Human infection with Halomonas venusta following fish bite.
Halomonas venusta, a moderately halophilic, nonfermentative gram-negative rod, is reported for the first time as a human pathogen in a wound that originated from a fish bite.
Animals↗
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Purification, characterization, and gene sequencing of a catalase from an alkali- and halo-tolerant bacterium, Halomonas sp. SK1.
An alkali- and halo-tolerant bacterium with high catalase activity was isolated and identified as a new species of the genus Halomonas. Its catalase (HktA) was simply purified by two steps of liquid chromatography. A 71.4% yield of the catalase was obtained with 97% purity on SDS-PAGE. The specific activity of HktA (57,900 U/mg protein) was two times higher than that of bovine liver catalase. The purified enzyme is inhibited by KCN, NH2OH, NaN3, and 3-amino-1,2,4-triazole, active at pH 5.0-11.0, thermo-sensitive, and KCl-tolerant. HktA is suggested to be a typical catalase, a homotetrameric protein containing heme groups in the active sites. The nucleotide sequence of the catalase gene (hktA) comprises 1,530 bp, encoding a protein of 509 amino acid residues. The deduced amino acid sequence of the hktA shares 99% identity with that of Vibrio rumoiensis S-1T.
Amino Acid Sequence↗
Alkali- and halo-tolerant catalase from Halomonas sp. SK1: overexpression in Escherichia 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.
Alkalies↗
Characterization of halophilic alkaline phosphatase from Halomonas sp. 593, a moderately halophilic bacterium.
A halophilic alkaline phosphatase was highly purified (about 510-fold with about 21% yield) from a moderate halophile, Halomonas sp. 593. The N-terminal 35 amino acid sequence of this enzyme was found to be more acidic than those previously isolated from Vibrio spp., and this enzyme was partially resistant to SDS. Several enzymatic properties demonstrated that it showed higher halophilicity than those enzymes from Vibrio spp.
Alkaline Phosphatase↗
Effect of exopolysaccharide V2-7, isolated from Halomonas eurihalina, on the proliferation in vitro of human peripheral blood lymphocytes.
The immunomodulatory activity of the exopolysaccharide V2-7, a sulfated polymer excreted by the moderately halophilic bacteria Halomonas eurihalina, was studied in vitro. [3H]thymidine incorporation and flow-cytometry measurements showed that this exopolysaccharide enhanced the unspecific proliferation of human lymphocytes in response to the presence of anti-CD3 monoclonal antibody. It was effective at concentrations of less than 1 microg/ml, maximum activity being achieved at 0.2 microg/ml.
Adjuvants, Immunologic↗
[Halomonas campisalis, an obligatorily alkaliphilic, nitrous oxide-reducing denitrifier with a Mo-cofactor-lacking nitrate reductase].
We isolated eight strains of denitrifying bacteria that reduce nitrate and nitrous oxide at pH 10 from Lake Magadi (Kenya). Despite certain differences between the strains, they are similar in terms of G+C content (66.1-68.1 mol %) and DNA-DNA homology (75-92%) and represent different morphotypes of the same species. Based on the results of partial 16S rRNA sequencing, strain Z-7398-2 was most closely related to the Halomonas campisalis isolate from Alkali Lake (USA). The DNA-DNA homology level between the tested strain and the type strain of H. campisalis 4A was 88%. These two strains were also similar phenotypically. However, the culture isolated by us was characterized by peculiar properties, such as obligate alkaliphily, which manifested itself in the culture dependence on carbonates and lack of growth at pH values below 7, a nitrous oxide-reducing capacity, and an unusual nitrate reductase that lacked molybdenum and a Mo cofactor.
Alkalies↗
Growth and physiological properties of wild type and mutants of Halomonas subglaciescola DH-1 in saline environment.
A halophilic bacterium was isolated from fermented seafood. The 16S rDNA sequence identity between the isolate and Halomonas subglaciescola AJ306801 was above 95%. The isolate that did not grow in the condition without NaCl or in the condition with other sodium (Na+) or chloride ions (Cl-) instead of NaCl was named H. subglaciescola DH-1. Two mutants capable of growing without NaCl were obtained by random mutagenesis, of which their total soluble protein profiles were compared with those of the wild type by two-dimensional electrophoresis. The external compatible solutes (betaine and choline) and cell extract of the wild type did not function as osmoprotectants, and these parameters within the mutants did not enhance their growth in the saline environment. In the proton translocation test, rapid acidification of the reactant was not detected for the wild type, but it was detected for the mutant in the condition without NaCl. From these results, we derived the hypothesis that NaCl may be absolutely required for the energy metabolism of H. subglaciescola DH-1 but not for its osmoregulation, and the mutants may have another modified proton translocation system that is independent of NaCl, except for those mutants with an NaCl-dependent system.
