Structural Study of the Cobetia marina Bacteriophage 1 (Carin-1) by Cryo-EM.

Most of studied bacteriophages (phages) are terrestrial viruses. However, marine phages are shown to be highly involved in all levels of oceanic regulation. They are, however, still largely overlooked by the scientific community. By inducing cell lysis on half of the bacterial population daily, their role and influence on the bacterial biomass and evolution, as well as their impact in the global biogeochemical cycles, is undeniable. Cobetia marina virus 1 (Carin-1) is a member of the Podoviridae family infecting the γ-protoabacteria C. marina. Here, we present the almost complete, nearly-atomic resolution structure of Carin-1 comprising capsid, portal, and tail machineries at 3.5 Å, 3.8 Å and 3.9 Å, respectively, determined by cryo-electron microscopy (cryo-EM). Our experimental results, combined with AlphaFold2 (AF), allowed us to obtain the nearly-atomic structure of Carin-1 by fitting and refining the AF atomic models in the high resolution cryo-EM map, skipping the bottleneck of de-novo manual building and speeding up the structure determination process. Our structural results highlighted the T7-like nature of Carin1, as well as several novel structural features like the presence of short spikes on the capsid, reminiscent those described for Rhodobacter capsulatus gene transfer agent (RcGTA). This is, to our knowledge, the first time such assembly is described for a bacteriophage, shedding light into the common evolution and shared mechanisms between gene transfer agents and phages. This first full structure determined for a marine podophage allowed to propose an infection mechanism different than the one proposed for the archetypal podophage T7. IMPORTANCE Oceans play a central role in the carbon cycle on Earth and on the climate regulation (half of the planet's CO2 is absorbed by phytoplankton photosynthesis in the oceans and just as much O2 is liberated). The understanding of the biochemical equilibriums of marine biology represents a major goal for our future. By lysing half of the bacterial population every day, marine bacteriophages are key actors of these equilibriums. Despite their importance, these marine phages have, so far, only been studied a little and, in particular, structural insights are currently lacking, even though they are fundamental for the understanding of the molecular mechanisms of their mode of infection. The structures described in our manuscript allow us to propose an infection mechanism that differs from the one proposed for the terrestrial T7 virus, and might also allow us to, in the future, better understand the way bacteriophages shape the global ecosystem.

Efficient flocculation pretreatment of coal gasification wastewater by halophilic bacterium Halovibrio variabilis TG-5.

The isolated halophilic bacterial strain Halovibrio variabilis TG-5 showed a good performance in the pretreatment of coal gasification wastewater. With the optimum culture conditions of pH = 7, a temperature of 46 °C, and a salinity of 15%, the chemical oxygen demand and volatile phenol content of pretreated wastewater were decreased to 1721 mg/L and 94 mg/L, respectively. The removal rates of chemical oxygen demand and volatile phenol were over 90% and 70%, respectively. At the optimum salinity conditions of 15%, the total yield of intracellular compatible solutes and the extracellular transient released yield under hypotonic conditions were increased to 6.88 g/L and 3.45 g/L, respectively. The essential compatible solutes such as L-lysine, L-valine, and betaine were important in flocculation mechanism in wastewater pretreatment. This study provided a new method for pretreating coal gasification wastewater by halophilic microorganisms, and revealed the crucial roles of compatible solutes in the flocculation process.

Genetic characterization of a novel Salinicola salarius isolate applied for the bioconversion of agro-industrial wastes into polyhydroxybutyrate.

