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Corals establish symbiotic relationships with microorganisms, especially endosymbiotic photosynthetic algae. Although other microbes have been commonly detected in coral tissues, their identity and beneficial functions for their host are unclear. Here, we confirm the beneficial outcomes of the inoculation of bacteria selected as probiotics and use fluorescence in situ hybridization (FISH) to define their localization in the coral Pocillopora damicornis . Our results show the first evidence of the inherent presence of Halomonas sp. and Cobetia sp. in native coral tissues, even before their inoculation. Furthermore, the relative enrichment of these coral tissue-associated bacteria through their inoculation in corals correlates with health improvements, such as increases in photosynthetic potential, and productivity. Our study suggests the symbiotic status of Halomonas sp. and Cobetia sp. in corals by indicating their localization within coral gastrodermis and epidermis and correlating their increased relative abundance through active inoculation with beneficial outcomes for the holobiont. This knowledge is crucial to facilitate the screening and application of probiotics that may not be transient members of the coral microbiome., Importance: Despite the promising results indicating the beneficial outcomes associated with the application of probiotics in corals and some scarce knowledge regarding the identity of bacterial cells found within the coral tissue, the correlation between these two aspects is still missing. This gap limits our understanding of the actual diversity of coral-associated bacteria and whether these symbionts are beneficial. Some researchers, for example, have been suggesting that probiotic screening should only focus on the very few known tissue-associated bacteria, such as Endozoicomonas sp., assuming that the currently tested probiotics are not tissue-associated. Here, we provide specific FISH probes for Halomonas sp. and Cobetia sp., expand our knowledge of the identity of coral-associated bacteria and confirm the probiotic status of the tested probiotics. The presence of these beneficial microorganisms for corals (BMCs) inside host tissues and gastric cavities also supports the notion that direct interactions with the host may underpin their probiotic role. This is a new breakthrough; these results argue against the possibility that the positive effects of BMCs are due to factors that are not related to a direct symbiotic interaction, for example, that the host simply feeds on inoculated bacteria or that the bacteria change the water quality., Competing Interests: The authors declare no conflict of interest.

Bacteria from the genus Halomonas hold promise in biotechnology as sources of salt-tolerant enzymes, biosurfactants, biopolymers, osmolytes, and as actors in bioremediation processes. In a previous work, we have identified Halomonas socia strain CKY01 having various hydrolase activities. Here, we aimed to study the survival strategies of marine bacteria. A deep genome sequencing study of H. socia CKY01 has revealed 4627 genes reaching 4,753,299 bp with 64 % of GC content. This strain produced polyhydroxybutyrate (PHB) having one gene clusters having phaC and phasin, and it has several genes responsible for the uptake, synthesis, and transport of the osmolytes such as betaine, choline, ectoine, carnitine, and proline in the bacterial genome. The addition of 60 mM glutamate, 60 mM proline and 60 mM ectoine enhanced growth 300.8 %, 294.2 % and 235.0 %, respectively, under 10 % saline conditions. In particular, ectoine and proline increased salt resistance and allowed cells to survive in more than 15 % NaCl. By combining experimental and genome sequencing data, we have investigated the importance of osmolytes on the survival of this Halomonas strain., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Two moderately halophilic strains SBS 10 T and SSO 06 were isolated from the saltern crystallizer ponds of the hypersaline Sambhar Salt Lake in India. Strains were aerobic, Gram-stain-negative, and rod shaped. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that two strains belong to the genus Halomonas in the Gammaproteobacteria, with highest 16S rRNA gene sequence similarities with Halomonas gudaonensis LMG 23610 T (98.2% similarity) and Halomonas campaniensis 5AG T (99.0% similarity). Strains grew optimally at 37 °C, pH 7.5-8.0 in the presence of 5-8% (w/v) NaCl. The major fatty acids of the strain SBS 10 T were C 18:1 ω7c (54.37%), C 16:0 (25.69%), C 16:1  × 7c/C 16:1  × 6c (13.28%), and C 12:0 (1.21%). The G+C content was 63.6 mol % (T m ). Phenotypic features, fatty acids profile, and DNA G+C content supported placement of the strain SBS 10 T in the genus Halomonas having distinct characteristics with related strains. Analysis of the housekeeping genes: gryB and rpoD and in silico DNA-DNA hybridization between the strain SBS 10 T and its type strain Halomonas gudaonensis (LMG 23610 T ) further revealed the strain SBS 10 T to be a distinct species. On the basis of the phenotypic, chemotaxonomic and phylogenetic analysis, the strain SBS 10 T is considered to represent a novel species for which the name Halomonas sambharensis is proposed. The type strain is SBS 10 T (= MTCC 12313 T  = LMG 30344 T ).

Two Gram-negative strains obtained from tank water in a scallop hatchery in Norway, were phenotypically and genotypically characterized in order to clarify their taxonomic position. On the basis of 16S rRNA gene sequence analysis, these isolates, ATF 5.2 T and ATF 5.4 T , were included in the genus Halomonas, being their closest relatives H. smyrnensis and H. taeanensis, with similarities of 98.9% and 97.7%, respectively. Sequence analysis of the housekeeping genes atpA, ftsZ, gyrA, gyrB, mreB, rpoB, rpoD, rpoE, rpoH, rpoN and rpoS clearly differentiated the isolates from the currently described Halomonas species, and the phylogenetic analysis using concatenated sequences of these genes located them in two robust and independent branches. DNA-DNA hybridization (eDDH) percentage, together with average nucleotide identity (ANI), were calculated using the complete genome sequences of the strains, and demonstrate that the isolates constitute two new species of Halomonas, for which the names of Halomonas borealis sp. nov. and Halomonas niordiana sp. nov. are proposed, with type strains ATF 5.2 T (=CECT 9780 T =LMG 31367 T ) and ATF 5.4 T (=CECT 9779 T =LMG 31227 T ), respectively., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The study aims to find out osmoadaptive mechanism used to overcome the salinity stress by Halomonas sp SBS 10 isolated from the saltern crystallizer ponds of the Sambhar Salt Lake and its taxonomic position using neighbor-joining algorithm. The strain SBS 10 was tested for accumulation of two major compatable solutes betaine and ectoine and was observed that osmoprotection in the strain SBS 10 is achieved by the accumulation of betaine or by the de-novo synthesis of betaine or ectoine. Amount of endogenous content of the betaine and ectoine per milligram of cell biomass was estimated to be 581 µg, 587 µg, 588 µg, 617 µg, and 761 µg for betaine and 1.52 µg, 2.74 µg, 3.14 µg, 3.50 µg, and 52.67 µg for ectoine, when exposed to 5, 10, 15, 20 and 25% of NaCl concentration. Results obtained from HPLC analysis showed that the betaine accumulation suppresses the de-novo synthesis of ectoine partially at low NaCl concentration in the growth medium. However, at a high NaCl concentration, the ectoine concentration increases abruptly as compared to the betaine. This indicates that the ectoine accumulation is transcriptionally up-regulated by the salinity stress. Phylogenetic analysis based on the neighbor-joining algorithm included the strain SBS 10 in the genus Halomonas of the family Halomonadaceae belonging to the class Gammaproteobacteria. Most closely related type strain was found to be Halomonas gudaonensis SL014B-69 T (98.2% similarity). Ultrastructure characteristics showed the strain to be non-spore forming rod, 0.3-0.4 × 0.75-1.65 μm in size and motile with the help of peritrichous flagella.

Ten Halomonas strains were screened from different Tunisian hypersaline environments for the production of exopolysaccharides (EPS), characterized and identified basing on 16S rRNA gene sequencing. EPS production was therefore studied using two different culture media M1 (complex medium) and M2 (semi-complex medium). Selected isolates produced different EPS amounts ranging from 86 to 170 mg L -1 and 26 to 105 mg L -1 when grown on M1 and M2, respectively. The use of M1 encouraged stronger bacterial growth associated with greater EPS production compared to M2. Nevertheless, the highest EPS yield (Y EPS/X ) was observed for strains grown on M2. When cultivated on M1, all isolates produced EPS exhibiting almost the same monosaccharide profile with mannose, glucose and arabinose being the main monomers. However, the produced EPS on M2 were characterized by heterogeneous monosaccharide profiles among the different species, mostly consisting of glucomannan that could be a versatile material used for many further applications., (Copyright © 2019. Published by Elsevier B.V.)

