member of the genus Halomonas. On the basis of 16S rRNA gene sequence similarity, the closest relatives were Halomonas campaniensis 5AGT, H. fontilapidosi 5CRT, H. korlensis XK1T and H. sinaiensis ALO SharmT, with similarities of 96.2-97.2 %. DNA-DNA hybridization with H. korlensis CGMCC 1.6981T (the nearest phylogenetic neighbour) and H. campaniensis DSM 15293T (the highest 16S rRNA gene sequence similarity) showed relatedness values of 53 and 38 %, respectively, demonstrating the separateness of the three taxa. The bacterium stained Gram-negative and the cells were motile and rod-shaped. The strain formed creamy-white colonies and grew under optimal conditions of 1.42 M Na+ (range 0.22-4.32 M Na+), pH 8.0-8.5 (range pH 6.0-10.0) and 39 °C (range 4-43 °C). The dominant fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c; 36.6 %), C16 : 0 (25.9 %) and summed feature 3 (C16 : 1ω7c/C16 : 1ω6c; 21.2 %). The dominant polar lipids were two unknown phospholipids, phosphatidylethanolamine and phosphatidylglycerol, and the main respiratory quinones were ubiquinone 9 (Q-9; 89 %) and ubiquinone 8 (Q-8; 10 %). The genomic DNA G+C content was 61.7 ± 0.8 mol% (Tm). On the basis of phenotypic, chemotaxonomic and phylogenetic features, strain BZ-SZ-XJ27T is proposed to represent a novel species, Halomonas urumqiensis sp. nov., within the genus Halomonas of the family Halomonadaceae. The type strain is BZ-SZ-XJ27T ( = JCM 30202T = CGMCC 1.12917T).

Genomic decay is a common feature of intracellular bacteria that have entered into symbiosis with plant sap-feeding insects. This study of the whitefly Bemisia tabaci and two bacteria (Portiera aleyrodidarum and Hamiltonella defensa) cohoused in each host cell investigated whether the decay of Portiera metabolism genes is complemented by host and Hamiltonella genes, and compared the metabolic traits of the whitefly symbiosis with other sap-feeding insects (aphids, psyllids, and mealybugs). Parallel genomic and transcriptomic analysis revealed that the host genome contributes multiple metabolic reactions that complement or duplicate Portiera function, and that Hamiltonella may contribute multiple cofactors and one essential amino acid, lysine. Homologs of the Bemisia metabolism genes of insect origin have also been implicated in essential amino acid synthesis in other sap-feeding insect hosts, indicative of parallel coevolution of shared metabolic pathways across multiple symbioses. Further metabolism genes coded in the Bemisia genome are of bacterial origin, but phylogenetically distinct from Portiera, Hamiltonella and horizontally transferred genes identified in other sap-feeding insects. Overall, 75% of the metabolism genes of bacterial origin are functionally unique to one symbiosis, indicating that the evolutionary history of metabolic integration in these symbioses is strongly contingent on the pattern of horizontally acquired genes. Our analysis, further, shows that bacteria with genomic decay enable host acquisition of complex metabolic pathways by multiple independent horizontal gene transfers from exogenous bacteria. Specifically, each horizontally acquired gene can function with other genes in the pathway coded by the symbiont, while facilitating the decay of the symbiont gene coding the same reaction., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

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.

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.

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)).

