Halomonas Sp Research Articles

High level synthesis of levan by a novel Halomonas species growing on defined media

A novel halophilic isolate from soil samples taken from Çamaltı Saltern area in Turkey, Halomonas sp. AAD6 (JCM 15723) strain, was found to produce high levels of exopolysaccharides (EPS), in the presence of sucrose in defined media, by flasks and bioreactor condition yielded 1.073 g L −1 and 1.844 g L −1, respectively. Sugar analysis, methylation studies and NMR analysis of EPS indicated the repeating unit of this polysaccharide was composed of β-(2,6)- d-fructofuranosyl residues. Hence with this work, Halomonas sp. has been described as a levan producer microorganism for the first time. Biocompatibility studies showed this EPS did not affect cellular viability and proliferation of osteoblasts and murine macrophages. The protective effect of the polymer against the toxic activity of avarol implied its additional use as an anti-cytotoxic agent. Halomonas sp. AAD6 could represent an alternative cheap source of levan polymer when grown on defined media hypothesizing its larger employment in industrial application being non pathogenic microorganism.

Loihichelins A−F, a Suite of Amphiphilic Siderophores Produced by the Marine Bacterium Halomonas LOB-5

A suite of amphiphilic siderophores, loihichelins A-F, were isolated from cultures of the marine bacterium Halomonas sp. LOB-5. This heterotrophic Mn(II)-oxidizing bacterium was recently isolated from the partially weathered surfaces of submarine glassy pillow basalts and associated hydrothermal flocs of iron oxides collected from the southern rift zone of Loihi Seamount east of Hawai'i. The loihichelins contain a hydrophilic headgroup consisting of an octapeptide comprised of D-threo-beta-hydroxyaspartic acid, D-serine, L-glutamine, L-serine, L-N(delta)-acetyl-N(delta)-hydroxyornithine, dehydroamino-2-butyric acid, D-serine, and cyclic N(delta)-hydroxy-D-ornithine, appended by one of a series of fatty acids ranging from decanoic acid to tetradecanoic acid. The structure of loihichelin C was determined by a combination of amino acid and fatty acid analyses, tandem mass spectrometry, and NMR spectroscopy. The structures of the other loihichelins were inferred from the amino acid and fatty acid analyses and tandem mass spectrometry. The role of these siderophores in sequestering Fe(III) released during basaltic rock weathering, as well as their potential role in the promotion of Mn(II) and Fe(II) oxidation, is of considerable interest.

Isolation and characterization ofHalomonassp. strain C2SS100, a hydrocarbon-degrading bacterium under hypersaline conditions

To isolate and characterize an efficient hydrocarbon-degrading bacterium under hypersaline conditions, from a Tunisian off-shore oil field. Production water collected from 'Sercina' petroleum reservoir, located near the Kerkennah island, Tunisia, was used for the screening of halotolerant or halophilic bacteria able to degrade crude oil. Bacterial strain C2SS100 was isolated after enrichment on crude oil, in the presence of 100 g l(-1) NaCl and at 37 degrees C. This strain was aerobic, Gram-negative, rod-shaped, motile, oxidase + and catalase +. Phenotypic characters and phylogenetic analysis based on the 16S rRNA gene of the isolate C2SS100 showed that it was related to members of the Halomonas genus. The degradation of several compounds present in crude oil was confirmed by GC-MS analysis. The use of refined petroleum products such as diesel fuel and lubricating oil as sole carbon source, under the same conditions of temperature and salinity, showed that significant amounts of these heterogenic compounds could be degraded. Strain C2SS100 was able to degrade hexadecane (C16). During growth on hexadecane, cells surface hydrophobicity and emulsifying activity increased indicating the production of biosurfactant by strain C2SS100. A halotolerant bacterial strain Halomonas sp. C2SS100 was isolated from production water of an oil field, after enrichment on crude oil. This strain is able to degrade hydrocarbons efficiently. The mode of hydrocarbon uptake is realized by the production of a biosurfactant which enhances the solubility of hydrocarbons and renders them more accessible for biodegradation. The biodegradation potential of the Halomonas sp. strain C2SS100 gives it an advantage for possibly application on bioremediation of water, hydrocarbon-contaminated sites under high-salinity level.