Chemotaxis can play an important role in bioremediation and substrate bioavailability. The bioremediation of hydrocarbons in saline environments can be carried out by technologies using halophilic bacteria. The aim of this study is to analyse chemotactic responses of the halophilic bacterium Halomonas anticariensis FP35 T to environmental pollutants, as well as its catabolic potential for biotechnological use in bioremediation processes under saline conditions. Chemotaxis was detected and quantified using a modified Adler capillary assay. PCR amplification with degenerate primers for genes encoding ring-cleaving enzymes was used to characterize the catabolic versatility of FP35 T . The results indicate that phenol (100-1,000 ppm) and naphthalene (100-500 ppm) are chemoattractants for H. anticariensis FP35 T in a dose-dependent manner. These hydrocarbons were observed to act as chemoattractants for FP35 T grown in a wide range of sea salt solutions (5-12.5% (w/v). However, the 7.5% (w/v) saline concentration was found to have the strongest chemotactic response. We also detected genes encoding ring-cleaving enzymes in the β-ketoadipate pathway for aromatic catabolism. These results suggest that H. anticariensis FP35 T has the potential to catabolize aromatic compounds and to be used in bioremediation processes under saline conditions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In most halophiles, K + generally acts as a major osmotic solute for osmotic adjustment and pH homeostasis. However, strains also need to extrude excessive intracellular K + to avoid its toxicity. In the halotolerant and alkaliphilic Halomonas sp. Y2, an Na + -induced K + extrusion process was observed when the cells were confronted with high extracellular K + pressure and supplementation by millimolar Na + ions. Among three mechanosensitive channels (KefA) and two K + /H + antiporters founded in the genome of the strain, ke1 displayed around 3-5-fold upregulation to ion stress at pH 8.0, while much higher upregulation of Ha-mrp was observed at pH 10.0. Compared to the growth of wild-type Halomonas sp. Y2, deletion of these genes from the strain resulted in different growth phenotypes in response to the osmotic pressure of potassium. In combination with the transcriptional response of these genes, we proposed that the KefA channel of Ke1 is the main contributor to the K + -extrusion process under weak alkalinity, while the Mrp system plays critical roles in alleviating K + contents at high pH. The combination of these strategies allows Halomonas sp. Y2 to grow over a range of extracellular pH and ion concentrations, and thus protect cells under high osmotic stress conditions.

Glutathione S-transferases are one of the most important antioxidant enzymes to protect against oxidative damage induced by reactive oxygen species. In this study, a novel gst gene, designated as hsgst , was derived from Antarctic sea ice bacterium Halomonas sp. ANT108 and expressed in Escherichia coli ( E. coli ) BL21. The hsgst gene was 603 bp in length and encoded a protein of 200 amino acids. Compared with the mesophilic EcGST, homology modeling indicated HsGST had some structural characteristics of cold-adapted enzymes, such as higher frequency of glycine residues, lower frequency of proline and arginine residues, and reduced electrostatic interactions, which might be in relation to the high catalytic efficiency at low temperature. The recombinant HsGST (rHsGST) was purified to apparent homogeneity with Ni-affinity chromatography and its biochemical properties were investigated. The specific activity of the purified rHsGST was 254.20 nmol/min/mg. The optimum temperature and pH of enzyme were 25 °C and 7.5, respectively. Most importantly, rHsGST retained 41.67% of its maximal activity at 0 °C. 2.0 M NaCl and 0.2% H₂O₂ had no effect on the enzyme activity. Moreover, rHsGST exhibited its protective effects against oxidative stresses in E. coli cells. Due to its high catalytic efficiency and oxidative resistance at low temperature, rHsGST may be a potential candidate as antioxidant in low temperature health foods.

Algicidal bacteria play an important role in mitigating harmful algal blooms (HABs). In the study, five bacterial strains were isolated from the East China Sea. One strain of algicidal bacterium, named DH-e, was found to selectively inhibit the motor ability of Prorocentrum donghaiense, Alexandrium tamarense (ATDH-47) and Karenia mikimotoi Hansen. Both 16S rDNA sequence analysis and morphological characteristics revealed that the algicidal DH-e bacterium belonged to Halomonas. Furthermore, results showed that the metabolites in the DH-e cell-free filtrate could kill algae directly, and the minimum inhibitory concentrations (MICs) of the bacterial metabolites on the cells of the three dinoflagellate species ranged from 35.0-70.0 μg/mL. Following short-term inhibitory tests, the dinoflagellates in mixed crude extract solution (0.7 mg/mL) ceased movement after 5 min. The algicidal mechanism of the metabolites was investigated through enzyme activities, including that of catalase (CAT), alkaline phosphatase (AKP), acetone peroxide (T-ATP) synthetase and nitrite reductase (NR). Results indicated that metabolites did not disrupt the energy or nutrient routes of the algae (P > 0.05), but did initiate an increase in free radicals in the algal cells, which might explain the subsequent death of sensitive algae. Thus, the metabolites of the DH-e bacterium showed promising potential for controlling HABs.

We previously used whole-genome sequencing and Tn5 transposon mutagenesis to identify 16 critical genes involved in the halotolerance of Halomonas beimenensis, a species in the phylum Proteobacteria. In this present study, we sought to determine if orthologous genes in another phylum are also critical for halotolerance. Virgibacillus spp. are halotolerant species that can survive in high-saline environments. Some Virgibacillus species are used in different aspects of food processing, compatible solute synthesis, proteinase production, and wastewater treatment. However, genomic information on Virgibacillus chiguensis is incomplete. We assembled a draft V. chiguensis strain NTU-102 genome based on high-throughput next-generation sequencing (NGS) and used transcriptomic profiling to examine the high-saline response in V. chiguensis. The V. chiguensis draft genome is approximately 4.09 Mbp long and contains 4,166 genes. The expression profiles of bacteria grown in 5% and 20% NaCl conditions and the corresponding Gene Ontology (GO) and clusters of orthologous groups (COG) categories were also analyzed in this study. We compared the expression levels of these 16 orthologs of halotolerance-related genes in V. chiguensis and H. beimenensis. Interestingly, the expression of 7 of the 16 genes, including trkA2, smpB, nadA, mtnN2, rfbP, lon, and atpC, was consistent with that in H. beimenensis, suggesting that these genes have conserved functions in different phyla. The omics data were helpful in exploring the mechanism of saline adaptation in V. chiguensis, and our results indicate that these 7 orthologs may serve as biomarkers for future screening of halotolerant species in the future., Competing Interests: The authors have declared that no competing interests exist.

The gastric microbiome is suspected to have a role in the causation of diseases by Helicobacter pylori. Reports on their relative abundance vis-à-vis H. pylori are available from various ethnic and geographic groups, but little is known about their interaction patterns. Endoscopic mucosal biopsy samples from the gastric antrum and corpus of 39 patients with suspected H. pylori infection were collected and microbiomes were analyzed by 16S rDNA profiling. Four groups of samples were identified, which harbored Helicobacter as well as a diverse group of bacteria including Lactobacillus, Halomonas and Prevotella. There was a negative association between the microbiome diversity and Helicobacter abundance. Network analyses showed that Helicobacter had negative interactions with members of the gastric microbiome, while other microbes interacted positively with each other, showing a higher tendency towards intra-cluster co-occurrence/co-operation. Cross-geographic comparisons suggested the presence of region-specific microbial abundance profiles. We report the microbial diversity, abundance variation and interaction patterns of the gastric microbiota of Indian patients with H. pylori infection and present a comparison of the same with the gastric microbial ecology in samples from different geographic regions. Such microbial abundance profiles and microbial interactions can help in understanding the pathophysiology of gastric ailments and can thus help in development of new strategies to curb it.

