Bacterial exopolysaccharides produced by newly discovered bacteria belonging to the genus Halomonas, isolated from hypersaline habitats in Morocco

We have studied the exopolysaccharides (EPS) from a new group of moderately halophilic bacteria belonging to the genus Halomonas. The quantity of EPS produced, its chemical composition and physical properties depend greatly upon the bacterial strain. The majority of the polymers produced viscous solutions and/or emulsified different hydrocarbon compounds. The most interesting strain, S-30, produced EPS at 2.8 g/l with a maximum viscosity of 23.5 Pa·5 and exhibited pseudoplastic behavior. This EPS emulsified five hydrocarbons more efficiently than did four control surfactants tested. Its monosaccharide composition was glucose:galactose:manose:glucuronic acid in equimolar ratio. Some two-thirds of the strains tested emulsified crude oil better than control surfactants did. There are many potential industrial applications for polysaccharides with these qualities. Journal of Industrial Microbiology & Biotechnology (2000)24, 374–378. Received 09 August 1999/ Accepted in revised form 23 March 2000     

Partial characterization of an extracellular polysaccharide produced by the moderately halophilic bacterium Halomonas xianhensis SUR308

A moderately halophilic bacterium, Halomonas xianhensis SUR308 (Genbank Accession No. KJ933394) was isolated from a multi-pond solar saltern at Surala, Ganjam district, Odisha, India. The isolate produced a significant amount (7.87 g l^?1) of extracellular polysaccharides (EPS) when grown in malt extract–yeast extract medium supplemented with 2.5% NaCl, 0.5% casein hydrolysate and 3% glucose. The EPS was isolated and purified following the conventional method of precipitation and dialysis. Chromatographic analysis (paper, GC and GC-MS) of the hydrolyzed EPS confirmed its heteropolymeric nature and showed that it is composed mainly of glucose (45.74 mol%), galactose (33.67 mol %) and mannose (17.83 mol%). Fourier-transform infrared spectroscopy indicated the presence of methylene and carboxyl groups as characteristic functional groups. In addition, its proton nuclear magnetic resonance spectrum revealed functional groups specific for extracellular polysaccharides. X-ray diffraction analysis revealed the amorphous nature (CI_xrd, 0.56) of the EPS. It was thermostable up to 250°C and displayed pseudoplastic rheology and remarkable stability against pH and salts. These unique properties of the EPS produced by H. xianhensis indicate its potential to act as an agent for detoxification, emulsification and diverse biological activities.     

Analysis of the genome of the gram-negative moderate halophiles Halomonas and Chromohalobacter by using pulsed-field gel electrophoresis

The genomes of 11 moderately halophilic bacteria belonging to the genera Halomonas and Chromohalobacter have been analyzed by restriction endonuclease digestion and pulsed-field gel electrophoresis (PFGE). By using the infrequently cutting restriction endonucleases SpeI and SwaI, highly characteristic fingerprintings were obtained for each of the isolates studied. On the basis of the lengths of the SpeI and SwaI fragments, separated by PFGE, the genome size of the 11 strains studied was estimated. The genome size for 8 Halomonas strains ranged from 1450 to 2830 kb, whereas for the 3 Chromohalobacter strains studied it ranged from 1770 to 2295 kb. Finally, we show that macrorestriction fingerprints could be a useful tool to elucidate the taxonomic position of bacteria belonging to the Halomonas–Deleya complex. Received: October 13, 1997 / Accepted: May 12, 1998     

Isolation and characterization of Halomonas sp. strain IMPC, a p-coumaric acid-metabolizing bacterium that decarboxylates other cinnamic acids under hypersaline conditions

A moderately halophilic, mesophilic, Gram-negative, motile, nonsporulating bacterium, designated strain IMPC, was isolated from a table-olive fermentation rich in aromatic compounds, after enrichment on p-coumaric acid under halophilic conditions. Strain IMPC was able to degrade p-coumaric acid. p-hydroxybenzaldehyde and p-hydroxybenzoic acid were detected as breakdown products from p-coumaric acid. Protocatechuic acid was identified as the final aromatic product of p-coumaric acid catabolism before ring fission. Strain IMPC transformed various cinnamic acids with substituent H, OH, CH(3) or OCH(3) in the para- and/or meta-position of the aromatic ring to the corresponding benzoic acids, indicating a specific selection. A beta-oxidation pathway was proposed for these transformations. Phylogenetic analysis of the 16S rRNA gene revealed that this isolate was a member of the genus Halomonas. Strain IMPC was closely related to Halomonas elongata ATCC 33173(T)and Halomonas eurihalina ATCC 49336(T).     