Background: The whitefly Bemisia tabaci is an important agricultural pest with global distribution. This phloem-sap feeder harbors a primary symbiont, "Candidatus Portiera aleyrodidarum", which compensates for the deficient nutritional composition of its food sources, and a variety of secondary symbionts. Interestingly, all of these secondary symbionts are found in co-localization with the primary symbiont within the same bacteriocytes, which should favor the evolution of strong interactions between symbionts., Results: In this paper, we analyzed the genome sequences of the primary symbiont Portiera and of the secondary symbiont Hamiltonella in the B. tabaci Mediterranean (MED) species in order to gain insight into the metabolic role of each symbiont in the biology of their host. The genome sequences of the uncultured symbionts Portiera and Hamiltonella were obtained from one single bacteriocyte of MED B. tabaci. As already reported, the genome of Portiera is highly reduced (357 kb), but has kept a number of genes encoding most essential amino-acids and carotenoids. On the other hand, Portiera lacks almost all the genes involved in the synthesis of vitamins and cofactors. Moreover, some pathways are incomplete, notably those involved in the synthesis of some essential amino-acids. Interestingly, the genome of Hamiltonella revealed that this secondary symbiont can not only provide vitamins and cofactors, but also complete the missing steps of some of the pathways of Portiera. In addition, some critical amino-acid biosynthetic genes are missing in the two symbiotic genomes, but analysis of whitefly transcriptome suggests that the missing steps may be performed by the whitefly itself or its microbiota., Conclusions: These data suggest that Portiera and Hamiltonella are not only complementary but could also be mutually dependent to provide a full complement of nutrients to their host. Altogether, these results illustrate how functional redundancies can lead to gene losses in the genomes of the different symbiotic partners, reinforcing their inter-dependency.

Whiteflies are important agricultural insect pests, whose evolutionary success is related to a long-term association with a bacterial endosymbiont, Candidatus Portiera aleyrodidarum. To completely characterize this endosymbiont clade, we sequenced the genomes of three new Portiera strains covering the two extant whitefly subfamilies. Using endosymbiont and mitochondrial sequences we estimated the divergence dates in the clade and used these values to understand the molecular evolution of the endosymbiont coding sequences. Portiera genomes were maintained almost completely stable in gene order and gene content during more than 125 Myr of evolution, except in the Bemisia tabaci lineage. The ancestor had already lost the genetic information transfer autonomy but was able to participate in the synthesis of all essential amino acids and carotenoids. The time of divergence of the B. tabaci complex was much more recent than previous estimations. The recent divergence of biotypes B (MEAM1 species) and Q (MED species) suggests that they still could be considered strains of the same species. We have estimated the rates of evolution of Portiera genes, synonymous and nonsynonymous, and have detected significant differences among-lineages, with most Portiera lineages evolving very slowly. Although the nonsynonymous rates were much smaller than the synonymous, the genomic dN/dS ratios were similar, discarding selection as the driver of among-lineage variation. We suggest variation in mutation rate and generation time as the responsible factors. In conclusion, the slow evolutionary rates of Portiera may have contributed to its long-term association with whiteflies, avoiding its replacement by a novel and more efficient endosymbiont., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

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., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

The infection density of symbionts is among the major parameters to understand their biological effects in host-endosymbionts interactions. Diaphorina citri harbors two bacteriome-associated bacterial endosymbionts (Candidatus Carsonella ruddii and Candidatus Profftella armatura), besides the intracellular reproductive parasite Wolbachia. In this study, the density dynamics of the three endosymbionts associated with the psyllid D. citri was investigated by real-time quantitative PCR (qPCR) at different developmental stages. Bacterial density was estimated by assessing the copy number of the 16S rRNA gene for Carsonella and Profftella, and of the ftsZ gene for Wolbachia. Analysis revealed a continuous growth of the symbionts during host development. Symbiont growth and rate curves were estimated by the Gompertz equation, which indicated a negative correlation between the degree of symbiont-host specialization and the time to achieve the maximum growth rate (t*). Carsonella densities were significantly lower than those of Profftella at all host developmental stages analyzed, even though they both displayed a similar trend. The growth rates of Wolbachia were similar to those of Carsonella, but Wolbachia was not as abundant. Adult males displayed higher symbiont densities than females. However, females showed a much more pronounced increase in symbiont density as they aged if compared to males, regardless of the incorporation of symbionts into female oocytes and egg laying. The increased density of endosymbionts in aged adults differs from the usual decrease observed during host aging in other insect-symbiont systems.