Studies on the halotolerance of bacteria are attractive to the fermentation industry. However, a lack of sufficient genomic information has precluded an investigation of the halotolerance of Halomonas beimenensis. Here, we describe the molecular mechanisms of saline adaptation in H. beimenensis based on high-throughput omics and Tn5 transposon mutagenesis. The H. beimenensis genome is 4.05 Mbp and contains 3,807 genes, which were sequenced using short and long reads obtained via deep sequencing. Sixteen Tn5 mutants with a loss of halotolerance were identified. Orthologs of the mutated genes, such as nqrA, trkA, atpC, nadA, and gdhB, have significant biological functions in sodium efflux, potassium uptake, hydrogen ion transport for energy conversion, and compatible solute synthesis, which are known to control halotolerance. Other genes, such as spoT, prkA, mtnN, rsbV, lon, smpB, rfbC, rfbP, tatB, acrR1, and lacA, function in cellular signaling, quorum sensing, transcription/translation, and cell motility also shown critical functions for promoting a halotolerance. In addition, KCl application increased halotolerance and potassium-dependent cell motility in a high-salinity environment. Our results demonstrated that a combination of omics and mutagenesis could be used to facilitate the mechanistic exploitation of saline adaptation in H. beimenensis, which can be applied for biotechnological purposes.

To engineer non-model organisms, suitable genetic parts must be available. However, biological parts are often host strain sensitive. It is therefore necessary to develop genetic parts that are functional regardless of host strains. Here we report several novel phage-derived expression systems used for transcriptional control in non-model bacteria. Novel T7-like RNA polymerase-promoter pairs were obtained by mining phage genomes, followed by in vivo characterization in non-model strains Halomonas spp TD01 and Pseudomonas entomophila. Three expression systems, namely, MmP1, VP4, and K1F, were developed displaying orthogonality (crosstalk<0.7%), tight regulation (3085-fold induction), and high efficiency (2.5-fold of P tac ) in Halomonas sp. TD01, a chassis strain with a high industrial value. The expression under the corresponding T7-like promoter libraries persisted with striking correlations (R 2 >0.94) between Escherichia coli and Halomonas sp. TD01, implying suitability of broad-host range. Three Halomonas TD strains were then constructed based upon these expression systems that enabled interchangeable and controllable gene expression. One of the strains termed Halomonas TD-MmP1 was used to express the cell-elongation cassette (minCD genes) and polyhydroxybutyrate (PHB) biosynthetic pathway, resulting in a 100-fold increase in cell lengths and high levels of PHB production (up to 92% of cell dry weight), respectively. We envision these T7-like expression systems to benefit metabolic engineering in other non-model organisms., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Physical and chemical boundaries for microbial multiplication on Earth are strongly influenced by interactions between environmental extremes. However, little is known about how interactions between multiple stress parameters affect the sensitivity of microorganisms to antibiotics. Here, we assessed how 12 distinct permutations of salinity, availability of an essential nutrient (iron) and atmospheric composition (aerobic or microaerobic) affect the susceptibility of a polyextremotolerant bacterium, Halomonas hydrothermalis, to ampicillin, kanamycin and ofloxacin. While salinity had a significant impact on sensitivity to all three antibiotics (as shown by turbidimetric analyses), the nature of this impact was modified by iron availability and the ambient gas composition, with differing effects observed for each compound. These two parameters were found to be of particular importance when considered in combination and, in the case of ampicillin, had a stronger combined influence on antibiotic tolerance than salinity. Our data show how investigating microbial responses to multiple extremes, which are more representative of natural habitats than single extremes, can improve our understanding of the effects of antimicrobial compounds and suggest how studies of habitability, motivated by the desire to map the limits of life, can be used to systematically assess the effectiveness of antibiotics., (© 2017 The Author(s).)

Fouling of marine surfaces has been a perpetual problem ever since the days of the early sailors. The tenacious attachment of seaweed and invertebrates to man-made surfaces, notably on ship hulls, has incurred undesirable economic losses. Graphene receives great attention in the materials world for its unique combination of physical and chemical properties. Herein, we present a novel 2-step synthesis method of graphene-silver nanocomposites which bypasses the formation of graphene oxide (GO), and produces silver nanoparticles supported on graphene sheets through a mild hydrothermal reduction process. The graphene-Ag (GAg) nanocomposite combines the antimicrobial property of silver nanoparticles and the unique structure of graphene as a support material, with potent marine antifouling properties. The GAg nanocomposite was composed of micron-scaled graphene flakes with clusters of silver nanoparticles. The silver nanoparticles were estimated to be between 72 and 86nm (SEM observations) while the crystallite size of the silver nanoparticles (AgNPs) was estimated between 1 and 5nm. The nanocomposite also exhibited the SERS effect. GAg was able to inhibit Halomonas pacifica, a model biofilm-causing microbe, from forming biofilms with as little as 1.3wt.% loading of Ag. All GAg samples displayed significant biofilm inhibition property, with the sample recording the highest Ag loading (4.9wt.% Ag) associated with a biofilm inhibition of 99.6%. Moreover, GAg displayed antiproliferative effects on marine microalgae, Dunaliella tertiolecta and Isochrysis sp. and inhibited the growth of the organisms by more than 80% after 96h. The marine antifouling properties of GAg were a synergy of the biocidal AgNPs anchored on the stable yet flexible graphene sheets, providing maximum active contact surface areas to the target organisms., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Algae-bacteria consortia may be potentially applied in wastewater treatment and environment remediation. In this study, in order to investigate effects of a symbiotic bacterium on the accumulation and transformation of arsenate[As(Ⅴ)] by Chlorella salina , we used batch cultures to determine the uptake, adsorption and transformation of As by axenic and non-axenic C. salina exposed to 0-750 μg·L -1 As(Ⅴ) for 7 d. The symbiotic bacterium of C. salina was confirmed to be Halomonas sp. after isolation, cultivation and 16S rRNA identification. The bacterial presence markedly increased the adsorption of As in C. salina , but it markedly reduced the absorption and the toxic effect of As(Ⅴ). Arsenate was the major arsenic species in the cells of axenic and non-axenic C. salina . The proportion of arsenite[As(Ⅲ)] was 8.99%-11.52% in the axenic microalga whereas a small quantity of monomethylarsonous acid (MMA) and dimethylarsinous acid (DMA) (0.02%-0.04%) were detected in the non-axenic counterpart. As(Ⅲ) dominated the As speciation in the bacterial culture and the percentage of As(Ⅴ) was 7.59%-26.80%, indicating that this symbiotic bacterium had a strong As(Ⅴ) reducing ability. The As removal rate (19.81%-41.08%) by non-axenic C. salina was higher than the bacterium alone (5.14%-14.62%) and axenic C. salina (14.98%-21.08%) after 7 d As(Ⅴ) exposure. The symbiotic Halomonas sp. promoted the accumulation of As by C. salina , indicating that algae-bacteria consortia might enhance the bioremediation of As contaminated water.

Although Halomonas phages belonging to the families Myoviridae and Siphoviridae have been reported, no virulent Halomonas siphoviruses are known. In this study, a virulent bacteriophage, QHHSV-1, of the family Siphoviridae that specifically infects H. ventosae QH52-2 was isolated from the Qiaohou salt mine. Restriction analysis indicated that QHHSV-1 is a dsDNA virus with a genome size of 33.5-39.5 kb. Transmission electron microscopy showed that QHHSV-1 is a typical representative of the Siphoviridae, with an icosahedral head (47 nm in diameter) and a non-contractile tail (75 nm in length). We also assessed the adsorption rate of QHHSV-1 for the host bacterium and found significant inhibition after the addition of 10 mM CaCl2. Based on a one-step growth curve, we determined a latent period of 30 min and a burst size of 73 PFU/infected cell. At the optimal pH of 8.0, 25.9 and 15.2 % of the phages survived after a 60-min incubation at 50 and 60 °C, respectively. Phage replication was possible at a wide range of salt concentrations, from 2.0 to 20 % (w/v), with an optimum concentration of 5 %. The survival of QHHSV-1 at different salt concentrations decreased with time and 25 % survival after 25 days at 30 % salt concentration.