一株耐热嗜盐菌的分离及16SrRNA基因序列分析

目的利用盐固体分离培养基,从西藏自治区澜沧江边康宁镇一个47℃的盐井样品中分离纯化到一株耐热嗜盐菌菌株YJ0232。方法通过形态观察、生理生化特性和16srRNA基因序列分析,鉴定嗜盐菌菌株YJ0232分类学地位。结果菌株YJ0232初步鉴定为中度嗜盐菌,属于盐单胞菌属（Halomonassp．）菌株。其16SrRNA基因序列已被GenBank数据库收录,序列号为EU029645。结论本研究对澜沧江高盐环境微生物资源进行了初步探索研究。可为今后研究同类极端环境中新的物种资源以及微生物多样性提供参考。     

Halomonas maura is a physiologically versatile bacterium of both ecological and biotechnological interest

Halomonas maura is a bacterium of great metabolic versatility. We summarise in this work some of the properties that make it a very interesting microorganism both from an ecological and biotechnological point of view. It plays an active role in the nitrogen cycle, is capable of anaerobic respiration in the presence of nitrate and has recently been identified as a diazotrophic bacterium. Of equal interest is mauran, the exopolysaccharide produced by H. maura, which contributes to the formation of biofilms and thus affords the bacterium advantages in the colonisation of its saline niches. Mauran is highly viscous, shows thixotropic and pseudoplastic behaviour, has the capacity to capture heavy metals and exerts a certain immunomodulator effect in medicine. All these attributes have prompted us to make further investigations into its molecular characteristics. To date we have described 15 open reading frames (ORF’s) related to exopolysaccharide production, nitrogen fixation and nitrate reductase activity among others.     

Chemical and UV mutagenesis in Zymomonas mobilis

Mutagenesis of the facultative anaerobe Zymomonas mobilis was accomplished by three different mutagens. Ultra-violet (UV) irradiation, whose effectiveness relies on misrepair of damaged DNA via an error-prone pathway, was a poor mutagen for this organism. Ethyl methane sulphonate (EMS) gave results very similar to UV-irradiation. N-methyl-N-nitro-N-nitrosoguanidine (MNNG), which is believed to act by multiple mutagenic mechanisms, was the most powerful mutagen, always resulting in a large number of mutants of all types examined (i.e. auxotrophs, antibiotic resistant, heavy metal resistant and ultraviolet sensitive). Reversion frequencies of MNNG-induced mutants were very low. Evidence is provided that mutagenesis of Z. mobilis is affected by photoreactivation, adaptive response and error-prone repair mechanisms. Moreover, cells treated with alkylating agents and allowed to recover under anaerobic conditions clearly demonstrated that anaerobiosis plays a significant role in repair, but not in the induction of mutants.     

中度嗜盐菌 Halomonas sp.BYS-1的渗透调节

Halomonassp BYS 1是一株从活性污泥中分离的中度嗜盐菌 ,它能在 0 1～ 2 6mol LNaCl的以苯乙酸为唯一碳源的基础培养基中生长。BYS 1在不同NaCl条件下生长时 ,胞内的Na 含量基本不发生变化 ;它主要通过积累K 、谷氨酸和甜菜碱来调节胞内外的渗透压平衡。当培养基中的NaCl浓度从 0 1mol L上升到 2 0mol L时 ,其胞内的K 、谷氨酸和甜菜碱分别提高了 1 9、 2 4和 1 3 6倍。     

Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion of transposon Tn5 and Tn1732 in Halomonas elongata

Abstract Molecular genetic studies of halophilic eubacteria have been limited by the lack of a suitable method for mutagenesis. To overcome this, we established a transposon mutagenesis procedure for the ectoine-producing, halophilic bacterium Halomonas elongata . We used suicide plasmids pSUP101 and pSUP102-Gm to introduce the transposons Tn5 and Tn7732 respectively into H. elongata via Escherichia coli SM10 mediated conjugation. Our finding that H. elongata is sensitive to aminoglycoside antibiotics at low salinity enabled us to apply transposons that mediate kanamycin resistance. The insertions of transposon In 1732 occurred at different sites in the chromosome of H. elongata , as proved by Southern hybridization analysis. Phenotypic analysis revealed that different auxotrophic and salt sensitive mutants were generated by mutagenesis with transposon Tn 1732 . To our knowledge this is the first report of a successful application of a transposon for direct generalized mutagenesis in a halophilic eubacterium.     