Effects of Dietary Administration of Probiotic Halomonas sp. B12 on the Intestinal Microflora, Immunological Parameters, and Midgut Histological Structure of Shrimp, Fenneropenaeus chinensis

Abstract The experiment was conducted to investigate the effects of oral administration of probiotic Halomonas sp. B12 (previously isolated from the intestine of Fenneropenaeus chinensis) on the intestinal microflora, immunological parameters, and midgut histological structure of F. chinensis. Shrimp (initial weight: 4.00 ± 0.10 g) were fed diets containing Halomonas sp. B12 at 0 (control), 3.68 × 107 (T1), and 7.18 × 1010 (T2) colony‐forming units per gram for 6 wk, respectively. The results showed that the total bacterial counts significantly increased (P < 0.05) with supplementation of dietary probiotic B12. However, Vibrio spp. counts significantly decreased (P < 0.05) in the intestine of shrimp with increasing dietary probiotic B12. Hemocyte counts in the shrimp fed the diets supplemented with probiotic B12 were significantly higher (P < 0.05) than the control group. Both phenoloxidase (PO) activity in plasma and hemocyte lysate supernatant were higher in the shrimp fed diets supplemented with probiotic B12 compared with the control group. No significant difference was found in PO activity in plasma between the T1 and the control (P > 0.05). PO activity in plasma was higher in T2 than that in T1. Cumulative shrimp mortality after 10‐d white spot syndrome virus (WSSV) challenge test significantly decreased with increasing dietary probiotic B12 (P < 0.05). No significant difference was found between T1 and control in cumulative shrimp mortality after challenge test (P > 0.05). The oral administration of probiotic B12 to F. chinensis was beneficial to protect the integrity of shrimp intestinal mucosal layer. In summary, even though the low dose (T1) had some effects on bacterial counts and immunological parameters, only the high dose (T2) significantly increased the resistance of the shrimp to WSSV.

Effect of salinity on vanadate biosorption by Halomonas sp. GT-83: Preliminary investigation on biosorption by micro-PIXE technique

Thirty-eight soil samples were collected from crude oil contaminated land in south of Iran. Initial screening of a total of 100 bacterial isolates, resulted in the selection of one isolate with maximum adsorption capacity of 52.7 mg vanadate/g dry weight. It was tentatively identified as Halomonas sp. according to morphological and biochemical properties and named strain GT-83. Removal of vanadate by biosorption with Halomonas sp. GT-83 was very sensitive to solution pH. Vanadate adsorption decreased with increasing pH, with maximum adsorption capacities achieved in at pH 3.0 in the absence and in the presence of increasing concentrations of salt. Vanadate-salt biosorption studies were also performed at this pH value. Equilibrium uptakes of vanadate increased with increasing vanadate concentration up to 600 mg/l. Maximum metal removal (91.8%) took place at pH 3.0 with initial vanadate concentration of 100 mg/l, which got reduced (84.8%) in the presence of 50 g/l salt. The equilibrium sorption data were analyzed by using Freundlich isotherm. The specific uptake of vanadate increased at low cell concentration and decreased when cell concentration exceeded 0.75 g/l. The paper also demonstrates the potential value of micro-PIXE in biosorption studies.

Effect of osmotic downshock treatment on the yield of ectione synthesized by Halomonas sp. EG6

Effect of osmotic downshock treatment on the yield of ectione synthesized by Halomonas sp. EG6

Disposal of seaweed wakame (Undaria pinnatifida) in composting process by marine bacterium Halomonas sp. AW4

A novel marine bacterium, identified as Halomonas sp. AW4 by partial 16S rDNA analysis, was isolated from the seaweeds in Awaji Island, Japan. Strain AW4 grew well even in the wide NaCl concentration ranges of 0-3 mol/L, where it showed an optimal growth in the presence of 0.5 mol/L NaCl. The organic components were reduced to 73.6% of initial dry weight after 168 h of composting by inoculation of AW4. The microbial community structure became complex after 72 h of composting. The initial content of alginate was 35.6%, which decreased to 15.6% after 168 h of composting. The decomposition of alginate mainly occurred at the late stage of composting, suggesting that the microbial community consisting of various types of microorganisms is effective in degrading alginate. The germination of plant Brassica campestris L. indicated the promotion effect of composted wakame.