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))., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Moss bugs (Coleorrhyncha: Peloridiidae) are members of the order Hemiptera, and like many hemipterans, they have symbiotic associations with intracellular bacteria to fulfill nutritional requirements resulting from their unbalanced diet. The primary endosymbiont of the moss bugs, Candidatus Evansia muelleri, is phylogenetically related to Candidatus Carsonella ruddii and Candidatus Portiera aleyrodidarum, primary endosymbionts of psyllids and whiteflies, respectively. In this work, we report the genome of Candidatus Evansia muelleri Xc1 from Xenophyes cascus, which is the only obligate endosymbiont present in the association. This endosymbiont possesses an extremely reduced genome similar to Carsonella and Portiera. It has crossed the borderline to be considered as an autonomous cell, requiring the support of the insect host for some housekeeping cell functions. Interestingly, in spite of its small genome size, Evansia maintains enriched amino acid (complete or partial pathways for ten essential and six nonessential amino acids) and sulfur metabolisms, probably related to the poor diet of the insect, based on bryophytes, which contains very low levels of nitrogenous and sulfur compounds. Several facts, including the congruence of host (moss bugs, whiteflies, and psyllids) and endosymbiont phylogenies and the retention of the same ribosomal RNA operon during genome reduction in Evansia, Portiera, and Carsonella, suggest the existence of an ancient endosymbiotic Halomonadaceae clade associated with Hemiptera. Three possible scenarios for the origin of these three primary endosymbiont genera are proposed and discussed.

Phenol is a toxic aromatic compound used or produced in many industries and as a result a common component of industrial wastewaters. Phenol containing waste streams are frequently hypersaline and therefore require halophilic microorganisms for efficient biotreatment without dilution. In this study three halophilic bacteria isolated from different saline environments and identified as Halomonas organivorans, Arhodomonas aquaeolei and Modicisalibacter tunisiensis were shown to be able to grow on phenol in hypersaline media containing 100 g/L of total salts at a concentration of 3 mM (280 mg/L), well above the concentration found in most waste streams. Genes encoding the aromatic dioxygenase enzymes catechol 1,2 dioxygenase and protocatechuate 3,4-dioxygenase were present in all strains as determined by PCR amplification using primers specific for highly conserved regions of the genes. The gene for protocatechuate 3,4-dioxygenase was cloned from the isolated H. organivorans and the translated protein was evaluated by comparative protein sequence analysis with protocatechuate 3,4-dioxygenase proteins from other microorganisms. Although the analysis revealed a wide range of sequence divergence among the protocatechuate 3,4-dioxygenase family, all of the conserved domain amino acid structures identified for this enzyme family are identical or conservatively substituted in the H. organivorans enzyme.

Biosurfactants are produced by hydrocarbon-degrading marine bacteria in response to the presence of water-insoluble hydrocarbons. This is believed to facilitate the uptake of hydrocarbons by bacteria. However, these diffusible amphiphilic surface-active molecules are involved in several other biological functions such as microbial competition and intra- or inter-species communication. We report the isolation and characterization of a marine bacterial strain identified as Cobetia sp. MM1IDA2H-1, which can grow using the sulfur-containing heterocyclic aromatic hydrocarbon dibenzothiophene (DBT). As with DBT, when the isolated strain is grown in the presence of a microbial competitor, it produces a biosurfactant. Because the obtained biosurfactant was formed by hydroxy fatty acids and extracellular lipidic structures were observed during bacterial growth, we investigated whether the biosurfactant at its critical micelle concentration can interfere with bacterial communication systems such as quorum sensing. We focused on Aeromonas salmonicida subsp. salmonicida, a fish pathogen whose virulence relies on quorum sensing signals. Using biosensors for quorum sensing based on Chromobacterium violaceum and Vibrio anguillarum, we showed that when the purified biosurfactant was mixed with N-acyl homoserine lactones produced by A. salmonicida, quorum sensing was inhibited, although bacterial growth was not affected. In addition, the transcriptional activities of A. salmonicida virulence genes that are controlled by quorum sensing were repressed by both the purified biosurfactant and the growth in the presence of Cobetia sp. MM1IDA2H-1. We propose that the biosurfactant, or the lipid structures interact with the N-acyl homoserine lactones, inhibiting their function. This could be used as a strategy to interfere with the quorum sensing systems of bacterial fish pathogens, which represents an attractive alternative to classical antimicrobial therapies in fish aquaculture., (© 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