Production and biochemical characterization of an alpha-amylase from the moderate halophile Halomonas meridiana

Extracellular amylase production by the moderate halophile Halomonas meridiana was optimized and the enzyme was characterized biochemically. The highest amylase production was achieved by growing H. meridiana cultures in media with 5% salts and starch, in the absence of glucose until the end of the exponential phase. The amylase exhibited maximal activity at pH 7.0, being relatively stable in alkaline conditions. Optimal temperature and salinity for activity were 37 degrees C and 10% NaCl, respectively. Moreover, activity at salinity as high as 30% salts was detected. Maltose and maltotriose were the main end products of starch hydrolysis, indicating an alpha-amylase activity.     

Glycoprotein emulsifiers from two marine Halomonas species: chemical and physical characterization

AIMS: To partially purify and characterize bioemulsifiers produced by two new marine Halomonas species, TG39 and TG67, and to compare their emulsifying activities with those of commercial emulsifiers. METHODS AND RESULTS: The production of emulsifiers HE39 and HE67 was achieved from glucose-supplemented marine broth, and recovered by cell removal, concentration by ultrafiltration, precipitation with salt and ethanol, and lyophilization. Purification and chemical analysis revealed both emulsifiers to be glycoproteins of high molecular weight with a notably high content of protein and uronic acids. Physical characterization showed both glycoproteins to effectively emulsify a wide range of food oils under both neutral and acidic pH conditions and withstand acid and high temperature treatment. CONCLUSIONS: The emulsifying activities of these two new glycoprotein emulsifiers were comparable and, under certain conditions, superior to those produced by commercial emulsifiers tested (xanthan gum, gum arabic and lecithin). They show the highest reported emulsifying activities derived from a Halomonas species. SIGNIFICANCE AND IMPACT OF THE STUDY: These strains, and the emulsifiers produced, appear to be promising candidates for further development in applications requiring emulsifiers that are natural and compatible to the existing commercial emulsifiers.     

Characteristics of bacteria showing high denitrification activity in saline wastewater

AIMS: Denitrification efficiency at 10% salinity was compared with that at 2% salinity. The characteristics of bacterial strains isolated from the denitrification system, where an improvement of denitrification efficiency was observed at a high salinity were investigated. METHODS AND RESULTS: Two continuous feeding denitrification systems for saline solutions of 2% and 10% salinity, were operated. Denitrification efficiency at 10% salinity was higher than that at 2% salinity. The bacterial strains were isolated using the trypticase soy agar (TSA) medium at 30 degrees C. The phylogenetic analysis of 16S rRNA gene sequences of isolates indicated that halophilic species were predominant at 10% salinity. CONCLUSIONS: The improvement of denitrification efficiency at a high salinity was demonstrated. The strains isolated from the denitrifying system with 10% salinity were halophilic bacteria, Halomonas sp. and Marinobacter sp., suggesting that these bacteria show a high denitrifying activity at 10% salinity. SIGNIFICANCE AND IMPACT OF THE STUDY: The long-term acclimated sludge used in this study resulted in high denitrification performance at a high salinity, indicating that the design of a high-performance denitrification system for saline wastewater will be possible.     

Application of flow cytometry for monitoring the production of poly(3‐hydroxybutyrate) by Halomonas boliviensis

In this study, a flow cytometry (FC) protocol was implemented to measure poly(3‐hydroxybutyrate) (PHB) content in a halophilic bacterium, to have a faster and easier control of the process. The halophilic bacterium Halomonas boliviensis was stained with BODIPY 493/503 and analyzed using FC. Bacterial polymer accumulation induced by two different nutrient limitations during the operation of a 2 L bioreactor was studied using traditional gas chromatography (GC) analysis and FC. The application of this rapid and straightforward method is useful to obtain complex and precise information about PHB accumulation that could be used for the monitoring, control and optimization of the production of PHB. A clear correlation between the PHB concentration determined by GC and the fluorescence signal obtained from stained bacteria by using FC was observed. Additionally, the heterogeneity of bacterial population as a function of PHB content was measured. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:276–284, 2017     

Bioconversion of tyrosol into hydroxytyrosol and 3,4-dihydroxyphenylacetic acid under hypersaline conditions by the new Halomonas sp. strain HTB24