Potential of bacterial indoleacetic acid to induce adventitious shoots in plant tissue culture

The main aim of this study was to investigate the possible role of indoleacetic acid (IAA) from bacteria to induce in vitro adventitious shoots in internodal explants of Brassica oleracea L. Culture supernatant of Halomonas sp. RE1 and Halomonas sp. HT1 that contain 21 and 40 microg ml(-1) IAA, respectively, was used to supplement Murashige and Skoog (MS) medium. Two combinations that were supplemented with bacterial supernatant (BS) are MS + BS and MS + BS + 10%CW (coconut water) while basal MS medium was used as control. The amounts of BS used in this experiment were 50, 100, 150 and 200 microl in 5 ml MS medium in each combination. In vitro-grown internodal explants of B. oleracea were inoculated on these media combinations and incubated in a growth chamber at 25 +/- 1 degrees C and exposed to 16-h cool fluorescent light. After 5-6 weeks of incubation adventitious shoot induction was observed in all treatments that were supplemented with BS as compared with the controls where very low response was observed. The frequency of shoot induction was high in media that were supplemented with 10%CW in the presence of bacterial auxin. It was concluded that IAA of microbial origin has the potential to induce adventitious shoots in internodal explants. IAA from bacteria can be effectively used in plant tissue culture; especially a combination of MS + BS + 10%CW is very cost-effective as compared with synthetic phytohormones for in vitro studies.

A novel moderately halophilic bacterium for decolorizing azo dye under high salt condition

Halomonas sp strain GTW was newly isolated from coastal sediments contaminated by chemical wastewater and was identified to be a member of the genus Halomonas by 16S rDNA sequence analysis and physical and biochemical tests. The optimal decolorization conditions were as follows: temperature 30 degrees C, pH 6.5.0-8.5, NaCl 10-20% (w/v) and the optimal carbon source was yeast exact. The results of experiments demonstrated that the bacteria could decolorize different azo dyes under high salt concentration conditions, and the decolorization rate of five tested azo dyes could be above 90% in 24 h. The exploitation of the salt-tolerant bacteria in the bio-treatment system would be a great improvement of conventional biological treatment systems and the bio-treatment concept.

Relationships between the osmoadaptation strategy, amino acid composition of bulk protein, and properties of certain enzymes of haloalkaliphilic bacteria

Haloalkaliphilic microorganisms isolated from soda lakes were compared in terms of the amino acid composition of total cellular protein and the reaction of a number of key enzymes to salts and pH of the medium. In the extremely halophilic bacterium Natroniella acetigena (salt-inside osmoadaptation strategy), acidic amino acids (glutamic and aspartic) made up 30.91 mol % of the total of cellular protein amino acids. In the moderate haloalkaliphiles Tindallia magadiensis, Halomonas campisalis, and Halomonas sp. AIR-1 (compatible-solutes osmoadaptation strategy), the proportion of acidic amino acids (24.36, 23.15, and 23.58 mol %, respectively) was lower than in N. acetigena but higher than in the freshwater Acetobacterium paludosum (20.77 mol %). The excess of acidic amino acids over basic amino acids (lysine and arginine) increased with the degree of halophily. The enzymes of haloalkaliphiles proved to be tolerant to salts and high pH values, although the degree of tolerance varied. The activity of N. acetigena CO dehydrogenase was maximum in the presence of 0.7 M NaCl, but it was virtually independent of the NaHCO3 concentration. The hydrogenase and CO dehydrogenase of T. magadiensis exhibited maximum activity in the absence of NaCl; the CO dehydrogenase was most active at 0.25 M NaHCO3, and hydrogenase activity was only weakly dependent on NaHCO3 in the concentration range of 0-1.2 M. The nitrate reductases of H. campisalis and Halomonas sp. AIR-2 were active in broad ranges of NaCl and KCl concentrations; the activity maxima were recorded at moderate concentrations of these salts. The pH optima of most of the studied enzymes of haloalkaliphiles were in the alkaline zone. Thus, it was shown that the amino acid composition of total cellular protein is determined by the osmoadaptation strategy employed by the bacterium. A correlation was found between the salt tolerance of enzymes and the proportion of acidic amino acids in the total cellular protein. The ability of enzymes to function at high pH values is one of the mechanisms of adaptation of microorganisms to high pH values.