Biogenic amines are identified as toxicological substances in foods and may have detrimental effects on consumers’ health. In recent years, the application of microorganisms that can degrade biogenic amines has become an emerging method for their reduction. The degradation characteristics and application potential of a salt-tolerant bacterium Halomonas shantousis SWA25 were investigated in this study. H. shantousis SWA25 exhibited degradation activity against eight biogenic amines at 10–40°C (optimum, 30–40°C) and pH 3.0–9.0 (optimum, 6.0–7.0) in the presence of 0–20% (w/v) NaCl (optimum, 0%). Specifically, H. shantousis SWA25 degraded all tryptamine (TRY) and tyramine (TYR) in 6 h, all phenethylamine (PHE) in 9 h, 66.7% of histamine (HIM), 52.4% of cadaverine (CAD), 48.0% of spermidine (SPD), 42.9% of putrescine (PUT) and 42.0% of spermine (SPM) in 20 h at 30°C and pH 7.0 with shaking at 120 r min−1. The enzymes from H. shantousis SWA25 responsible for degradation of biogenic amines were mainly amine oxidases located on the cell membrane. Further studies showed that H. shantousis SWA25 effectively degraded TRY, PHE, PUT, CAD, HIM and TYR in commercial fish sauce and soy sauce samples. Nevertheless, significant SPD and SPM degradation were not observed due to low initial concentrations. Therefore, H. shantousis SWA25 can be applied as a potential biogenic amines degradation bacterium in foods.

A moderately halophilic bacterium, designated strain 9-2(T), was isolated from saline and alkaline soil collected in Lindian county, Heilongjiang province, China. The strain was observed to be strictly aerobic, Gram-negative, rod-shaped, oxidase-positive, catalase-positive and motile. It was found to require NaCl for growth and to grow at NaCl concentrations of 0.5-14 % (w/v) (optimum, 7-10 %, w/v), at temperatures of 10-45 °C (optimum 25-30 °C) and at pH 5.0-10.0 (optimum pH 8.0). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 9-2(T) is a member of the genus Halomonas and is closely related to Halomonas desiderata DSM 9502(T) (96.68 %), Halomonas campaniensis DSM 1293(T) (96.46 %), Halomonas ventosae DSM 15911(T) (96.27 %) and Halomonas kenyensis DSM 17331(T) (96.27 %). The DNA-DNA hybridization value was 38.9 ± 0.66 % between the novel isolate 9-2(T) and H. desiderata DSM 9502(T). The predominant ubiquinones were identified as Q9 (75.1 %) and Q8 (24.9 %). The major fatty acids were identified as C16:0 (22.0 %), Summed feature 8 (C18:1 ω6c/C18:1 ω7c, 19.6 %), Summed feature 3 (C16:1 ω6c/C16:1 ω7c, 12.6 %), C12:0 3-OH (12.0 %) and C10:0 (11.7 %). The DNA G+C content was determined to be 69.7 mol%. On the basis of the evidence presented in this study, strain 9-2(T) is considered to represent a novel species of the genus Halomonas, for which the name Halomonas heilongjiangensis sp. nov. is proposed. The type strain is 9-2(T) (=DSM 26881(T) = CGMCC 1.12467(T)).

Lake Vanda is a perennially ice-covered and stratified lake in the McMurdo Dry Valleys, Antarctica. The lake develops a distinct chemocline at about a 50-m depth, where the waters transition from cool, oxic, and fresh to warm, sulfidic, and hypersaline. The bottom water brine is unique, as the highly chaotropic salts CaCl2 and MgCl2 predominate, and CaCl2 levels are the highest of those in any known microbial habitat. Enrichment techniques were used to isolate 15 strains of heterotrophic bacteria from the Lake Vanda brine. Despite direct supplementation of the brine samples with different organic substrates in primary enrichments, the same organism, a relative of the halophilic bacterium Halomonas (Gammaproteobacteria), was isolated from all depths sampled. The Lake Vanda (VAN) strains were obligate aerobes and showed broad pH, salinity, and temperature ranges for growth, consistent with the physicochemical properties of the brine. VAN strains were halophilic and quite CaCl2 tolerant but did not require CaCl2 for growth. The fact that only VAN strain-like organisms appeared in our enrichments hints that the highly chaotropic nature of the Lake Vanda brine may place unusual physiological constraints on the bacterial community that inhabits it., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Biofilm development is characterized by distinct stages of initial attachment, microcolony formation and maturation (sessile cells), and final detachment (dispersal of new, planktonic cells). In this work we examined the influence of polyhydroxyalkanoate (PHA) accumulation on bacterial surface properties and biofilm formation on polystyrene in detached vs. planktonic cells of an environmental strain isolated from microbial mats, Halomonas venusta MAT28. This strain was cultured either in an artificial biofilm in which the cells were immobilized on alginate beads (sessile) or as free-swimming (planktonic) cells. For the two modes of growth, conditions allowing or preventing PHA accumulation were established. Cells detached from alginate beads and their planktonic counterparts were used to study cell surface properties and cellular adhesion on polystyrene. Detached cells showed a slightly higher affinity than planktonic cells for chloroform (Lewis-acid) and a greater hydrophobicity (affinity for hexadecane and hexane). Those surface characteristics of the detached cells may explain their better adhesion on polystyrene compared to planktonic cells. Adhesion to polystyrene was not significantly different between H. venusta cells that had accumulated PHA vs. those that did not. These observations suggest that the surface properties of detached cells clearly differ from those of planktonic cells and that for at least the first 48 h after detachment from alginate beads H. venusta retained the capacity of sessile cells to adhere to polystyrene and to form a biofilm., (Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.)

In this study we analyzed the diversity of the halophilic bacteria community from Rambla Salada during the years 2006 and 2007. We collected a total of 364 strains, which were then identified by means of phenotypic tests and by the hypervariable V1-V3 region of the 16S rRNA sequences (around 500 bp). The ribosomal data showed that the isolates belonged to Proteobacteria (72.5%), Firmicutes (25.8%), Actinobacteria (1.4%), and Bacteroidetes (0.3%) phyla, with Gammaproteobacteria the predominant class. Halomonas was the most abundant genus (41.2% isolates) followed by Marinobacter (12.9% isolates) and Bacillus (12.6% isolates). In addition, 9 strains showed <97% sequence identity with validly described species and may well represent new taxa. The diversity of the bacterial community analyzed with the DOTUR package determined 139 operational taxonomic units at 3% genetic distance level. Rarefaction curves and diversity indexes demonstrated that our collection of isolates adequately represented all the bacterial community at Rambla Salada that can be grown under the conditions used in this work. We found that the sampling season influenced the composition of the bacterial community, and bacterial diversity was higher in 2007; this fact could be related to lower salinity at this sampling time.

The halophile Halomonas TD01 and its derivatives have been successfully developed as a low-cost platform for the unsterile and continuous production of chemicals. Therefore, to increase the genetic engineering stability of this platform, the DNA restriction/methylation system of Halomonas TD01 was partially inhibited. In addition, a stable and conjugative plasmid pSEVA341 with a high-copy number was constructed to contain a LacI(q)-Ptrc system for the inducible expression of multiple pathway genes. The Halomonas TD01 platform, was further engineered with its 2-methylcitrate synthase and three PHA depolymerases deleted within the chromosome, resulting in the production of the Halomonas TD08 strain. The overexpression of the threonine synthesis pathway and threonine dehydrogenase made the recombinant Halomonas TD08 able to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV consisting of 4-6 mol% 3-hydroxyvalerate or 3 HV, from various carbohydrates as the sole carbon source. The overexpression of the cell division inhibitor MinCD during the cell growth stationary phase in Halomonas TD08 elongated its shape to become at least 1.4-fold longer than its original size, resulting in enhanced PHB accumulation from 69 wt% to 82 wt% in the elongated cells, further promoting gravity-induced cell precipitations that simplify the downstream processing of the biomass. The resulted Halomonas strains contributed to further reducing the PHA production cost., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