BACKGROUND: Polyhydroxybutyrate (PHB) has emerged as a promising eco-friendly alternative to traditional petrochemical-based plastics. In the present study, we isolated and characterized a new strain of Salinicola salarius, a halophilic bacterium, from the New Suez Canal in Egypt and characterized exclusively as a potential PHB producer. Further genome analysis of the isolated strain, ES021, was conducted to identify and elucidate the genes involved in PHB production. RESULTS: Different PHB-producing marine bacteria were isolated from the New Suez Canal and characterized as PHB producers. Among the 17 bacterial isolates, Salinicola salarius ES021 strain showed the capability to accumulate the highest amount of PHB. Whole genome analysis was implemented to identify the PHB-related genes in Salinicola salarius ES021 strain. Putative genes were identified that can function as phaCAB genes to produce PHB in this strain. These genes include fadA, fabG, and P3W43_16340 (encoding acyl-CoA thioesterase II) for PHB production from glucose. Additionally, phaJ and fadB were identified as key genes involved in PHB production from fatty acids. Optimization of environmental factors such as shaking rate and incubation temperature, resulted in the highest PHB productivity when growing Salinicola salarius ES021 strain at 30°C on a shaker incubator (110 rpm) for 48 h. To maximize PHB production economically, different raw materials i.e., salted whey and sugarcane molasses were examined as cost-effective carbon sources. The PHB productivity increased two-fold (13.34 g/L) when using molasses (5% sucrose) as a fermentation media. This molasses medium was used to upscale PHB production in a 20 L stirred-tank bioreactor yielding a biomass of 25.12 g/L, and PHB of 12.88 g/L. Furthermore, the produced polymer was confirmed as PHB using Fourier-transform infrared spectroscopy (FTIR), gas chromatography-mass spectroscopy (GC-MS), and nuclear magnetic resonance spectroscopy (NMR) analyses. CONCLUSIONS: Herein, Salinicola salarius ES021 strain was demonstrated as a robust natural producer of PHB from agro-industrial wastes. The detailed genome characterization of the ES021 strain presented in this study identifies potential PHB-related genes. However, further metabolic engineering is warranted to confirm the gene networks required for PHB production in this strain. Overall, this study contributes to the development of sustainable and cost-effective PHB production strategies.

Lysine provisioning by horizontally acquired genes promotes mutual dependence between whitefly and two intracellular symbionts.

Horizontal gene transfer is widespread in insects bearing intracellular symbionts. Horizontally transferred genes (HTGs) are presumably involved in amino acid synthesis in sternorrhynchan insects. However, their role in insect-symbiont interactions remains largely unknown. We found symbionts Portiera, Hamiltonella and Rickettsia possess most genes involved in lysine synthesis in the whitefly Bemisia tabaci MEAM1 although their genomes are reduced. Hamiltonella maintains a nearly complete lysine synthesis pathway. In contrast, Portiera and Rickettsia require the complementation of whitefly HTGs for lysine synthesis and have lysE, encoding a lysine exporter. Furthermore, each horizontally transferred lysine gene of ten B. tabaci cryptic species shares an evolutionary origin. We demonstrated that Hamiltonella did not alter the titers of Portiera and Rickettsia or lysine gene expression of Portiera, Rickettsia and whiteflies. Hamiltonella also did not impact on lysine levels or protein localization in bacteriocytes harboring Portiera and ovaries infected with Rickettsia. Complementation with whitefly lysine synthesis HTGs rescued E. coli lysine gene knockout mutants. Silencing whitefly lysA in whiteflies harboring Hamiltonella reduced lysine levels, adult fecundity and titers of Portiera and Rickettsia without influencing the expression of Hamiltonella lysA. Furthermore, silencing whitefly lysA in whiteflies lacking Hamiltonella reduced lysine levels, adult fecundity and titers of Portiera and Rickettsia in ovarioles. Therefore, we, for the first time, demonstrated an essential amino acid lysine synthesized through HTGs is important for whitefly reproduction and fitness of both obligate and facultative symbionts, and it illustrates the mutual dependence between whitefly and its two symbionts. Collectively, this study reveals that acquisition of horizontally transferred lysine genes contributes to coadaptation and coevolution between B. tabaci and its symbionts.

Larsenimonas rhizosphaerae sp. nov., isolated from a tidal mudflat.

