A nuclease domain fused to the Snf2 helicase confers antiphage defence in coral-associated Halomonas meridiana.

The coral reef microbiome plays a vital role in the health and resilience of reefs. Previous studies have examined phage therapy for coral pathogens and for modifying the coral reef microbiome, but defence systems against coral-associated bacteria have received limited attention. Phage defence systems play a crucial role in helping bacteria fight phage infections. In this study, we characterized a new defence system, Hma (HmaA-HmaB-HmaC), in the coral-associated Halomonas meridiana derived from the scleractinian coral Galaxea fascicularis. The Swi2/Snf2 helicase HmaA with a C-terminal nuclease domain exhibits antiviral activity against Escherichia phage T4. Mutation analysis revealed the nickase activity of the nuclease domain (belonging to PDD/EXK superfamily) of HmaA is essential in phage defence. Additionally, HmaA homologues are present in ~1000 bacterial and archaeal genomes. The high frequency of HmaA helicase in Halomonas strains indicates the widespread presence of these phage defence systems, while the insertion of defence genes in the hma region confirms the existence of a defence gene insertion hotspot. These findings offer insights into the diversity of phage defence systems in coral-associated bacteria and these diverse defence systems can be further applied into designing probiotics with high-phage resistance.

Molecular identification of Halomonas AZ07 and its multifunctional enzymatic activities to degrade Pyropia yezoensis under high-temperature condition.

BACKGROUND: Pyropia yezoensis a commercially important red seaweed species, is susceptible to various microorganisms infections, among which bacterial infections are the most prominent ones. Pyropia yezoensis is often affected by harmful bacterial communities under high temperatures that can lead to its degradation and economic losses. The current study aimed to explore Pyropia yezoensis-associated microbiota and further identify potential isolates, which can degrade Pyropia yezoensis under high-temperature conditions. METHODS AND RESULTS: The 16S rRNA gene sequencing was used to identify the agarolytic bacterial species. The results showed that Chromohalobacter sp. strain AZ6, Pseudoalteromonas sp. strain AZ, Psychrobacter sp. strain AZ3, Vibrio sp. strain AZ, and Halomonas sp. strain AZ07 exhibited algicidal properties as these strains were more abundant at high temperature (25 °C). Among the five isolated strains, the potential isolate Halomonas sp. strain AZ07 showed high production of agarolytic enzymes, including lipase, protease, cellulase, and amylase. This study confirmed that the isolated strain could produce these four different enzymes. The strain Halomonas AZ07 was co-treated with Pyropia yezoensis cells under two different temperature environments, including 13 °C and 25 °C. The degradation of Pyropia yezoensis occurred at the optimum temperature of 25 °C and effectively degraded their cell wall, proteins, lipids, and carbohydrates. CONCLUSION: The successful cultivation of Pyropia yezoensis in coastal farm environments is dependent on specific temperature and environmental factors, and lower temperatures have been observed to be particularly beneficial for the survival and growth of Pyropia yezoensis. The temperature below 13 °C was confirmed to be the best niche for the symbiotic relationship of microbiota associated with Pyropia yezoensis for its growth, development, and production.

Characterization of the surface-active exopolysaccharide produced by Halomonas sp TGOS-10: Understanding its role in the formation of marine oil snow.

In this study, we characterize the exopolymer produced by Halomonas sp. strain TGOS-10 -one of the organisms found enriched in sea surface oil slicks during the Deepwater Horizon oil spill. The polymer was produced during the early stationary phase of growth in Zobell's 2216 marine medium amended with glucose. Chemical and proton NMR analysis showed it to be a relatively monodisperse, high-molecular-mass (6,440,000 g/mol) glycoprotein composed largely of protein (46.6% of total dry weight of polymer). The monosaccharide composition of the polymer is typical to that of other marine bacterial exopolymers which are generally rich in hexoses, with the notable exception that it contained mannose (commonly found in yeast) as a major monosaccharide. The polymer was found to act as an oil dispersant based on its ability to effectively emulsify pure and complex oils into stable oil emulsions-a function we suspect to be conferred by the high protein content and high ratio of total hydrophobic nonpolar to polar amino acids (52.7:11.2) of the polymer. The polymer's chemical composition, which is akin to that of other marine exopolymers also having a high protein-to-carbohydrate (P/C) content, and which have been shown to effect the rapid and non-ionic aggregation of marine gels, appears indicative of effecting marine oil snow (MOS) formation. We previously reported the strain capable of utilising aromatic hydrocarbons when supplied as single carbon sources. However, here we did not detect biodegradation of these chemicals within a complex (surrogate Macondo) oil, suggesting that the observed enrichment of this organism during the Deepwater Horizon spill may be explained by factors related to substrate availability and competition within the complex and dynamic microbial communities that were continuously evolving during that spill.