A novel halophilic bacterium, strain RHS90(T), was isolated from marine sediments from the Gulf of Lions, in the Mediterranean Sea. Its metabolic and physiological characteristics were examined under various cultural conditions, including exposure to stressful ones (oligotrophy, high pressure and high concentrations of metals). Based on phylogenetic analysis of the 16S rRNA gene, the strain was found to belong to the genus Halomonas in the class Gammaproteobacteria. Its closest relatives are Halomonas axialensis and Halomonas meridiana (98% similarity). DNA-DNA hybridizations indicated that the novel isolate is genotypically distinct from these species. The DNA G + C content of the strain is 54.4 mol%. The main fatty acids (C18:1ω7c, 2-OH iso-C15:0, C16:0 and/or C19:0 cyclo ω8c), main polar lipids (diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unidentified phosphoglycolipid) and major respiratory quinone (ubiquinone Q9) were determined. The novel isolate is heterotrophic, mesophilic, euryhaline (growth optimum ranging from 2 to 8% w/v NaCl) and is able to grow under stressful conditions. The strain accumulates poly-β-hydroxyalkanoates granules and compatible solutes. Based on genotypic, chemotaxonomic and phenotypic distinctiveness, this isolate is likely to represent a novel species, for which the name Halomonas lionensis is proposed. The type strain of H. lionensis is RHS90(T) (DSM 25632(T) = CIP 110370(T) = UBOCC 3186(T))., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Two Gram-stain negative, moderately halophilic, aerobic, motile bacteria, designated strains YIM QH88(T) and YIM QH103, were isolated from the Qiaohou salt mine in Yunnan, southwest China. Cells of the strains were observed to be rod-shaped and produce creamy-coloured colonies. Growth of the two strains was observed at 10-45 °C (optimum 25-37 °C), at pH 6.0-10.0 (optimum 7.0-8.0), and in the presence of 0.5-20 % (w/v) NaCl (optimum 2-6 %). The two strains were found to contain summed feature 8 (C18:1 ω7c/ω6c), C19:0 cyclo ω8c and C16:0 as the major cellular fatty acids. The polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and unknown phospholipid. The G+C content of the genomic DNA of strains YIM QH88(T) and YIM QH103 were determined to be 64.6 and 64.2 mol%, respectively, and the predominant respiratory quinone detected was ubiquinone 9. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strains YIM QH88(T) and YIM QH103 formed a distinct lineage within the genus Halomonas and were most closely related to Halomonas pantelleriensis DSM 9661(T) with 97.3 and 97.5 % of 16S rRNA sequence similarity respectively. The DNA-DNA hybridization relatedness value for strains YIM QH88(T) and YIM QH103 was 95.2 ± 0.8 %. The levels of DNA-DNA relatedness between each of these two strains and the type strains of phylogenetically closely related Halomonas species were clearly below 70 %. On the basis of their phylogenetic analysis, DNA-DNA hybridization relatedness, phenotypic and chemotaxonomic characteristics, strains YIM QH88(T) and YIM QH103 should be classified as a novel species of the genus Halomonas, for which the name Halomonas qiaohouensis sp. nov. is proposed. The type strain is YIM QH88(T) (=DSM 26770(T) =CCTCC AB 2012965(T)).

The quorum sensing (QS) dependent behaviour of micro-organisms, in particular expression of virulence genes, biofilm formation and dispersal, have provided impetus for investigating practical approaches to interfere with microbial QS. This study tests Halomonas pacifica and Marinobacter hydrocarbonoclasticus, two halophilic marine micro-organism, for their AI-2 dependent QS signalling and the effect of two well-known quorum-sensing inhibitors (QSIs), patulin and penicillic acid, on biofilm formation. We report, for the first time, the successful amplification of a putative luxS gene in H. pacifica using degenerated primers and AI-2 dependent QS as well as inhibition using QSIs. Penicillic acid had a strong inhibitory effect on AI-2 induction of H. pacifica at non-growth inhibitory concentrations, while patulin has an adverse effect only at the highest concentration (25 μM). QSIs effect on biofilm forming capability was isolate specific, with maximum inhibition at 25 μM of patulin in H. pacifica. In M. hydrocarbonoclasticus, no adverse effects were noted at any tested concentration of either QSIs. Detection of bioluminescence and the presence of a putative luxS gene provide biochemical and genetic evidence for the production of a signalling molecule(s) which is the essential first step in characterizing H. pacifica QS. This study highlights the importance of AI-2 dependent QS in a marine setting, not previously reported. It further suggests that QSI compounds must be selected in the specific system in which they are to function, and they cannot easily be transferred from one QS system to another.

Halomonas has been organized as a genus since 1980, and comprises halophilic and/or halotolerant Gram-negative aerobic bacteria, typically found in saline environments. The genus is enlarging: at present, 76 species are taxonomically recognized, with more to be added. Increasing industrial uses have been found, largely in bioremediation and the production of desirable compounds. Originally seen as environmental contaminants, pathogenicity was initially not recognized; however, disease in algae, animals and humans has now been described. As the biotechnological use of these species increases, and the ability to isolate and recognize them improves, one might expect further pathogenic encounters with humans to be described.

A slightly halophilic bacterium (strain NEAU-ST10-25(T)) was isolated from saline-alkaline soils in Zhaodong City, Heilongjiang Province, China. The strain is a Gram-negative, aerobic motile rod. It accumulates poly-β-hydroxyalkanoate and produces exopolysaccharide. It produces beige-yellow colonies. Growth occurs at NaCl concentrations (w/v) of 0-15 % (optimum 3 %), at temperatures of 4-60 °C (optimum 35 °C) and at pH 6-12 (optimum pH 9). Its G+C content is 53.8 mol%. Phylogenetic analyses based on the separate 16S rRNA gene and concatenation of the 16S rRNA, gyrB and rpoD genes indicate that it belongs to the genus Halomonas in the class Gammaproteobacteria. The most phylogenetically related species is Halomonas alkaliphila DSM 16354(T), with which strain NEAU-ST10-25(T) showed 16S rRNA, gyrB and rpoD gene sequence similarities of 99.2, 82.3 and 88.2 %, respectively. The results of DNA-DNA hybridization assays showed 60.47 ± 0.69 % DNA relatedness between strain NEAU-ST10-25(T) and H. alkaliphila DSM 16354(T), 42.43 ± 0.37 % between strain NEAU-ST10-25(T) and Halomonas venusta DSM 4743(T) and 30.62 ± 0.43 % between strain NEAU-ST10-25(T) and Halomonas hydrothermalis DSM 15725(T). The major fatty acids are C18:1 ω7c (62.3 %), C16:0 (17.6 %), C16:1 ω7c/C16:1 ω6c (7.7 %), C14:0 (2.9 %), C12:0 3-OH (2.8 %), C10:0 (2.1 %) and C18:1 ω9c (1.6 %) and the predominant respiratory quinone is ubiquinone 9 (Q-9). The proposed name is Halomonas zhaodongensis, NEAU-ST10-25(T) (=CGMCC 1.12286(T) = DSM 25869(T)) being the type strain.

A novel Gram-negative, aerobic, slightly halophilic, yellow-pigmented, oxidase-negative, Voges-Proskauer positive, non-spore-forming bacterium, designated YIM M 13059(T), was isolated from a sediment sample collected from the South China Sea at a depth of 310 m. Optimal growth was found to occur at 28-30 °C, pH 7.0 and in the presence of 3-4 % (w/v) NaCl. Cells were observed to be rod-shaped and motile by peritrichous flagella. The polar lipids of strain YIM M 13059(T) were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a ninhydrin-positive phospholipid, one glycolipid and two unknown phospholipids. The predominant respiratory quinone was determined to be Q-9. The major fatty acids were identified as C18:1 ω7c, C16:1 ω6c/C16:1 ω7c, C16:0 and C12:0 3-OH. The genomic DNA G+C content was determined to be 54.4 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the isolate belongs to the genus Halomonas in the family Halomonadaceae. The 16S rRNA gene sequence similarities between strain YIM M 13059 (T) and the type strains of members of the genus Halomonas were in the range 93.3-98.3 %. However, the levels of DNA-DNA relatedness values between YIM M 13059 and the type strains of the most closely related species, Halomonas zhangjiangensis, Halomonas variabilis, Halomonas neptunia, Halomonas boliviensis and Halomonas sulfadieris were 50.2 ± 0.68 %, 46.8 ± 1.9 %, 28.5 ± 0.74 %, 42.9 ± 0.55 % and 37.1 ± 0.68 %, respectively. Based on phylogenetic, chemotaxonomic and phenotypic data, the strain YIM M 13059(T) is proposed to represent a novel member of the genus Halomonas, with the name Halomonas nanhaiensis sp. nov. The type strain is YIM M 13059(T) (=JCM 18142(T) =CCTCC AB 2012911(T)).