Betaine↗
Salt-Sensitive and Auxotrophic Mutants of Halomonas elongata and H. meridiana by Use of Hydroxylamine Mutagenesis
The killing action and induced mutagenicity inhydroxylamine (HA)-treated cells of two moderately halophilic species of thegenus Halomonas, H. elongata and H. meridiana, wereinvestigated. A high sensitivity of H. elongata and especially ofH. meridiana to HA was found. The efficiency of the mutagenicityobtained with the HA treatment was tested at different salinities. Optimalexperimental conditions for HA mutagenesis of these two moderate halophileswere determined. A clear, efficient mutagenicity of both H. elongataand H. meridiana after HA mutagenesis was achieved. The optimalprocedures yielded a number of useful auxotrophic mutants of H.elongata as well as different salt-sensitive mutants of H.elongata and H. meridiana for further studies. Some of theselatter mutants appeared to be affected in the synthesis of compatiblesolutes.
Journal Article↗
Analysis of the replication region of the cryptic plasmid pHE1 from the moderate halophile Halomonas elongata.
The basic replicon of the narrow-host-range plasmid pHE1 from the moderately halophilic bacterium Halomonas elongata ATCC 33174 has been identified and characterized. The replicon consists of a 1.7-kb DNA fragment, which contains the genetic information required for autonomous replication and stable maintenance. Analysis of its sequence revealed the presence of two ORFs which seem to form one transcription unit. ORF1 encodes a replication initiator protein (RepA), which has a high degree of homology to the theta-replicase (RepA) protein of ColE2 plasmid and to the RepA proteins of a family of replicons from gram-positive and gram-negative bacteria, also related to ColE2. The product encoded by ORF2 showed a certain similarity to the RepB proteins of the same family of replicons and perhaps represents the pHE1 RepB function. Deletion analysis suggests that the pHE1 origin of replication (ori) is located in an 800-bp region upstream of repA. A third putative gene, incA, was found on the complementary strand to the leader region of the repA mRNA. This, together with the presence in the 5' untranslated region of the repA mRNA of inverted repeats that could form stable stem-loop structures, suggests that the incA gene encodes a small antisense RNA. A possible control mechanism for pHE1 replication is proposed, involving an RNA molecule which sequesters the translational initiation region of the replication protein RepA. The basic replicon characterized here shows very interesting properties that should allow it to be used in the construction of cloning and expression vectors for moderate halophiles.
Amino Acid Sequence↗
Biomineralization of carbonates by Halomonas eurihalina in solid and liquid media with different salinities: crystal formation sequence.
Carbonate precipitation by 20 strains of the moderately halophilic species Halomonas eurihalina in both solid and liquid media was studied. The influence of salinity and temperature on the quantity and type of crystals precipitated was also investigated. Some strains of H. eurihalina formed crystals in all conditions tested. The mineral phases precipitated were magnesium calcite, aragonite and monohydrocalcite in variable proportions depending on various factors such as the type of growth medium employed and its salinity. Scanning electron microscopy and X-ray dispersive energy microanalysis were used to investigate the crystal formation sequence. The process of biolith formation was sequential. It started with chains or filaments of bacteria, giving way to discs which finally produced spherical forms of approximately 50 microns in diameter. We suggest a mechanism of carbonate crystal formation by H. eurihalina.
Carbonates↗
New aminophenoxazinones from a marine Halomonas sp.: fermentation, structure elucidation, and biological activity.
The addition of anthranilic acid to the culture medium of the marine derived Halomonas sp. strain GWS-BW-H8hM completely altered the secondary metabolite pattern relative to the standard conditions. The red-orange color of the culture filtrate extract was the result of the production of 2-aminophenoxazin-3-one (1), chandrananimycin C (5) and three new derivatives of 1 with a previously unknown substitution pattern: 2-amino-, 2-amino-8-benzoyl-, and 2-amino-8-(4-hydroxybenzoyl)-6-hydroxyphenoxazin-3-one (2-4). The compounds were determined to have antibacterial and cytotoxic activities; a mode of action other than DNA intercalation is discussed.
Anti-Bacterial Agents↗
Isolation and characterization of salt-sensitive mutants of the moderate halophile Halomonas elongata and cloning of the ectoine synthesis genes.
The moderate halophile Halomonas elongata Deustche Sammlung für Mikroorganismen 3043 accumulated ectoine, hydroxyectoine, glutamate, and glutamine in response to osmotic stress (3 M NaCl). Two Tn1732-induced mutants, CHR62 and CHR63, that were severely affected in their salt tolerance were isolated. Mutant CHR62 could not grow above 0.75 M NaCl, and CHR63 did not grow above 1.5 M NaCl. These mutants did not synthesize ectoine but accumulated ectoine precursors, as shown by 13C NMR and mass spectroscopy. Mutant CHR62 accumulated low levels of diaminobutyric acid, and mutant CHR63 accumulated high concentrations of N-gamma-acetyldiaminobutyric acid. These results suggest that strain CHR62 could be defective in the gene for diaminobutyric acid acetyltransferase (ectB), and strain CHR63 could be defective in the gene for the ectoine synthase (ectC). Salt sensitivity of the mutants at 1.5-2.5 M NaCl could be partially corrected by cytoplasmic extracts of the wild-type strain, containing ectoine, and salt sensitivity of strain CHR62 could be partially repaired by the addition of extracts of strain CHR63, which contained N-gamma-acetyldiaminobutyric acid. This is the first evidence for the role of N-gamma-acetyldiaminobutyric acid as osmoprotectant. Finally, a cosmid from the H. elongata genomic library was isolated which complemented the Ect- phenotype of both mutants, indicating that it carried at least the genes ectB and ectC of the biosynthetic pathway of ectoine.