A Gram-reaction-negative, aerobic, motile, rod-shaped bacterium, designated GH3-8T, was isolated from rhizosphere mudflats of halophytes on the seashore of Gangwha Island, Republic of Korea. Growth was observed at pH 4-10 (optimum, pH 7-8), at 4-40 °C (optimum, 37 °C) and in the presence of 0.5-20 % (w/v) NaCl (optimum, 4 %). The predominant respiratory quinone was Q-9. The major fatty acids were C18 : 1 ω7c, C16 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and C12 : 0 3OH. The polar lipids contained phosphatidylethanolamine, phosphatidylglycerol, an unidentified phosphoglycolipid, an unidentified phosphoglycoaminolipid, an unidentified glycoaminolipid, two unidentified phospholipids and two unidentified lipids. Phylogenetic analysis based on 16S rRNA gene sequences exhibited that the isolate belonged to the family Halomonadaceae, with the most closely related species, Larsenimonas suaedae (98.1 % sequence similarity) and Larsenimonas salina (97.9 %). Sequence similarity values between the isolate and other representatives of the family Halomonadaceae were all below 95.3 %. The values of average nucleotide identity between strain GH3-8T and members of the genus Larsenimonas were 73.42 % with L. salina CCM 8464T and 72.38 % with L. suaedae DSM 22428T. Strain GH3-8T showed digital DNA-DNA hybridization values of 18.5-18.6 % with members of the genus Larsenimonas. Based on phenotypic and chemotaxonomic distinctiveness together with low overall genomic relatedness indices and phylogenetic data, the isolate is considered to represent a new species of the genus Larsenimonas, for which the name Larsenimonas rhizosphaerae sp. nov. is proposed, with the type strain GH3-8T (=KCTC 62127T=NBRC 113214T).

Autophagy Regulates Whitefly-Symbiont Metabolic Interactions.

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.

A novel microRNA regulates cooperation between symbiont and a laterally acquired gene in the regulation of pantothenate biosynthesis within Bemisia tabaci whiteflies.

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.

Immobilization of Halomonas halodurans and Bacillus halodurans in packed bed bioreactor for continuous removal of phenolic impurities in waste water.

Phenol is an organic contaminant widely distributed in wastewater. Biodegradation is one of the suitable methods used to remove phenol from the wastewater. In this study, the bacterial laccase and pectinase were analyzed and phenol degradation potential was studied. A total of six bacterial strains were selected and their phenol degrading potentials were studied. Laccase and pectinase producers were screened on substrate agar plates and several strains produced these enzymes in submerged fermentation. Among these enzyme producing strains, strain PD8 and PD22 exhibited potent phenol degrading ability than other strains. These two bacterial strains (Halomonas halodurans PD8 and Bacillus halodurans PD22) exhibited maximum growth in phenol-supplemented culture medium. These two organisms grown well at wide pH values (pH 3.0 and 10.0), survive well between 20 °C and 50 °C, and showed growth between 1 and 10% sodium chloride concentration. The lyophilized enzyme from PD8 and PD22 were immobilized with alginate beads cross liked with divalent cations. At 1% alginate, the binding efficiency was 40.2 ± 2.9% and it improved up to 2.0% concentration (67.5 ± 4.2%) and further increase on alginate concentration affected binding efficiency. Phenol degradation was maximum within 10 h of treatment in the immobilized packed bed column reactor (83.1 ± 3.2%) and colour removal efficiency was maximum at 12 h treatment (82.1 ± 3.9%). After four successive experimental trials more than 40% efficiency was achieved.

Difference in calcium ion precipitation between free and immobilized Halovibrio mesolongii HMY2.

Biomineralization has become a research focus in wastewater treatment due to its much lower costs compared to traditional methods. However, the low sodium chloride (NaCl)-tolerance of bacteria limits applications to only water with low NaCl concentrations. Here, calcium ions in hypersaline wastewater (10% NaCl) were precipitated by free and immobilized Halovibrio mesolongii HMY2 bacteria and the differences between them were determined. The results show that calcium ions can be transformed into several types of calcium carbonate with a range of morphologies, abundant organic functional groups (C-H, C-O-C, C=O, etc), protein secondary structures (ß-sheet, α-helix, 310 helix, and ß-turn), P=O and S-H indicated by P2p and S2p, and more negative δ13CPDB (‰) values (-16.8‰ to -18.4‰). The optimal conditions for the immobilized bacteria were determined by doing experiments with six factors and five levels and using response surface method. Under the action of two groups of immobilized bacteria prepared under the optimal conditions, by the 10th day, Ca2+ ion precipitation ratios had increased to 79%-89% and 80%-88% with changes in magnesium ion cencentrations. Magnesium ions can significantly inhibit the calcium ion precipitation, and this inhibitory effect can be decreased under the action of immobilized bacteria. Minerals induced by immobilized bacteria always aggregated together, had higher contents of Mg, P, and S, lower stable carbon isotope values and less well-developed protein secondary structures. This study demonstrates an economic and eco-friendly method for recycling calcium ions in hypersaline wastewater, providing an easy step in the process of desalination.