Two moderately halophilic, facultatively aerobic, motile bacteria with flagella, designated strains 10-C-3(T) and 30-C-3, were isolated from jeotgal, a traditional Korean fermented seafood. Cells of the strains were observed to be ovoid-rods showing catalase- and oxidase-positive reactions and production of creamy-pink pigments. Growth of strain 10-C-3(T) was observed at 15-35 °C (optimum, 25-30 °C), at pH 5.5-9.0 (optimum, pH 7.0-7.5), and in the presence of 3-15 % (w/v) salts (optimum: 5-10 %). The two strains were found to contain C(18:1) ω7c, C(16:0), summed feature 3 (as defined by the MIDI system, comprising C(16:1) ω7c and/or C(16:1) ω6c), and C(12:0) 3-OH as the major cellular fatty acids. The G+C contents of the genomic DNA of strains 10-C-3(T) and 30-C-3 were determined to be 63.2 and 63.1 mol%, respectively and the respiratory quinone detected was ubiquinone 9 (Q-9) only. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strains 10-C-3(T)and 30-C-3 formed a distinct phyletic lineage within the genus Halomonas and are most closely related to Halomonas fontilapidosi 5CR(T) with 95.2 % of 16S rRNA sequence similarity. Strains 10-C-3(T)and 30-C-3 shared 99.2 % of 16S rRNA gene sequence similarity and their DNA-DNA relatedness value was 96.6 ± 0.9 %. On the basis of phenotypic, chemotaxonomic and molecular features, strains 10-C-3(T)and 30-C-3 represent a novel species of the genus Halomonas, for which the name Halomonas cibimaris sp. nov. is proposed. The type strain is 10-C-3(T) (= KACC 14932(T) = JCM 16914(T)).

A multisensory, hybrid histidine kinase (HK) and a response regulator (RR), which together may well constitute a two-component regulatory system (TCS), have been located in Halomonas anticariensis FP35(T) by transposon mutagenesis. This TCS is homologous to the GacS/GacA system described for many Gram-negative bacteria. An analysis of crude N-acylhomoserine lactone (AHL) extracts from cultures of FP35gacS and FP35gacA mutants showed that they produced lower quantities of AHLs than the wild-type strain. In addition, RT-PCR analysis revealed a considerable decrease in the expression of the quorum-sensing (QS) genes hanR and hanI compared with the wild-type strain. This result indicates that the GacS/GacA TCS exerts a positive effect upon the QS HanR/HanI system and suggests its integral involvement in the intercellular communication strategies of this bacterium. We have also demonstrated the influence of GacS and GacA upon exopolysaccharide production and biofilm formation, in which this regulatory machinery appears to play a key role in an overall system that co-ordinates gene expression and behaviour in H. anticariensis FP35(T) in response to environmental conditions.

Extremophilic bacterial polysaccharide based biocompatible nanofibers were produced for the first time via electrospinning technique. Mauran (MR), an extremophilic sulfated exopolysaccharide was extracted from moderately halophilic bacterium, Halomonas maura and characterized for the application of nanofiber synthesis. Thin-uniform MR nanofibers were produced using homogenous solutions of poly (vinyl alcohol) (PVA) blended with different concentrations of MR. Characterization of complex MR/PVA nanofibers were performed using scanning electron microscope and analyzed for the cytotoxicity using mouse fibroblast cells as well as mesenchymal stem cells. An average of 120 nm sized nanofibers were produced and tested for an enhanced cell growth under in vitro conditions in comparison with control. MR and MR/PVA nanofibers were found to be an excellent biomaterial for the migration, proliferation and differentiation of mammalian cells, which was confirmed by cell adhesion studies and confocal microcopy. Interestingly, biological and physicochemical properties of MR hasten the application of MR based nanofibers for various biomedical applications like tissue engineering and drug delivery., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Microbial mats are complex but stable, multi-layered and multi-functional biofilms, which are the most frequent bacterial formations in nature. The functional strategies and physiological versatility of the bacterial populations growing in microbial mats allow bacteria to resist changing conditions within their environment. One of these strategies is the accumulation of carbon- and energy-rich polymers that permit the recovery of metabolic activities when favorable conditions are restored. In the present study, we systematically screened microbial mats for bacteria able to accumulate large amounts of the ester carbon polymers polyhydroxyalkanoates (PHA). Several of these strains were isolated from Ebro Delta microbial mats and their ability to accumulate PHA up to 40-60% of their dry weight was confirmed. According to two identification approaches (16S rRNA and rpoD genes), these strains were identified as Halomonas alkaliphila (MAT-7, -13, -16), H. neptunia (MAT-17), and H. venusta (MAT-28). To determine the mode of growth yielding maximum PHA accumulation, these three different species were cultured in an artificial biofilm in which the cells were immobilized on alginate beads. PHA accumulation by cells that had detached from the biofilm was compared with that of their planktonic counterparts. Experiments in different culture media showed that PHA accumulation, measured as the relative fluorescence intensity after 48 h of incubation at 30 degrees C, was higher in immobilized than in planktonic cells, with the exception of cells growing in 5% NaCl, in which PHA accumulation was drastically lower in both. Therefore, for obtaining high PHA concentrations, the use of immobilized cells may be a good alternative to the PHA accumulation by bacteria growing in the classical, planktonic mode. From the ecological point of view, increased PHA accumulation in detached cells from biofilms would be a natural strategy to improve bacterial dispersion capacity and, consequently, to increase survival in stressed environments.

Microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was used to screen for potential polyphosphate-accumulating organisms (PAOs) in full-scale enhanced biological phosphorus removal (EBPR) plants. Clone library analyses and application of MAR-FISH using newly designed probes revealed that small rods related to uncultured Halomonas within the gammaproteobacterial family Halomonadaceae were actively involved in uptake of orthophosphate. Although deeply branched in the Gammaproteobacteria, they were not targeted by the gammaproteobacterial probe (GAM42a). A part of them were also not targeted with the general bacterial probes (EUBmix). They could take up short-chain fatty acids (e.g. acetate and propionate) and ethanol under both anaerobic and aerobic conditions. Polyhydroxyalkanoate storage was observed under anaerobic conditions. There was no indication of a denitrifying capability. A survey of the occurrence of these Halomonas-PAOs in 23 full-scale EBPR plants revealed that they made up 0.5-5.7% of all bacteria in the plants, and were often in higher abundance than the well-described PAOs 'Candidatus Accumulibacter phosphatis'. This indicates a potentially important role for these uncultured Halomonas bacteria in the EBPR process in full-scale plants and we propose to name them 'Candidatus Halomonas phosphatis'., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Halomonas smyrnensis AAD6(T) is a Gram-negative, aerobic, exopolysaccharide-producing, and moderately halophilic bacterium that produces levan, a fructose homopolymer with many potential uses in various industries. We report the draft genome sequence of H. smyrnensis AAD6(T), which will accelerate research on the rational design and optimization of microbial levan production.

In the present study, osmoadaptive mechanism of Halomonas sp. AAD12 was studied through analysis of changes in its proteome maps and osmolyte accumulation strategy to understand how this euryhaline microorganism masters osmotic stress of saline environments. Under salt stress, there were significant variations in the expression of proteins involved in osmoregulation, stress response, energy generation and transport. This was accompanied by an increase in proline and hydroxyectoine but a decrease in ectoine accumulation. The major osmolyte at high salinity was proline. Unexpectedly the size of the total ectoines' pool was smaller at elevated salinity. Experimental findings were then integrated with a metabolic model to get insight into carbon trafficking during osmoadaptation. Simulations predicted that the total flux through energy generating pathways, namely gluconeogenesis and the pentose phosphate pathway, was significantly lower and carbon source that entered the system as citrate was mainly diverted to osmolyte synthesis at high salinity. Overall these results suggested that the moderately halophilic Halomonas sp. AAD12 pursued a different osmoregulatory strategy than the two well known moderate halophiles, Chromohalobacter salexigens and Halobacillus halophilus. The climbing value of osmolytes such as ectoine in health care and skin care products places significant attention to halophilic microorganisms hence an understanding of the osmoadaptive mechanism and osmolyte accumulation strategy of this isolate is very valuable to be able to manipulate its metabolism towards desired goals., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

A Gram-negative, aerobic, short rod-shaped and non-motile bacterium, strain A-1(T), was isolated from a saline soil contaminated with crude oil in Xianhe, Shangdong Province, China. Strain A-1(T) formed yellow colonies, was moderately halophilic and grew with 0.05-27.5% (w/v) total salts (optimum 5-8%), at 10-42 °C (optimum 30 °C) and at pH 5.5-9.0 (optimum pH 7.2). The dominant fatty acids (>5%) were C(16:0), summed feature 3 (comprising C(16:1)ω7c and/or iso-C(15:0) 2-OH), C(18:1)ω7c, C(19:0) cyclo ω8c and C(12:0) 3-OH and the predominant ubiquinone was Q-9. The genomic DNA G+C content was 67.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A-1(T) belonged to the genus Halomonas in the class Gammaproteobacteria. The closest relatives were Halomonas lutea YIM 91125(T) (97.7% 16S rRNA gene sequence similarity), H. muralis LMG 20969(T) (95.6%), H. pantelleriensis AAP(T) (95.5%) and H. kribbensis BH843(T) (95.2%). DNA-DNA relatedness between strain A-1(T) and H. lutea CCTCC AB 206093(T) was 27±3%. On the basis of phenotypic, chemotaxonomic and phylogenetic features, strain A-1(T) should be placed in the genus Halomonas as a representative of a novel species. The name Halomonas xianhensis sp. nov. is proposed, with strain A-1(T) (=CGMCC 1.6848(T) =JCM 14849(T)) as the type strain.