Osmoadaptation in Representatives of Haloalkaliphilic Bacteria from Soda Lakes

The adaptation of microorganisms to life in brines allows two strategies: the accumulation of organic osmoregulators in the cell (as in many moderate halophiles, halomonads in particular) or the accumulation of inorganic ions at extremely high intracellular concentrations (as, for example, in haloanaerobes). To reveal the regularities of osmoregulation in haloalkaliphiles developing in soda lakes, Halomonas campisalis Z-7398-2 and Halomonas sp. AIR-2 were chosen as representatives of halomonads, and Natroniella acetigena, as a representative of haloanaerobes. It was established that, in alkaliphilic halomonads, the intracellular concentrations of inorganic ions are insufficient for counterbalancing the environmental osmotic pressure and balance is attained due to the accumulation of organic osmoregulators, such as ectoine and betaine. On the contrary, the alkaliphilic haloanaerobe N. acetigena employs K+, Na+, and Cl- ions for osmoregulation. High intracellular salt concentrations increasing with the content of Na+ in the medium were revealed in this organism. At a concentration of 1.91 M Na+ in the medium, N. acetigena accumulated 0.83 M K+, 0.91 M Na+, and 0.29 M Cl- in cells, and, with an increase in the Na+ content in the medium to 2.59 M, it accumulated 0.94 M K+, 1.98 M Na+, and 0.89 M Cl-, which counterbalanced the external osmotic pressure and provided for cell turgor. Thus, it was shown that alkaliphilic microorganisms use osmoregulation strategies similar to those of halophiles and these mechanisms are independent of the mechanism of pH homeostasis.

Characterization of Molybdenum-Free Nitrate Reductase from Haloalkalophilic Bacterium Halomonas sp. Strain AGJ 1-3

Nitrate reductase from the haloalkalophilic denitrifying bacterium Halomonas sp. strain AGJ 1-3 was isolated and purified to homogeneity. The isolated enzyme belongs to a novel family of molybdenum-free nitrate reductases. It presents as a 130-140 kD monomeric protein with specific activity of 250 micromol/min per mg protein. The enzyme reduces not only nitrate, but also other anions, thus showing polyoxoanion reductase activity. Enzyme activity was maximal at pH 7.0 and 70-80 degrees C.

Carbon and energy fluxes during haloadaptation of Halomonas sp. EF11 growing on phenol.

The haloalkaliphile Halomonas sp. EF11 can grow on phenol as sole source for carbon and energy, while maintaining an osmotic equilibrium predominantly by adjusting levels of a certain compatible solute. To determine the energy costs of haloadaptation and the fate of substrate-carbon, the strain was grown continuously in an isothermal compensation calorimeter, keeping all conditions constant except salinity. As salinity increased, slight linear reductions in exothermic heat flow and biomass formation occurred, and 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) synthesis increased linearly. However, beyond a certain salinity threshold the stationary phenol concentration increased exponentially, while heat flow fell sharply, indicating intoxication or wash-out. The clear transition point between the phases, where ectoine formation peaked, suggests that calorimetric measurements could be used to control the conversion of growth-inhibiting substrates (like phenol) into ectoine and to optimize the process. Enthalpy balance and chemical determinations revealed acetate and formate were formed as side products when the C/N ratio in the feed was low, while 2-muconic acid semialdehyde and formate were produced when the ratio was high. These findings indicate that phenol assimilation occurs via the meta pathway. However, enzyme assays implied that assimilation occurs via the ortho and meta pathways at a low C/N ratio and exclusively via the meta pathway at a high C/N ratio.

Alkali- and Halo-tolerant Catalase fromHalomonassp. SK1: Overexpression inEscherichia coli, Purification, Characterization, and Genetic Modification

A catalase gene, ohktA, from an alkali- and halo-tolerant bacterium, Halomonas sp. SK1, on the pKK223-3, was expressed in the catalase-lacking Escherichia coli strain UM2. Highly purified catalase showing a single band on SDS-PAGE was obtained by two liquid chromatography steps on DEAE-Toyopear1 and Chelating-Sepharose Fast Flow. The enzyme, oHktA, shows high catalase activity with a pH optimum at 10, and the activity was stable in 4 M KC1. This enzyme is thermo-sensitive, showing a significant loss of activity within 5 minutes at 37 degrees C. To modify the stability of the catalase, the addition of domain II of the heat stable Mn catalase from Thermus thermophilus to the C-terminus was made. When coexpressed with a chaperone (PhFKBP29) gene product, peptidyl-prolyl cis-trans isomerase, from a thermophilic bacterium, a chimeric catalase was produced in the soluble fraction. The stability of this catalase in the range of 37 degrees -45 degrees C was improved and it was stable for more than 1 h at 37 degrees C.