This paper reports the characterization of a Halomonas sp. strain (named HTB24) isolated from olive-mill wastewater and capable of transforming tyrosol into hydroxytyrosol (HT) and 3,4-dihydroxyphenylacetic acid (DHPA) in hypersaline conditions. This is the first time that a halophile has been shown to perform such reactions. The potent natural antioxidant HT was obtained through a C3 hydroxylation on the ring cycle, whereas DHPA was synthesized via the 4-hydroxyphenylacetic acid (HPA) pathway, which has been well described from other bacterial sources. HT was produced first, and then DHPA was detected in the medium accompanied by traces of HPA. HPA involved another pathway resulting from the activity of an aryl-dehydrogenase, which is suggested to be responsible for both tyrosol and hydroxytyrosol oxidation. Maximal HT content (2.30 mM) and maximal DHPA (5.15+/-0.42 mM) were obtained from a culture inoculated in the presence of 20 mM tyrosol and 0.5 g L(-1) yeast extract. Following this, DHPA was quickly degraded into 5-carboxymethyl-2-hydroxymuconic semialdehyde by a 2,3-dioxygenase, finally resulting in succinate and pyruvate. Phylogenetic analysis of the 16S rRNA gene revealed that this isolate was a member of the genus Halomonas. Strain HTB24, with a G+C content of 55.3 mol%, is closely related to Halomonas neptunia DSM 15720(T), 'Halomonas alkaliantarctica' DSM 15686(T) and Halomonas boliviensis DSM 15516(T).     

Effect of cations, pH and sulfate content on the viscosity and emulsifying activity of the Halomonas eurihalina exopolysaccharide

The effects of monovalent and divalent cations on the rheological behavior of Halomonas eurihalina exopolysaccharide (EPS) were studied. Sodium, potassium, magnesium and calcium were added and the relative abilities to increase viscosity were as follows: KCl > NaCl > MgCl₂ > CaCl₂. The highest viscosity value was measured in acidic 10⁻⁴ M KCl, in which a gel formed. A loss of sulfate content seemed to correlate with the increase of viscosity. H. eurihalina produced EPS in all growth media. Addition of hydrophobic substrates to culture media produced changes in chemical composition and emulsifying activity of the EPS. Xylene was the most effectively emulsified substance and the EPS produced on tetradecane and on corn oil the most active emulsifier. Received 25 July 1997/ Accepted in revised form 30 January 1998     

Dimeric structure of nucleoside diphosphate kinase from moderately halophilic bacterium: contrast to the tetrameric Pseudomonas counterpart

Light scattering and chemical cross-linking analyses of nucleoside diphosphate kinase (NDK) from moderate halophile, Halomonas sp. 593 (HaNDK), unambiguously demonstrated that this enzyme formed a dimeric structure, in contrast to the Pseudomonas NDK (PaNDK), a nonhalophilic counterpart, and other NDKs from Gram-negative bacteria, which all formed a tetrameric structure. Comparison of HaNDK and PaNDK showed that the HaNDK was less thermally stable than the PaNDK: the optimum temperature of PaNDK enzyme activity was 20 degrees C higher than that of HaNDK. However, the HaNDK readily refolded and reassembled back to the active dimeric structure, upon heat denaturation at 0.2 M NaCl, as soon as the temperature was lowered. On the contrary, the thermally more stable PaNDK was irreversibly denatured at its melting temperature.     

Halomonas magadii sp. nov., a new member of the genus Halomonas, isolated from a soda lake of the East African Rift Valley

A number of novel alkaliphilic organotrophic bacteria have been isolated from several saline and alkaline East African soda lakes. The new isolates grow at pH values between 7.0 and 11.0, with pH optima for growth between 9.0 and 10.0. Growth occurs at total salts concentration between 0% and 20% (w/v) with optimum at 0%–7% (w/v). Phylogenetic analyses based on 16S rDNA sequence comparison indicate that these isolates are related (>96% similarity) to members of the Halomonadaceae within the γ-3 subdivision of the Proteobacteria. These analyses indicate that existing species within the Halomonadaceae fell within three main groups, one group comprising the type species of Halomonas, Halomonas elongata, and a number of other known species including one soda lake isolate. A second group constituting most of the remaining known species of Halomonas and related Chromohalobacter spp. includes 3 soda lake isolates with high DNA–DNA homologies. The third group included Halomonas halodenitrificans, Halomonas desiderata, Halomonas cupida, and 13 soda lake isolates. Phenotypic comparisons indicated that the majority of soda lake strains shared similar morphological, phenotypic, and chemotaxonomic properties to known strains of Halomonas but grew under alkaline conditions. The 3 soda lake isolates with high DNA–DNA homologies were, however, significantly different in antibiotic sensitivity pattern and in the utilization of several substrates, were unable to reduce nitrite, and showed low DNA–DNA homologies with known halomonads in the same group. We propose that these isolates comprise a new species of the genus Halomonas that we name Halomonas magadii sp. nov. The type strain is strain 21 MI (NCIMB 13595). Received: July 20, 1999 / Accepted: October 29, 1999