Quorum sensing is a cell density-dependent gene expression mechanism found in many Gram-negative bacteria which involves the production of signal molecules such as N-acylhomoserine lactones (AHLs). One significant group of micro-organisms in which quorum sensing has not been previously studied, however, are the moderate halophiles. We describe here the results of our studies of the quorum-sensing system in Halomonas anticariensis FP35(T), which is composed of luxR/luxI homologues: hanR (the putative transcriptional regulator gene) and hanI (the autoinducer synthase gene). To understand how the hanR/hanI system is organized and regulated we conducted RT-PCR and quantitative real-time PCR assays. Transcriptional analysis indicated that the hanR and hanI genes are on the same transcript and that their transcription is growth phase-dependent. HanI seems to be the only autoinducer synthase responsible for the synthesis of AHLs by the bacterium, since the inactivation of hanI resulted in the complete loss of its AHLs. We also found that the hanI gene appears to be transcribed from its own promoter and that its expression does not depend upon HanR. This finding was supported by the fact that the FP35hanR mutant showed AHL-producing activity and hanI expression similar to that of the wild-type strain, the latter being measured by RT-PCR. Moreover, hanR is expressed from its own promoter and appears to be independent of the AHL signalling molecules produced by HanI.

A moderately halophilic bacterial strain 15-13(T), which was isolated from soda meadow saline soil in Daqing City, Heilongjiang Province, China, was subjected to a polyphasic taxonomic study. The cells of strain 15-13 were found to be Gram-negative, rod-shaped, and motile. The required growth conditions for strain 15-13(T) were: 1-23% NaCl (optimum, 7%), 10-50°C (optimum, 35°C), and pH 7.0-11.0 (optimum, pH 9.5). The predominant cellular fatty acids were C(18:1) ω7c (60.48%) and C(16:0) (13.96%). The DNA G+C content was 67.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that strain 15-13(T) clustered within a branch comprising species of the genus Halomonas. The closest phylogenetic neighbor of strain 15-13(T) was Halomonas pantelleriensis DSM 9661(T) (98.9% 16S rRNA gene sequence similarity). The level of DNA-DNA relatedness between the novel isolated strain and H pantelleriensis DSM 9661(T) was 33.8%. On the basis of the phenotypic and phylogenetic data, strain 15-13(T) represents a novel species of the genus Halomonas, for which the name Halomonas alkalitolerans sp. nov. is proposed. The type strain for this novel species is 15-13(T) (=CGMCC 1.9129(T) =NBRC 106539(T)).

Composting of the Undaria pinnatifida (wakame) seaweed was conducted after inoculation with 6×10(8) CFU g(-1)Halomonas sp. AW4 and the alginate-degrading bacterium Gracilibacillus sp. A7. Inoculation with strains A7 and AW4 resulted in 27.8% and 24.7% degradation of U. pinnatifida dry mass after 168 h, whereas only 17.5% degradation occurred in the uninoculated control. The C/N ratio decreased in the A7 and AW4 inoculated compost by 7.0% and 9.2% after 72 h, but increased by 11.5% in the control. Inoculation with A7 resulted in 2.8 times faster degradation of alginate and 1.2 and 1.6 times higher levels of reducing sugars and unsaturated sugars than inoculation with AW4. The compost produced from the inoculation with A7 had low plant toxicity as measured by germination experiment. The results suggest that inoculation of wakame with alginate-degrading bacteria not only shortened the length of composting but also created seaweed compost with good fertilizer qualities., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Photosynthetic microbial mats are sources of microbial diversity and physiological strategies that reflect the physical and metabolic interactions between their resident species. This study focused on the diversity and activity of polyhydroxyalkanoate-producing and -degrading bacteria and their close partnership with cyanobacteria in an estuarine and a hypersaline microbial mat. The aerobic heterotrophic population was characterized on the basis of lipid biomarkers (respiratory quinones, sphingoid bases), polyhydroxyalkanoate determination, biochemical analysis of the isolates, and interaction assays. Most of the polyhydroxyalkanoate-producing isolates obtained from an estuarine mat belonged to the Halomonas and Labrenzia genera, while species of Sphingomonas and Bacillus were more prevalent in the hypersaline mat. Besides, the characterization of heterotrophic bacteria coisolated with filamentous cyanobacteria after selection suggested a specific association between them and diversification of the heterotrophic partner belonging to the Halomonas genus. Preliminary experiments suggested that syntrophic associations between strains of the Pseudoalteromonas and Halomonas genera explain the dynamics of polyhydroxyalkanoate accumulation in some microbial mats. These metabolic interactions and the diversity of the bacteria that participate in them are most likely supported by the strong mutual dependence of the partners., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Wheat straw black liquor is a notorious pulp mill wastewater with very high pH and pollution load. Two halotolerant and alkaliphilic bacteria, designated as Halomonas sp. 19-A and Y2, were isolated from wheat straw black liquor and shown to be able to use guaiacol, vanillin, dibenzo-p-dioxin, biphenyl and fluorene, as sole carbon and carbazole as sole carbon and nitrogen source at pH 9.5 and in the presence of 10% NaCl. The two strains produced carboxymethylcellulase (CMCase), xylanase, lipase, amylase, and pullulanase. High activities of CMCase, xylanase, and amylase were observed at pH 5.0-11.0 and NaCl concentrations of 0-15%. The metabolic versatility of these Halomonas strains even under extreme pH and salinity conditions makes them promising agents for bioremediation and industrial processes., ((c) 2010 Elsevier Ltd. All rights reserved.)

We have carried out a polyphasic taxonomic characterization of the type strains of the species with the recently validated name Salinicola socius, together with two species that were phylogenetically closely related, Halomonas salaria and Chromohalobacter salarius. 16S rRNA gene sequence analyses showed that they constituted a coherent cluster, with sequence similarities between 98.7 and 97.7 %. We have determined the almost complete 23S rRNA gene sequences of these three type strains, and the percentage of similarity between them was 99.2-97.6 %. Phylogenetic trees based on the 16S rRNA and 23S rRNA gene sequences, obtained by using three different algorithms, were consistent and showed that these three species constituted a cluster separated from the other species of the genera of the family Halomonadaceae, supporting their placement in a single genus. All three species have ubiquinone 9 as the major respiratory quinone, and showed similar fatty acid and polar lipid profiles. The level of DNA-DNA hybridization between Salinicola socius DSM 19940(T), Halomonas salaria DSM 18044(T) and Chromohalobacter salarius CECT 5903(T) was 41-21 %, indicating that they are different species of the genus Salinicola. A comparative phenotypic study of these strains following the proposed minimal standards for describing new taxa of the family Halomonadaceae has been carried out. The phenotypic data are consistent with the placement of these three species in a single genus and support their differentiation at the species level. On the basis of these data we have emended the description of the species Salinicola socius and we propose to transfer the species Halomonas salaria and Chromohalobacter salarius to the genus Salinicola, as Salinicola salarius comb. nov. (type strain M27(T) =KCTC 12664(T) =DSM 18044(T)) and Salinicola halophilus nom. nov. (type strain CG4.1(T) =CECT 5903(T) =LMG 23626(T)), respectively.