Tolerance to Cadmium of Free-Living and Associated with Marine Animals and Eelgrass Marine Gamma-Proteobacteria

The tolerance to Cd2+ and possible mechanisms of Cd2+ detoxification by 178 free-living bacteria isolated from sea water, associated with marine animals (a mussel Crenomytilus grayanus, a scallop Patinopecten yessoensis), and eelgrass Zostera marina collected in The Sea of Japan and The Sea of Okhotsk have been studied. The concentrations of 25 and 50 mg Cd2+/L were highly toxic and inhibited the growth from 54% to 78% of the total bacteria studied. The free-living bacteria isolated from seawater samples (up to 50%) were tolerant to high concentrations of cadmium. Marine gamma-proteobacteria tolerated Cd2+ by the activation of different detoxifying mechanisms. The strain Halomonas sp. KMM 734 isolated from seawater prevented the uptake of Cd2+ into bacterial cells. The chromosomal cadmium resistance system of Pseudoalteromonas citrea KMM 461 and Marinobacter sp. KMM 181 was found to be similar to class III metallothioneins (also known as phytochelatins).

Expression of the 2,4-D degradative pathway of pJP4 in an alkaliphilic, moderately halophilic soda lake isolate, Halomonas sp. EF43.

The broad host range plasmid pJP4, which carries genes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid, and 3-chlorobenzoic acid, was used in conjugation experiments with mixed cultures enriched from water and sediment samples from an alkaline pond in the area of Szegedi Fehértó, a soda lake in south Hungary. pJP4-encoded mercury resistance was used as a selection marker. One of the transconjugants, the alkaliphilic, moderately halophilic strain EF43, stably maintained the plasmid and was able to degrade 2,4-D and 3-chlorobenzoate under alkaline conditions in the presence of an additional carbon source such as pyruvate, benzoate, or alpha-ketoglutarate, indicating that the degradative genes of pJP4 were expressed in this strain. However, it was unable to grow on these chloroaromatic substrates when the substrate was the sole source of carbon and energy. Chemostat cultivation experiments revealed that the 2,4-D degradation rate during growth on benzoate or pyruvate was limited by the low activity of chlorocatechol-degrading enzymes, particularly chloromuconate cycloisomerase. Strain EF43 was identified as Halomonas sp. on the basis of 16S rRNA sequencing and additional taxonomic studies. 16S rRNA sequence analysis revealed that strain EF43 is closely related to typical soda lake isolates belonging to the genus Halomonas.

The role of the outer membrane in formaldehyde tolerance in Escherichia coli VU3695 and Halomonas sp. MAC

To investigate the mechanism of formaldehyde tolerance in Gram-negative bacteria, two formaldehyde-tolerant strains, Escherichia coli VU3695 and Halomonas sp. MAC (DSM 7328), and formaldehyde-sensitive revertants obtained by ethidium bromide or novobiocin treatment were studied. The presence of high levels of formaldehyde dehydrogenase activity alone proved insufficient to confer tolerance to high formaldehyde concentrations, as shown by high activity displayed by formaldehyde-sensitive revertants of Halomonas MAC. Moreover, formaldehyde-tolerant strains also proved to be tolerant to high concentrations of acetaldehyde and glutaraldehyde, which are not oxidized by formaldehyde dehydrogenase. Treatment with sublethal concentrations of EDTA rendered the resistant strains highly sensitive to formaldehyde without affecting the activity of formaldehyde dehydrogenase. Comparison of the outer membrane proteins of formaldehyde-resistant strains with those of their sensitive revertants showed the presence of at least one additional high molecular mass protein in the tolerant strains. It is concluded that formaldehyde tolerance in the bacteria studied depends on the composition and structure of the outer membrane.

Limnology of Organic Lake, Antarctica, a meromictic lake that contains high concentrations of dimethyl sulfide

Organic Lake in the Vestfold Hills, Antarctica, is a shallow (7.5 m), meromictic, hypersaline lake that contains a microflora of low species diversity. The lake monimolimnion is anoxic but contains no H2S. Organic Lake has the highest concentrations of dimethyl sulfide (DMS) as yet recorded in a natural aquatic ecosystem. The greatest concentration of DMS in the lake, 97 �g 1-1, occurs just above the oxic-anoxic interface. Its presence coincides with maximal numbers of the alga Dunaliella sp. and maximal numbers of bacteria. Analysis of head space samples from axenic cultures indicates that Dunaliella sp. is not directly involved in DMS production. A bacterial strain that was isolated from Organic Lake and that produced DMS from sulfur-containing amino acids was presumptively identified as a Halomonas sp.