A Gram stain-negative, aerobic and rod-shaped bacterium, strain DY22(T), was isolated from a deep-sea sediment collected from the east Pacific Ocean. The isolate was found to grow in the presence of 0-20.0 % (w/v) NaCl and at pH 4.5-8.5; optimum growth was observed with 0.5-2.0 % (w/v) NaCl and at pH 5.0-7.0. Chemotaxonomic analysis showed the presence of ubiquinone-9 as predominant respiratory quinone and C16:0, C19:0 ω8c cyclo and C12:0 3-OH as major cellular fatty acids. The genomic DNA G+C content was determined to be 59.6 mol%. Comparative 16S rRNA gene sequence analysis revealed that the novel isolate belongs to the genus Salinicola. Strain DY22(T) exhibited the closest phylogenetic affinity to the type strain of Salinicola salarius with 97.2 % sequence similarity and less than 97 % sequence similarity with respect to other Salinicola species with validly published names. The DNA-DNA reassociation values between strain DY22(T) and S. salarius DSM 18044(T) was 52 ± 4 %. On the basis of phenotypic, chemotaxonomic and genotypic data, strain DY22(T) represents a novel species of the genus Salinicola, for which the name Salinicola peritrichatus sp. nov. (type strain DY22(T) = CGMCC 1.12381(T) = JCM 18795(T)) is proposed.

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we conferred the ability to ferment cellulosic materials directly on Zymobacter palmae by co-expressing foreign endoglucanase and β-glucosidase genes. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, the six genes encoding the cellulolytic enzymes (CenA, CenB, CenD, CbhA, CbhB, and Cex) from Cellulomonas fimi were introduced and expressed in Z. palmae. Of these cellulolytic enzyme genes cloned, CenA degraded carboxymethylcellulose and phosphoric acid-swollen cellulose (PASC) efficiently. The extracellular CenA catalyzed the hydrolysis of barley β-glucan and PASC to liberate soluble cello-oligosaccharides, indicating that CenA is the most suitable enzyme for cellulose degradation among those cellulolytic enzymes expressed in Z. palmae. Furthermore, the cenA gene and β-glucosidase gene (bgl) from Ruminococcus albus were co-expressed in Z. palmae. Of the total endoglucanase and β-glucosidase activities, 57.1 and 18.1 % were localized in the culture medium of the strain. The genetically engineered strain completely saccharified and fermented 20 g/l barley β-glucan to ethanol within 84 h, producing 79.5 % of the theoretical yield. Thus, the production and secretion of CenA and BGL enabled Z. palmae to efficiently ferment a water-soluble cellulosic polysaccharide to ethanol.

"Candidatus Portiera aleyrodidarum" is the primary endosymbiont of whiteflies. We report two complete genome sequences of this bacterium from the worldwide invasive B and Q biotypes of the whitefly Bemisia tabaci. Differences in the two genome sequences may add insights into the complex differences in the biology of both biotypes.

The taxonomic status of a moderately halophilic bacterium, strain N4(T), isolated from soil of a chicken farm in China was determined. It was Gram-negative, non-spore-forming, motile, and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequence indicated that this strain belonged to the genus Salinicola, as it showed the highest sequence similarities to Salinicola salaries M27(T) (98.3 %), Salinicola socius SMB35(T) (98.1 %), and Salinicola halophilus CG4.1(T) (98.1 %). The major cellular fatty acids were C(16:0) (25.6 %), C(18:1)ω7c (35.0 %), and C(19:0) cyclo ω8c (11.9 %), which are properties shared by members of the genus Salinicola. The DNA G+C content of strain N4(T) was 69.1 mol %. The level of DNA-DNA relatedness between strain N4(T) and the other three type strains of the genus of Salinicola salaries M27(T), Salinicola socius SMB35(T), and Salinicola halophilus CG4.1(T) were 34.3, 28.7, and 26.9 %, respectively. Based on the results of phenotypic, chemotaxonomic, DNA-DNA relatedness, and phylogenetic analysis, strain N4(T) should be classified as a novel species of the genus Salinicola, for which the name Salinicola zeshunii sp. nov. is proposed, with strain N4(T) (=KACC 16602(T) = CCTCC AB 2012912(T)) as the type strain.