We have made a comprehensive study of the denitrifying species of the genus Halomonas, evaluating both the phylogenetic and phenotypic relationships amongst them and other species of Halomonas. The phylogenetic analysis was based on the 16S rRNA gene sequence as well as those of the three genes essential to a complete denitrification process: narH, nirS and nosZ. The main aim of the phenotypic study was to improve our knowledge of some of the species in question. To this end we investigated the type strain of each species, although in the case of Halomonas cerina we also studied strains R53 and 15CR. In addition to this we investigated some other strains phylogenetically related to Halomonas ventosae, Halomonas denitrificans and Halomonas koreensis that were isolated during this study. We also looked into the conditions under which all these bacteria denitrify. Our results indicate that these denitrifying species of Halomonas are all closely related. A numerical analysis of the phenotypic data demonstrates a high phenotypic similarity (73%) between most of them. In addition, all the denitrifying strains have a high G+C content of between 63 and 74.3 mol%. The results of the phylogenetic study point to two evolutionary lineages for the process. Although phenotypic similarity does not always reflect phylogenetic relatedness, we have found significant congruence between both features in Halomonas, making it clear that denitrifying ability should be considered as an important phenotypic and phylogenetic discriminatory marker within this genus., (Copyright 2009 Elsevier GmbH. All rights reserved.)

A novel bacterium, TRM 0175(T), belonging to the genus Halomonas, was isolated from a soil sample taken from a salt lake in Xinjiang Province, north-west China. The isolate was Gram-negative, aerobic, rod-shaped and motile by means of peritrichous flagella. It was catalase-positive and oxidase-negative. Growth occurred at NaCl concentrations of 0-20 % (optimum at 10-13 %), at 15-50 degrees C (optimum at 37 degrees C) and at pH 6.0-9.0 (optimum at pH 7.0). Metabolism was respiratory with oxygen as terminal electron acceptor. Acid was produced from D-ribose, D- and L-arabinose, D-xylose, D-galactose, D-mannose, L-rhamnose, cellobiose, maltose, trehalose and D- and L-fucose and was produced weakly from aesculin. The predominant ubiquinone was Q-9. The major fatty acids were C(18 : 1)omega7c and C(19 : 0) cyclo omega8c. The G+C content of the genomic DNA was 60.0 mol%. The affiliation of strain TRM 0175(T) with the genus Halomonas was confirmed by 16S rRNA gene sequence comparisons. The most closely related species was Halomonas anticariensis; 16S rRNA gene sequence similarity between H. anticariensis FP35(T) and strain TRM 0175(T) was 95.3 %. Phenotypically, some characteristics of TRM 0175(T) differed from those of H. anticariensis. On the basis of data from this polyphasic study, strain TRM 0175(T) represents a novel species of the genus Halomonas, for which the name Halomonas xinjiangensis sp. nov. is proposed; the type strain is TRM 0175(T) (=CCTCC AB 208329(T) =KCTC 22608(T)).

Streptococcus pyogenes is the frequent cause of purulent infections in humans. Formation of a biofilm is one of the important aspects of its pathogenicity. Streptococcus pyogenes biofilm communities tend to exhibit significant tolerance to antimicrobial challenge during infections. Exploring novel targets against biofilm-forming pathogens is therefore an important alternative treatment measure. We attempted to screen marine bacteria, especially coral-associated bacteria (CAB), for antibiofilm activity against streptococcal biofilm formation. The bacterial biofilms were quantified by crystal violet staining. Of 43 CAB isolates, nine clearly demonstrated antibiofilm activity. At biofilm inhibitory concentrations (BIC), biofilm formation was reduced up to 80%, and sub-BIC (0.5 and 0.25 BIC) significantly reduced biofilm formation by up to 60% and 40-60%, respectively. Extracts of Bacillus horikoshii (E6) displayed efficient antibiofilm activity. As quorum sensing (QS) and cell surface hydrophobicity (CSH) are crucial factors for biofilm formation in S. pyogenes, the CAB were further screened for QS inhibition properties and CSH reduction properties. This study reveals the antibiofilm and QS inhibition property of CAB.

Colonization of host mucus surfaces is one of the first steps in the establishment of coral-associated microbial communities. Coral mucus contains a sulfated glycoprotein (in which oligosaccharide decorations are connected to the polypeptide backbone by a mannose residue) and molecules that result from its degradation. Mucus is utilized as a growth substrate by commensal and pathogenic organisms. Two representative coral commensals, Photobacterium mandapamensis and Halomonas meridiana, differed from a white pox pathogen Serratia marcescens PDL100 in the pattern with which they utilized mucus polymers of Acropora palmata. Incubation with the mucus polymer increased mannopyranosidase activity in S. marcescens, suggestive of its ability to cleave off oligosaccharide side chains. With the exception of glucosidase and N-acetyl galactosaminidase, glycosidases in S. marcescens were subject to catabolite regulation by galactose, glucose, arabinose, mannose and N-acetyl-glucosamine. In commensal P. mandapamensis, at least 10 glycosidases were modestly induced during incubation on coral mucus. Galactose, arabinose, mannose, but not glucose or N-acetyl-glucosamine had a repressive effect on glycosidases in P. mandapamensis. Incubation with the mucus polymers upregulated 3 enzymatic activities in H. meridiana; glucose and galactose appear to be the preferred carbon source in this bacterium. Although all these bacteria were capable of producing the same glycosidases, the differences in the preferred carbon sources and patterns of enzymatic activities induced during growth on the mucus polymer in the presence of these carbon sources suggest that to establish themselves within the coral mucus surface layer commensals and pathogens rely on different enzymatic activities.

We isolated a moderately halophilic bacterium with high level of tolerance to two toxic oxyanions, selenite and tellurite, from hypersaline soil in Garmsar, Iran. 16s rRNA sequence analysis revealed that the isolate, strain MAM, had 98% similarity with Halomonas elongate, and is closely related to other species of the genus Halomonas. We observed that the tolerance to tellurite and its removal increased significantly when both selenite and tellurite were added to the culture media, suggesting a positive synergism of selenite on tellurite tolerance and removal. We applied a proteomic approach to study the proteome response of Halomonas sp. strain MAM to selenite, tellurite, and selenite + tellurite. Out of approximately 800 protein spots detected on 2-DE gels, 208 spots were differentially expressed in response to at least one of treatments. Of them, 70 CBB stained spots were analyzed by MALDI TOF/TOF mass spectrometry, leading to identification of 36 proteins. Our results revealed that several mechanisms including fatty acid synthesis, energy production, cell transport, oxidative stress detoxification, DNA replication, transcription and translation contributed in bacterial response and/or adaptation. These results provided new insights into the general mechanisms on the tolerance of halophilic bacteria to these two toxic oxyanions and the use of them for bioremediation of contaminated saline soils and wastes discharge sites.

We have made a polyphasic taxonomic study of strain 5CR(T), isolated from Fuente de Piedra, Málaga, southern Spain. The strain is a moderately halophilic, Gram-negative rod, oxidase-positive and motile by a single polar flagellum. It does not produce acids from sugars and shows respiratory metabolism, using oxygen, nitrate and nitrite as terminal electron acceptors. It requires NaCl and grows best with 5-7.5 % w/v at temperatures of between 32 and 45 degrees C within a pH range of 6-8. Its 16S rRNA gene sequence indicates that strain 5CR(T) belongs to the genus Halomonas in the class Gammaproteobacteria. Its closest relatives are Halomonas alimentaria, H. nitroreducens, H. shengliensis and H. ventosae, with the type strains of which our strain showed 16S rRNA gene sequence similarity values of 96.7-97.8 %. DNA-DNA hybridization studies between strain 5CR(T) and H. ventosae CECT 5797(T), the phylogenetically nearest type strain, showed 40 % relatedness. Its G+C content is 65.7 mol%. Its major fatty acids are C(18 : 1)omega7c (31.36 %), C(16 : 0) (25.55 %), C(16 : 1)omega7c/iso-C(15 : 0) 2-OH (23.23 %), C(19 : 0) cyclo omega8c (8.14 %), C(12 : 0) 3-OH (5.76 %) and C(10 : 0) (2.22 %) and the predominant respiratory lipoquinone is ubiquinone with nine isoprene units (Q-9). The proposed name for the novel species is Halomonas fontilapidosi sp. nov., strain 5CR(T) (=CECT 7341(T) =LMG 24455(T)) being the type strain.