In-Depth Genome Characterization and Pan-Genome Analysis of Strain KMM 296, a Producer of Highly Active Alkaline Phosphatase; Proposal for the Reclassification of Cobetia litoralis and Cobetia pacifica as the Later Heterotypic Synonyms of Cobetia amphilecti and Cobetia marina, and Emended Description of the Species Cobetia amphilecti and Cobetia marina.

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 0815T. 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 C18:1 ω7c, C12:0 3-OH, C18:1 ω7c, C12:0, and C17:0 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 0815T and C. litoralis NRIC 0814T showed their belonging to a single species. In addition, the high similarity between the C. pacifica NRIC 0813T and C. marina LMG 2217T 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.

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.

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.

Preparation and potential antitumor activity of alginate oligosaccharides degraded by alginate lyase from Cobetia marina.

It is of great significance to develop marine resources and study its potential biological activity by using alginate lyase produced by marine psychrophilic bacteria. In the previous study, a new marine psychrophilic bacterium (Cobetia marina HQZ08) was screened from the growth area of Laminaria japonica, and it was found that the strain could efficiently produce alginate-degrading enzyme (Aly30). In this paper, the ability of Aly30 to degrade alginate was optimized and the optimal degradation conditions were obtained. It was found that the main degradation product of alginate oligosaccharides was trisaccharide. In vitro cell experiments showed that the antitumor activity of low molecular weight alginate oligosaccharides was better than that of high molecular weight alginate oligosaccharides. In summary, Aly30 had the potential to produce alginate oligosaccharides with low degree of polymerization and antitumor activity, which provided a reference for the enzymatic preparation and application of alginate oligosaccharides.

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).

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.

Plant-mediated rifampicin treatment of Bemisia tabaci disrupts but does not eliminate endosymbionts.

Whiteflies are among the most important global insect pests in agriculture; their sustainable control has proven challenging and new methods are needed. Bacterial symbionts of whiteflies are poorly understood potential target of novel whitefly control methods. Whiteflies harbour an obligatory bacterium, Candidatus Portiera aleyrodidarum, and a diverse set of facultative bacterial endosymbionts. Function of facultative microbial community is poorly understood largely due to the difficulty in their selective elimination without removal of the primary endosymbiont. Since the discovery of secondary endosymbionts, antibiotic rifampicin has emerged as the most used tool for their manipulation. Its effectiveness is however much less clear, with contrasting reports on its effects on the endosymbiont community. The present study builds upon most recent method of rifampicin application in whiteflies and evaluates its ability to eliminate obligatory Portiera and two facultative endosymbionts (Rickettsia and Arsenophnus). Our results show that rifampicin reduces but does not eliminate any of the three endosymbionts. Additionally, rifampicin causes direct negative effect on whiteflies, likely by disrupting mitochondria. Taken together, results signify the end of a rifampicin era in whitefly endosymbiont studies. Finally, we propose refinement of current quantification and data analysis methods which yields additional insights in cellular metabolic scaling.

Structure and antiproliferative activity of the polysaccharide from Halomonas aquamarina related to Cobetia pacifica.

Here, the results of the structure and the activity of capsular polysaccharides isolated from the Halomonas aquamarina EG27S8QL and Cobetia pacifica KMM3878 have been described. Both polysaccharides were studied by spectroscopic and chemical methods and were found to be structurally related sulfated galactans differing in the position of the sulfate group: →6)-ß-D-Galp3S-(1 â†’ 4)-ß-D-Galp3S-(1 â†’ 6)-ß-D-Galp3,4(S-Pyr)-(1 â†’ [H. aquamarina EG27S8QL] →6)-ß-D-Gal-(1 â†’ 4)-ß-D-Gal2,3S-(1 â†’ 6)-ß-D-Gal3,4(S-Pyr)-(1 â†’ [C. pacifica KMM3878] Structure of the CPS from H. aquamarina EG27S8QL has not been hitherto reported, whereas the CPS from C. pacifica KMM3878 was identical to the previously studied O-polysaccharide. The CPSs exhibited an antiproliferative effect and suppressed the colony formation of DLD-1 and MCF-7 cells in a different manner.

A Novel Bioflocculant Produced by Cobetia marina MCCC1113: Optimization of Fermentation Conditions by Response Surface Methodology and Evaluation of Flocculation Performance when Harvesting Microalgae.

A preliminary study was carried out to optimize the culture medium conditions for producing a novel microbial flocculant from the marine bacterial species Cobetia marina. The optimal glucose, yeast extract, and glutamate contents were 30, 10, and 2 g/l, respectively, while the optimal initial pH of the culture medium was determined to be 8. Following response surface optimization, the maximum bioflocculant production level of 1.36 g/l was achieved, which was 43.40% higher than the original culture medium. Within 5 min, a 20.0% (v/v) dosage of the yielded bioflocculant applied to algal cultures resulted in the highest flocculating efficiency of 93.9% with Spirulina platensis. The bioflocculant from C. marina MCCC1113 may have promising application potential for highly productive microalgae collection, according to the findings of this study.

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

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

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.

Turning mannitol-rich agricultural waste to poly(3-hydroxybutyrate) with Cobetia amphilecti fermentation and recovery with methyl levulinate as a green solvent.

The present study explored the use of mannitol and mannitol-rich agro-industrial wastes as substrates for PHB production by Cobetia amphilecti isolated from the green Ulva sp. seaweed. Cultivation of C. amphilecti on mannitol, celery, and olive leaves (OLs) waste led to 4.20, 6.00, and 5.16 g L-1 of cell dry mass (CDM), 76.3, 25.5, and 12.0% of PHB content in CDM and 3.2, 1.53, and 0.62 g L-1 of PHB concentration, respectively; which suggested that they can be exploited as carbon substrates for the production of PHB. Extraction of PHB from C. amphilecti cultures by solubilization in the green solvent methyl levulinate (ML) (2% w/w, 140 °C, 1 h) indicated that the recovery yield and purity of PHB are above 97 and 90% w/w, respectively. The use of ML could be an attractive method for the recovery of PHB when safe and non-toxic solvents are required.

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.

Genomic and transcriptomic analyses reveal metabolic complementarity between whiteflies and their symbionts.

Nutritional mutualism between insects and symbiotic bacteria is widespread. The various sap-feeding whitefly species within the Bemisia tabaci complex associate with the same obligate symbiont (Portiera) and multiple secondary symbionts. It is often assumed that some of the symbionts residing in the whiteflies play crucial roles in the nutritional physiology of their insect hosts. Although effort has been made to understand the functions of the whitefly symbionts, the metabolic complementarity offered by these symbionts to the hosts is not yet well understood. We examined two secondary symbionts, Arsenophonus and Wolbachia, in two species of the B. tabaci whitefly complex, provisionally named as Asia II 3 and China 1. Genomic sequence analyses revealed that Arsenophonus and Wolbachia retained genes responsible for the biosynthesis of B vitamins. We then conducted transcriptomic surveys of the bacteriomes in these two species of whiteflies together with that in another species named MED of this whitefly complex previously reported. The analyses indicated that several key genes in B vitamin syntheses from the three whitefly species were identical. Our findings suggest that, similar to another secondary symbiont Hamiltonella, Arsenophonus and Wolbachia function in the nutrient provision of host whiteflies. Although phylogenetically distant species of symbionts are associated with their respective hosts, they have evolved and retained similar functions in biosynthesis of some B vitamins. Such metabolic complementarity between whiteflies and symbionts represents an important feature of their coevolution.

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.

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.

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.

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.

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

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