Control of Marine Bacteria and Diatom Biofouling by Constant and Alternating Potentials.

The application of electrochemical potentials to surfaces is an easy and direct way to alter surface charge density, the structure of the electrochemical double layer, and the presence of electrochemically activated species. On such electrified interfaces the formation of biofilms is reduced. Here we investigate how applied potentials alter the colonization of surfaces by the marine bacterium Cobetia marina and the marine diatom Navicula perminuta. Different constant potentials between -0.8 and 0.6 V as well as regular switching between two potentials were investigated, and their influence on the attachment of the two biofilm-forming microorganisms on gold-coated working electrodes was quantified. Reduced bacteria and diatom attachment were found when negative potentials and alternating potentials were applied. The results are discussed on the basis of the electrochemical processes occurring at the working electrode in artificial seawater as revealed by cyclic voltammetry.

Kushneria phosphatilytica sp. nov., a phosphate-solubilizing bacterium isolated from a solar saltern.

A Gram-stain-negative, motile, rod-shaped, non-endospore-forming, aerobic and halophilic bacterium, designated strain YCWA18T, was isolated from the sediment of Jimo-Daqiao saltern in China. This strain was able to grow at NaCl concentrations in the range 0.5-20 % (w/v) with optimum growth at 6 % (w/v) NaCl. Growth occurred at temperatures of 4-40 °C (optimum 28 °C) and pH 4.0-9.0 (optimum 7.0). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YCWA18T belonged to the genus Kushneria and shared the highest sequence similarity of 98.7 % with Kushneria sinocarnis DSM 23229T. Moreover, the phylogenetic analysis based on the 23S rRNA gene sequence also confirmed the phylogenetic position of this novel strain. The predominant fatty acids were C16 : 0, C17 : 0 cyclo and C12 : 0 3-OH. The major isoprenoid quinone was Q-9 (94.2 %) and the polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), an unidentified aminolipid (AL), an unidentified phospholipids (PL) and two unidentified lipids (L). The complete genome of strain YCWA18T consisted of a single, circular chromosome of 3 624 619 bp, with an average G+C content of 59.1 mol%. A genome-based phylogenetic tree constructed using an up-to-date bacterial core gene set (UBCG) showed that strain YCWA18T formed a clade with K. sinocarnis DSM 23229T. However, the level of the ANI and dDDH values between strain YCWA18T and K. sinocarnis DSM 23229T were 82.3 and 24.6 %, respectively, which were low enough to distinguish strain YCWA18T from K. sinocarnis DSM 23229T. Overall, based on the phenotypic, chemotaxonomic, phylogenetic and genomic analyses, strain YCWA18T represents a novel species of genus Kushneria. The name Kushneria phosphatilytica sp. nov. is proposed, with the type strain YCWA18T (=CGMCC 1.9149T=NCCB 100306T).

High natural PHA production from acetate in Cobetia sp. MC34 and Cobetia marina DSM 4741T and in silico analyses of the genus specific PhaC2 polymerase variant.

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 4741T 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 4741T produced 2.4 g PHB/L and 3.7 g PHBV/L. Cobetia marina DSM 4741T 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 (phaC2) was found in Cobetia sp. MC34, in contrast to two copies (phaC1 and phaC2) in C. marina DSM 4741T. In silico analyses based on phaC genes show that the PhaC2 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 4741T 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 PhaC2 variant in DNA-binding and the potential implications remains to be addressed by in vitro- or in vivo methods.

Halovibrio salipaludis sp. nov., Isolated from Saline-Alkaline Soil.