Active prophages in coral-associated Halomonas capable of lateral transduction.

Temperate phages can interact with bacterial hosts through lytic and lysogenic cycles via different mechanisms. Lysogeny has been identified as the major form of bacteria-phage interaction in the coral-associated microbiome. However, the lysogenic-to-lytic switch of temperate phages in ecologically important coral-associated bacteria and its ecological impact have not been extensively investigated. By studying the prophages in coral-associated Halomonas meridiana, we found that two prophages, Phm1 and Phm3, are inducible by the DNA-damaging agent mitomycin C and that Phm3 is spontaneously activated under normal cultivation conditions. Furthermore, Phm3 undergoes an atypical lytic pathway that can amplify and package adjacent host DNA, potentially resulting in lateral transduction. The induction of Phm3 triggered a process of cell lysis accompanied by the formation of outer membrane vesicles (OMVs) and Phm3 attached to OMVs. This unique cell-lysis process was controlled by a four-gene lytic module within Phm3. Further analysis of the Tara Ocean dataset revealed that Phm3 represents a new group of temperate phages that are widely distributed and transcriptionally active in the ocean. Therefore, the combination of lateral transduction mediated by temperate phages and OMV transmission offers a versatile strategy for host-phage coevolution in marine ecosystems.

Rational engineering of Halomonas salifodinae to enhance hydroxyectoine production under lower-salt conditions.

Hydroxyectoine is an important compatible solute that holds potential for development into a high-value chemical with broad applications. However, the traditional high-salt fermentation for hydroxyectoine production presents challenges in treating the high-salt wastewater. Here, we report the rational engineering of Halomonas salifodinae to improve the bioproduction of hydroxyectoine under lower-salt conditions. The comparative transcriptomic analysis suggested that the increased expression of ectD gene encoding ectoine hydroxylase (EctD) and the decreased expressions of genes responsible for tricarboxylic acid (TCA) cycle contributed to the increased hydroxyectoine production in H. salifodinae IM328 grown under high-salt conditions. By blocking the degradation pathway of ectoine and hydroxyectoine, enhancing the expression of ectD, and increasing the supply of 2-oxoglutarate, the engineered H. salifodinae strain HS328-YNP15 (ΔdoeA::PUP119-ectD p-gdh) produced 8.3-fold higher hydroxyectoine production than the wild-type strain and finally achieved a hydroxyectoine titer of 4.9 g/L in fed-batch fermentation without any detailed process optimization. This study shows the potential to integrate hydroxyectoine production into open unsterile fermentation process that operates under low-salinity and high-alkalinity conditions, paving the way for next-generation industrial biotechnology. KEY POINTS: • Hydroxyectoine production in H. salifodinae correlates with the salinity of medium • Transcriptomic analysis reveals the limiting factors for hydroxyectoine production • The engineered strain produced 8.3-fold more hydroxyectoine than the wild type.

Unlocking growth potential in Halomonas bluephagenesis for enhanced PHA production with sulfate ions.

The mutant strain Halomonas bluephagenesis (TDH4A1B5P) was found to produce PHA under low-salt, non-sterile conditions, but the yield was low. To improve the yield, different nitrogen sources were tested. It was discovered that urea was the most effective nitrogen source for promoting growth during the stable stage, while ammonium sulfate was used during the logarithmic stage. The growth time of H. bluephagenesis (TDH4A1B5P) and its PHA content were significantly prolonged by the presence of sulfate ions. After 64 hr in a 5-L bioreactor supplemented with sulfate ions, the dry cell weight (DCW) of H. bluephagenesis weighed 132 g/L and had a PHA content of 82%. To promote the growth and PHA accumulation of H. bluephagenesis (TDH4A1B5P), a feeding regimen supplemented with nitrogen sources and sulfate ions with ammonium sodium sulfate was established in this study. The DCW was 124 g/L, and the PHA content accounted for 82.3% (w/w) of the DCW, resulting in a PHA yield of 101 g/L in a 30-L bioreactor using the optimized culture strategy. In conclusion, stimulating H. bluephagenesis (TDH4A1B5P) to produce PHA is a feasible and suitable strategy for all H. bluephagenesis.