Amino Acids, Diamino↗
Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion of transposon Tn5 and Tn1732 in Halomonas elongata.
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 Tn1732 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 Tn1732 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 Tn1732. To our knowledge this is the first report of a successful application of a transposon for direct generalized mutagenesis in a halophilic eubacterium.
Blotting, Southern↗
Identification of a promoter region on the Halomonas elongata cryptic plasmid pHE1 employing the inaZ reporter gene of Pseudomonas syringae.
A native promoter located on the cryptic plasmid pHE1 from the moderate halophile Halomonas elongata was identified employing a promoterless ice nucleation gene inaZ of Pseudomonas syringae by direct subcloning and assaying for ice nucleation activity. The presence of the promoter was verified by inserting the corresponding intact or deleted pHE1 fragment in the promoter analysis vector pKK232-8 upstream of the promoterless cat or inaZ gene. Only constructs carrying the intact pHE1 fragment gave CAT phenotype (chloramphenicol resistance) or ice nucleation activity, respectively. Comparative evaluation of the sequence analysis data of the intact and deleted fragment suggested the localization of an Escherichia coli-type promoter region.
Base Sequence↗
Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis.
Using transposon mutagenesis we generated a salt-sensitive mutant of the halophilic eubacterium Halomonas elongata impaired in the biosynthesis of the compatible solute ectoine. HPLC determinations of the cytoplasmic solute content showed the accumulation of a biosynthetic precursor of ectoine, L-2,4-diaminobutyric acid. Ectoine and hydroxyectoine were not detectable. This mutant failed to grow in minimal medium with NaCl concentrations exceeding 4%. However, when supplemented with organic osmolytes, the ability to grow in high-salinity medium (15% and higher) was regained. We cloned and sequenced the regions flanking the transposon insertion in the H. elongata chromosome. Sequence comparisons with known proteins revealed significant similarity of the mutated gene to the L-2,4-diaminobutyric acid acetyltransferase from the ectoine biosynthetic pathway in Marinococcus halophilus. Analysis of a PCR product demonstrated that the ectoine biosynthetic genes (ectABC) follow the same order as in M. halophilus.
Amino Acids, Diamino↗
Synthesis of glycine betaine from exogenous choline in the moderately halophilic bacterium halomonas elongata
The role of choline in osmoprotection in the moderate halophile Halomonas elongata has been examined. Transport and conversion of choline to betaine began immediately after addition of choline to the growth medium. Intracellular accumulation of betaine synthesized from choline was salt dependent up to 2.5 M NaCl. Oxidation of choline was enhanced at 2.0 M NaCl in the presence or absence of externally provided betaine. This indicates that the NaCl concentration in the growth medium has major effects on the choline-betaine pathway of H. elongata.
Journal Article↗
Cell wall and phospholipid composition and their contribution to the salt tolerance of Halomonas elongata.
The salt-tolerant bacterium Halomonas elongata makes a variety of physiological adaptations in response to increases in the salt concentration of its growth medium. The cell walls become more compact and internally coherent. The overall lipid pattern shows an increased amount of negatively charged lipids. In addition, the peptidoglycan composition of H. elongata, although not changing in response to increased NaCl, contains the hydrophobic amino acid leucine which is unique among bacterial species. The results suggest that H. elongata is able to live in a wide variety of salt concentrations because it alters its cell physiology in ways which increase both structural integrity and the amount of less-mobile, "structured" cell water, making the cells less susceptible to NaCl-induced dehydration.
Acclimatization↗
Changes in the hydrophobic-hydrophilic cell surface character of Halomonas elongata in response to NaCl.
Phase-partitioning studies of the euryhaline bacterium Halomonas elongata demonstrated that the hydrophobic-hydrophilic nature of the cell surface changed as the bacterium grew in different NaCl concentrations. Mid-log-phase cells grown in a high (3.4 M) NaCl concentration were more hydrophilic than were cells grown in a low (0.05 M) NaCl concentration. Mid-log-phase cells from defined medium containing 3.4 M NaCl normally produced a hydrophobicity reading of only 14 (hexadecane hydrophobicity = 100), while corresponding cells from defined medium containing 0.05M NaCl gave a hydrophobicity reading of 90. Compared with cells grown in low salt concentrations, cells grown in high salt concentrations were more hydrophilic at all stages of growth. Rapid suspension of log-phase cells grown in 1.37 M NaCl into a 0.05 or 3.4 M NaCl solution produced no detectable rapid changes in surface hydrophobicity. These data suggest that as H. elongata adapts to different NaCl concentrations, it alters the affinity of its outermost cell surface to water.
Cell Membrane↗