Many insects depend on ancient associations with intracellular bacteria to perform essential metabolic functions. These endosymbionts exhibit striking examples of convergence in genome architecture, including a high degree of structural stability that is not typical of their free-living counterparts. However, the recently sequenced genome of the obligate whitefly endosymbiont Portiera revealed features that distinguish it from other ancient insect associates, such as a low gene density and the presence of perfectly duplicated sequences. Here, we report the comparative analysis of Portiera genome sequences both within and between host species. In one whitefly lineage (Bemisia tabaci), we identify large-scale structural polymorphisms in the Portiera genome that exist even within individual insects. This variation is likely mediated by recombination across identical repeats that are maintained by gene conversion. The complete Portiera genome sequence from a distantly related whitefly host (Trialeurodes vaporarium) confirms a history of extensive genome rearrangement in this ancient endosymbiont. Using gene-order-based phylogenetic analysis, we show that the majority of rearrangements have occurred in the B. tabaci lineage, coinciding with an increase in the rate of nucleotide substitutions, a proliferation of short tandem repeats (microsatellites) in intergenic regions, and the loss of many widely conserved genes involved in DNA replication, recombination, and repair. These results indicate that the loss of recombinational machinery is unlikely to be the cause of the extreme structural conservation that is generally observed in obligate endosymbiont genomes and that large, repetitive intergenic regions are an important substrate for genomic rearrangements.

A group of five Gram-negative, aerobic, halotolerant, non-pigmented bacteria isolated from shallow sediment samples and invertebrate specimens collected from the Gulf of Alaska and the Sea of Japan was subjected to taxonomic study. On the basis of 16S rRNA gene sequence analysis, the novel isolates were affiliated to the genus Cobetia, sharing the highest sequence similarity of 99.3-99.9 % with Cobetia marina DSM 4741(T). DNA-DNA hybridization experiments between and among the novel strains and C. marina DSM 4741(T) and Cobetia crustatorum JCM 15644(T) revealed that the five strains represent three separate genospecies, which could be differentiated in their morphological, physiological and biochemical characteristics. Halomonas halodurans NBRC 15607(T) was included in this study as it has recently been reported to exhibit high 16S rRNA gene sequence similarity to C. marina DSM 4741(T), and it showed a high DNA relatedness value of 96 % with C. marina DSM 4741(T), indicating that they belong to the same species. On the basis of phylogenetic analysis, DNA-DNA hybridization and phenotypic characterization, three novel species are proposed, named Cobetia amphilecti sp. nov. (type strain KMM 1561(T) = NRIC 0815(T) = CCUG 49560(T)), Cobetia litoralis sp. nov. (type strain KMM 3880(T) =NRIC 0814(T) =CCUG 49563(T)) and Cobetia pacifica sp. nov. (type strain KMM 3879(T) = NRIC 0813(T) = CCUG 49562(T)). It is also proposed that Halomonas halodurans is a later heterotypic synonym of Cobetia marina, and emended descriptions of the genus Cobetia and the species Cobetia marina are provided.