Establishment of low-cost production platforms of polyhydroxyalkanoate bioplastics from Halomonas cupida J9.

Microbial production of polyhydroxyalkanoate (PHA) is greatly restricted by high production cost arising from high-temperature sterilization and expensive carbon sources. In this study, a low-cost PHA production platform was established from Halomonas cupida J9. First, a marker-less genome-editing system was developed in H. cupida J9. Subsequently, H. cupida J9 was engineered to efficiently utilize xylose for PHA biosynthesis by introducing a new xylose metabolism module and blocking xylonate production. The engineered strain J9UΔxylD-P8xylA has the highest PHA yield (2.81 g/L) obtained by Halomonas with xylose as the sole carbon source so far. This is the first report on the production of short- and medium-chain-length (SCL-co-MCL) PHA from xylose by Halomonas. Interestingly, J9UΔxylD-P8xylA was capable of efficiently utilizing glucose and xylose as co-carbon sources for PHA production. Furthermore, fed-batch fermentation of J9UΔxylD-P8xylA coupled to a glucose/xylose co-feeding strategy reached up to 12.57 g/L PHA in a 5-L bioreactor under open and unsterile condition. Utilization of corn straw hydrolysate as the carbon source by J9UΔxylD-P8xylA reached 7.0 g/L cell dry weight (CDW) and 2.45 g/L PHA in an open fermentation. In summary, unsterile production in combination with inexpensive feedstock highlights the potential of the engineered strain for the low-cost production of PHA from lignocellulose-rich agriculture waste.

Purification, structural characterization, and neuroprotective effect of 3,6-diisobutyl-2,5-piperazinedione from Halomonas pacifica CARE-V15 against okadaic acid-induced neurotoxicity in zebrafish model.

Halomonas pacifica CARE-V15 was isolated from the southeastern coast of India to determine its genome sequence. Secondary metabolite gene clusters were identified using an anti-SMASH server. The concentrated crude ethyl acetate extract was evaluated by GC-MS. The bioactive compound from the crude ethyl acetate extract was fractionated by gel column chromatography. HPLC was used to purify the 3,6-diisobutyl-2,5-piperazinedione (DIP), and the structure was determined using FTIR and NMR spectroscopy. Purified DIP was used in an in silico molecular docking analysis. Purified DIP exhibits a stronger affinity for antioxidant genes like glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GSR). Using in silco molecular docking analysis, the protein-ligand binding affinities of GSR (-4.70 kcal/mol), GST (-5.27 kcal/mol), and GPx (-5.37 kcal/mol) were measured. The expression of antioxidant genes were investigated by qRT-PCR. The in vivo reactive oxygen species production, lipid peroxidation, and cell death levels were significantly (p ≤ 0.05) increased in OA-induced group, but all these levels were significantly (p ≤ 0.05) decreased in the purified DIP pretreated group. Purified DIP from halophilic bacteria could thus be a useful treatment for neurological disorders associated with oxidative stress.

Metabolic modeling of Halomonas campaniensis improves polyhydroxybutyrate production under nitrogen limitation.

Poly-hydroxybutyrate (PHB) is an environmentally friendly alternative for conventional fossil fuel-based plastics that is produced by various microorganisms. Large-scale PHB production is challenging due to the comparatively higher biomanufacturing costs. A PHB overproducer is the haloalkaliphilic bacterium Halomonas campaniensis, which has low nutritional requirements and can grow in cultures with high salt concentrations, rendering it resistant to contamination. Despite its virtues, the metabolic capabilities of H. campaniensis as well as the limitations hindering higher PHB production remain poorly studied. To address this limitation, we present HaloGEM, the first high-quality genome-scale metabolic network reconstruction, which encompasses 888 genes, 1528 reactions (1257 gene-associated), and 1274 metabolites. HaloGEM not only displays excellent agreement with previous growth data and experiments from this study, but it also revealed nitrogen as a limiting nutrient when growing aerobically under high salt concentrations using glucose as carbon source. Among different nitrogen source mixtures for optimal growth, HaloGEM predicted glutamate and arginine as a promising mixture producing increases of 54.2% and 153.4% in the biomass yield and PHB titer, respectively. Furthermore, the model was used to predict genetic interventions for increasing PHB yield, which were consistent with the rationale of previously reported strategies. Overall, the presented reconstruction advances our understanding of the metabolic capabilities of H. campaniensis for rationally engineering this next-generation industrial biotechnology platform. KEY POINTS: A comprehensive genome-scale metabolic reconstruction of H. campaniensis was developed. Experiments and simulations predict N limitation in minimal media under aerobiosis. In silico media design increased experimental biomass yield and PHB titer.