A Gram-staining-negative, halophilic, aerobic, oval-shaped or vibrio-shaped, motile by a polar flagellum strain, designated YL5-2T, was isolated from natural saline-alkaline wetland soil of Binhai new district, Tianjin, China. Strain YL5-2T grew optimally at 35 °C, pH 7.5-8.0, and in the presence of 10-25% (w/v) NaCl on MA medium. Phylogenetic analyses based on 16S rRNA gene sequences showed that the isolate belonged to the genus Halovibrio and exhibited high sequence similarity of 97.7% to Halovibrio variabilis DSM 3050T. The sole respiratory ubiquinone of strain YL5-2T is Q-9, and the dominant fatty acids were C18:1ω9c, C16:0, C19:0 cycloω8c, and Summed Feature 8. The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), and lipid (L). The DNA G+C content of the strain was 62.1 mol%. The average nucleotide identity (ANI) based on whole genome sequences of strain YL5-2T and Halovibrio variabilis DSM 3050T was 93.85%, and the dDDH value between these two strains was determined to be 52.0%. Phenotypic, chemotaxonomic, phylogenetic, and genomic analyses suggested that strain YL5-2T represent a novel species of the genus Halovibrio, for which the name Halovibrio salipaludis sp. nov. is proposed. The type strain is YL5-2T (=KCTC 52852T=ACCC 19971T).

Are the Closely Related Cobetia Strains of Different Species?

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.

Phytohalomonas tamaricis gen. nov., sp. nov., an endophytic bacterium isolated from Tamarix ramosissima roots growing in Kumtag desert.

A gram-stain-negative, aerobic, non-spore-forming, rod-shaped, non-motile bacterium strain R4HLG17T 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 R4HLG17T belonged to the family Halomonadaceae and was most closely related to Halomonas lutea DSM 23508T(95.1%), followed by Halotalea alkalilenta AW-7T(94.8%), Salinicola acroporae S4-41T(94.8%), Salinicola halophilus CG4.1T(94.6%), and Larsenimonas salina M1-18T(94.4%). Multilocus sequence analysis (MLSA) based on the partial sequences of 16S rRNA, atpA, gyrB, rpoD, and secA genes indicated that the strain R4HLG17T 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 R4HLG17T was 3.6 Mb with a total G + C content of 55.1%. The average nucleotide identity to Halomonas lutea DSM 23508T was 83.5%. Q-9 was detected as the major respiratory quinone and summed feature 8 (C18:1ω7c/C18:1ω6c), summed feature 3 (C16:1ω7c/C16:1ω6c), and C16:0 as predominant cellular fatty acids. On the basis of chemotaxonomic, phylogenetic, and phenotypic evidence, strain R4HLG17T 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 R4HLG17T (=ACCC 19929T=KCTC 52415T).

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).

Effect of ozone stress on the intracellular metabolites from Cobetia marina.

A GCxGC-MS system was employed with a non-polar × mid-polar column set for the metabolic non-target analysis of Cobetia marina, the model bacteria for marine biofouling. C. marina was treated with ozone to investigate the intracellular metabolic state change under oxidative stress. A minimal inhibitory concentration test was involved to guarantee that the applied ozone dosages were not lethal for the cells. In this study, non-target analyses were performed to identify the metabolites according to the NIST database. As a result, over 170 signals were detected under normal living conditions including 35 potential metabolites. By the comparison of ozone-treated and non-treated samples, five compounds were selected to describe observed trends of signals in the contour plots. Oleic acid exhibited a slight growth by increasing ozone dosage. In contrast, other metabolites such as the amino acid L-proline showed less abundance after ozone treatment, which was more evident once ozone dosage was raised. Thus, this work could provide a hint for searching for up/downregulating factors in such environmental stress conditions for C. marina. Graphical abstract.

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).

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.

A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium Cobetia amphilecti KMM 296.

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 Co2+, Mg2+, Ca2+, or Fe3+ at a concentration of 2 mM and exhibits maximum activity in the presence of both Co2+ and Fe3+ ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn2+, Cu2+, and Mn2+, 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 (Km), Vmax, and efficiency (kcat/Km) 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 Fe3+ and two Ca2+ ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.