Although carotenoids serve important biological functions, animals are generally unable to synthesize these pigments and instead obtain them from food. However, many animals, such as sap-feeding insects, may have limited access to carotenoids in their diet, and it was recently shown that aphids have acquired the ability to produce carotenoids by lateral transfer of fungal genes. Whiteflies also contain carotenoids but show no evidence of the fungus-derived genes found in aphids. Because many sap-feeding insects harbour intracellular bacteria, it has long been hypothesized that these endosymbionts could serve as an alternative source of carotenoid biosynthesis. We sequenced the genome of the obligate bacterial endosymbiont Portiera from the whitefly Bemisia tabaci. The genome exhibits typical signatures of obligate endosymbionts in sap-feeding insects, including extensive size reduction (358.2 kb) and enrichment for genes involved in essential amino acid biosynthesis. Unlike other sequenced insect endosymbionts, however, Portiera has bacterial homologues of the fungal carotenoid biosynthesis genes in aphids. Therefore, related lineages of sap-feeding insects appear to have convergently acquired the same functional trait by distinct evolutionary mechanisms-bacterial endosymbiosis versus fungal lateral gene transfer.

"Candidatus Portiera aleyrodidarum" is the obligate primary endosymbiotic bacterium of whiteflies, including the sweet potato whitefly Bemisia tabaci, and provides essential nutrients to its host. Here we report two complete genome sequences of this bacterium from the B and Q biotypes of B. tabaci.

The genome of "Candidatus Portiera aleyrodidarum," the primary endosymbiont of the whitefly Bemisia tabaci (Mediterranean species), is reported. It presents a reduced genome (357 kb) encoding the capability to synthetize, or participate in the synthesis of, several amino acids and carotenoids, being the first insect endosymbiont capable of supplying carotenoids.

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we developed a tool for cell surface display of cellulolytic enzymes on the ethanologenic bacterium Zymobacter palmae. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, to express and display heterologous cellulolytic enzymes on the Z. palmae cell surface, we utilized the cell-surface display motif of the Pseudomonas ice nucleation protein Ina. The gene encoding Ina from Pseudomonas syringae IFO3310 was cloned, and its product was comprised of three functional domains: an N-terminal domain, a central domain with repeated amino acid residues, and a C-terminal domain. The N-terminal domain of Ina was shown to function as the anchoring motif for a green fluorescence protein fusion protein in Escherichia coli. To express a heterologous cellulolytic enzyme extracellularly in Z. palmae, we fused the N-terminal coding sequence of Ina to the coding sequence of an N-terminal-truncated Cellulomonas endoglucanase. Z. palmae cells carrying the fusion endoglucanase gene were shown to degrade carboxymethyl cellulose. Although a portion of the expressed fusion endoglucanase was released from Z. palmae cells into the culture broth, we confirmed the display of the protein on the cell surface by immunofluorescence microscopy. The results indicate that the N-terminal anchoring motif of Ina from P. syringae enabled the translocation and display of the heterologous cellulase on the cell surface of Z. palmae.

Background: Detection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci., Results: In this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA) substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers) while the other a secondary endosymbiont Arsenophonus (and present in less numbers). Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe., Conclusion: By employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction about their specific distribution within samples.

Multilocus sequence analysis (MLSA) protocols have been developed for species circumscription for many taxa. However, at present, no studies based on MLSA have been performed within any moderately halophilic bacterial group. To test the usefulness of MLSA with these kinds of micro-organisms, the family Halomonadaceae, which includes mainly halophilic bacteria, was chosen as a model. This family comprises ten genera with validly published names and 85 species of environmental, biotechnological and clinical interest. In some cases, the phylogenetic relationships between members of this family, based on 16S rRNA gene sequence comparisons, are not clear and a deep phylogenetic analysis using several housekeeping genes seemed appropriate. Here, MLSA was applied using the 16S rRNA, 23S rRNA, atpA, gyrB, rpoD and secA genes for species of the family Halomonadaceae. Phylogenetic trees based on the individual and concatenated gene sequences revealed that the family Halomonadaceae formed a monophyletic group of micro-organisms within the order Oceanospirillales. With the exception of the genera Halomonas and Modicisalibacter, all other genera within this family were phylogenetically coherent. Five of the six studied genes (16S rRNA, 23S rRNA, gyrB, rpoD and secA) showed a consistent evolutionary history. However, the results obtained with the atpA gene were different; thus, this gene may not be considered useful as an individual gene phylogenetic marker within this family. The phylogenetic methods produced variable results, with those generated from the maximum-likelihood and neighbour-joining algorithms being more similar than those obtained by maximum-parsimony methods. Horizontal gene transfer (HGT) plays an important evolutionary role in the family Halomonadaceae; however, the impact of recombination events in the phylogenetic analysis was minimized by concatenating the six loci, which agreed with the current taxonomic scheme for this family. Finally, the findings of this study also indicated that the 16S rRNA, gyrB and rpoD genes were the most suitable genes for future taxonomic studies using MLSA within the family Halomonadaceae.