Localization and symbiotic status of probiotics in the coral holobiont.

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.

Effect of Halomonas titanicae on fluctuating water-line corrosion of EH40 steel.

The fluctuating water-line corrosion of EH40 steel in sterile and biotic media was investigated with a wire beam electrode. When the coupons were partially immersed in the sterile medium, the position of the low water-line acted as the cathodic zone and the area below the low water-line constantly served as the main anodic zone. The thin electrolyte layers with uneven thickness promoted the galvanic current of the region below the low water-line. Different from the sterile environment, the metabolism of Halomonas titanica with oxygen as the final electron acceptor reduced the dissolved oxygen concentration, which resulted in the position of the low water-line acting as the anodic zone.

Temporal dynamics of stress response in Halomonas elongata to NaCl shock: physiological, metabolomic, and transcriptomic insights.

BACKGROUND: The halophilic bacterium Halomonas elongata is an industrially important strain for ectoine production, with high value and intense research focus. While existing studies primarily delve into the adaptive mechanisms of this bacterium under fixed salt concentrations, there is a notable dearth of attention regarding its response to fluctuating saline environments. Consequently, the stress response of H. elongata to salt shock remains inadequately understood. RESULTS: This study investigated the stress response mechanism of H. elongata when exposed to NaCl shock at short- and long-time scales. Results showed that NaCl shock induced two major stresses, namely osmotic stress and oxidative stress. In response to the former, within the cell's tolerable range (1-8% NaCl shock), H. elongata urgently balanced the surging osmotic pressure by uptaking sodium and potassium ions and augmenting intracellular amino acid pools, particularly glutamate and glutamine. However, ectoine content started to increase until 20 min post-shock, rapidly becoming the dominant osmoprotectant, and reaching the maximum productivity (1450 ± 99 mg/L/h). Transcriptomic data also confirmed the delayed response in ectoine biosynthesis, and we speculate that this might be attributed to an intracellular energy crisis caused by NaCl shock. In response to oxidative stress, transcription factor cysB was significantly upregulated, positively regulating the sulfur metabolism and cysteine biosynthesis. Furthermore, the upregulation of the crucial peroxidase gene (HELO_RS18165) and the simultaneous enhancement of peroxidase (POD) and catalase (CAT) activities collectively constitute the antioxidant defense in H. elongata following shock. When exceeding the tolerance threshold of H. elongata (1-13% NaCl shock), the sustained compromised energy status, resulting from the pronounced inhibition of the respiratory chain and ATP synthase, may be a crucial factor leading to the stagnation of both cell growth and ectoine biosynthesis. CONCLUSIONS: This study conducted a comprehensive analysis of H. elongata's stress response to NaCl shock at multiple scales. It extends the understanding of stress response of halophilic bacteria to NaCl shock and provides promising theoretical insights to guide future improvements in optimizing industrial ectoine production.

Ectoine hyperproduction by engineered Halomonas bluephagenesis.

Ectoine, a crucial osmoprotectant for salt adaptation in halophiles, has gained growing interest in cosmetics and medical industries. However, its production remains challenged by stringent fermentation process in model microorganisms and low production level in its native producers. Here, we systematically engineered the native ectoine producer Halomonas bluephagenesis for ectoine production by overexpressing ectABC operon, increasing precursors availability, enhancing product transport system and optimizing its growth medium. The final engineered H. bluephagenesis produced 85 g/L ectoine in 52 h under open unsterile incubation in a 7 L bioreactor in the absence of plasmid, antibiotic or inducer. Furthermore, it was successfully demonstrated the feasibility of decoupling salt concentration with ectoine synthesis and co-production with bioplastic P(3HB-co-4HB) by the engineered H. bluephagenesis. The unsterile fermentation process and significantly increased ectoine titer indicate that H. bluephagenesis as the chassis of Next-Generation Industrial Biotechnology (NGIB), is promising for the biomanufacturing of not only intracellular bioplastic PHA but also small molecular compound such as ectoine.