Alkaline phosphatase catalyzes the hydrolysis of phosphomonoesters and is widely used in molecular biology techniques and clinical diagnostics. We expressed a recombinant alkaline phosphatase of the marine bacterium, Cobetia marina, in Escherichia coli BL21 (DE3). The recombinant protein was purified with a specific activity of 12,700 U/mg protein, which is the highest activity reported of any bacterial alkaline phosphatase studied to date. The molecular mass of the recombinant protein was 55-60 kDa, as determined by SDS-PAGE, and was observed to be a dimer by gel filtration analysis. The enzyme was optimally active at 45°C and the recombinant alkaline phosphatase efficiently hydrolyzed a phosphoric acid ester in luminescent and fluorescent substrates. Therefore, this enzyme can be considered to be extremely useful as a label conjugated to an antibody.

Halophilic gammaproteobacteria of the family Halomonadaceae (including the genera Aidingimonas, Carnimonas, Chromohalobacter, Cobetia, Halomonas, Halotalea, Kushneria, Modicisalibacter, Salinicola, and Zymobacter) have current and promising applications in biotechnology mainly as a source of compatible solutes (powerful stabilizers of biomolecules and cells, with exciting potentialities in biomedicine), salt-tolerant enzymes, biosurfactants, and extracellular polysaccharides, among other products. In addition, they display a number of advantages to be used as cell factories, alternative to conventional prokaryotic hosts like Escherichia coli or Bacillus, for the production of recombinant proteins: (1) their high salt tolerance decreases to a minimum the necessity for aseptic conditions, resulting in cost-reducing conditions, (2) they are very easy to grow and maintain in the laboratory, and their nutritional requirements are simple, and (3) the majority can use a large range of compounds as a sole carbon and energy source. In the last 15 years, the efforts of our group and others have made possible the genetic manipulation of this bacterial group. In this review, the most relevant and recent tools for their genetic manipulation are described, with emphasis on nucleic acid isolation procedures, cloning and expression vectors, genetic exchange mechanisms, mutagenesis approaches, reporter genes, and genetic expression analyses. Complementary sections describing the influence of salinity on the susceptibility of these bacteria to antimicrobials, as well as the growth media most routinely used and culture conditions, for these microorganisms, are also included.

Urmia Lake is one of the most permanent hypersaline lakes in the world which is threatened by hypersalinity and serious dryness. In spite of its importance no paper has been published regarding bacterial community of this lake. Accordingly, the present study aimed to investigate the halophilic bacteria in the aforementioned lake. In so doing, thirty seven strains were isolated on six different culture media. The isolated strains were characterized using phenotypic and genotypic methods. Growth of the strains occurred at 2535 degrees C, pH 6-9 and 7 to 20% (w/v) NaCl indicating that most of the isolates were moderately halophiles. Catalase, oxidase and urease activities were found to be positive for the majority of the isolates. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolated bacteria belonged to two major taxa: Gammaproteobacteria (92%, including Salicola [46%], Pseudomonas [13.5%], Marinobacter [ 11%], Idiomarina [11%], and Halomonas [8%]) and Firmicutes (8%, including Bacillus [5%] and Halobacillus [3%]). In addition, a novel bacterium whose 16S rRNA gene sequence showed almost 98% sequence identity with the taxonomically troubled DSM 3050T, Halovibrio denitrificans HGD 3T and Halospina denitrificans HGD 1-3T, each, was isolated. 16S rRNA gene similarity levels along with phenotypic characteristics suggest that some of the isolated strains could be regarded as potential type strain for novel species, on which further studies are recommended.