Production of polyhydroxyalkanoates by engineered Halomonas bluephagenesis using starch as a carbon source.

A novel α-amylase Amy03713 was screened and cloned from the starch utilization strain Vibrio alginolyticus LHF01. When heterologously expressed in Escherichia coli, Amy03713 exhibited the highest enzyme activity at 45 °C and pH 7, maintained >50 % of the enzyme activity in the range of 25-75 °C and pH 5-9, and sustained >80 % of the enzyme activity in 25 % (w/v) of NaCl solution, thus showing a wide range of adapted temperatures, pH, and salt concentrations. Halomonas bluephagenesis harboring amy03713 gene was able to directly utilize starch. With optimized amylase expression, H. bluephagenesis could produce poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB). When cultured for PHB production, recombinant H. bluephagenesis was able to grow up to a cell dry weight of 11.26 g/L, achieving a PHB titer of 6.32 g/L, which is the highest titer that has been reported for PHB production from starch in shake flasks. This study suggests that Amy03713 is an ideal amylase for PHA production using starch as the carbon source in H. bluephagenesis.

Halomonas citrativorans sp. nov., Halomonas casei sp. nov. and Halomonas colorata sp. nov., isolated from French cheese rinds.

Eight Gram-stain-negative bacterial strains were isolated from cheese rinds sampled in France. On the basis of 16S rRNA gene sequence analysis, all isolates were assigned to the genus Halomonas. Phylogenetic investigations, including 16S rRNA gene studies, multilocus sequence analysis, reconstruction of a pan-genome phylogenetic tree with the concatenated core-genome content and average nucleotide identity (ANI) calculations, revealed that they constituted three novel and well-supported clusters. The closest relative species, determined using the whole-genome sequences of the strains, were Halomonas zhanjiangensis for two groups of cheese strains, sharing 82.4 and 93.1 % ANI, and another cluster sharing 92.2 % ANI with the Halomonas profundi type strain. The strains isolated herein differed from the previously described species by ANI values <95 % and several biochemical, enzymatic and colony characteristics. The results of phenotypic, phylogenetic and chemotaxonomic analyses indicated that the isolates belonged to three novel Halomonas species, for which the names Halomonas citrativorans sp. nov., Halomonas casei sp. nov. and Halomonas colorata sp. nov. are proposed, with isolates FME63T (=DSM 113315T=CIRM-BIA2430T=CIP 111880T=LMG 32013T), FME64T (=DSM 113316T=CIRM-BIA2431T=CIP 111877T=LMG 32015T) and FME66T (=DSM 113318T=CIRM-BIA2433T=CIP 111876T=LMG 32014T) as type strains, respectively.

Proteomic insights into the response of Halomonas sp. MNB13 to excess Mn(â ¡) and the role of H2S in Mn(â ¡) resistance.

Halomonas spp. are moderately halophilic bacteria with the ability to tolerate various heavy metals. However, the role of basic cellular metabolism, particularly amino acid metabolism, has not been investigated in Halomonas spp. under excess Mn(Ⅱ). The strain Halomonas sp. MNB13 was isolated from a deep-sea ferromanganese nodule and can tolerate 80 mM Mn(Ⅱ). To comprehensively explore the mechanisms underlying its resistance to excess Mn(Ⅱ), we conducted a comparative proteome analysis. The data revealed that both 10 mM and 50 mM Mn(Ⅱ) significantly up-regulated the expression of proteins involved in Mn(Ⅱ) transport (MntE), oxidative stress response (alkyl hydroperoxide reductase and the Suf system), and amino acid metabolism (arginine, cysteine, methionine, and phenylalanine). We further investigated the role of cysteine metabolism in Mn(Ⅱ) resistance by examining the function of its downstream product, H2S. Consistent with the up-regulation of cysteine desulfurase, we detected an elevated level of H2S in Halomonas sp. MNB13 cells under Mn(Ⅱ) stress, along with increased intracellular levels of H2O2 and O2•-. Upon exogenous addition of H2S, we observed a significant restoration of the growth of Halomonas sp. MNB13. Moreover, we identified decreased intracellular levels of H2O2 and O2•- in MNB13 cells, which coincided with a decreased formation of Mn-oxides during cultivation. In contrast, in cultures containing NaHS, the residual Mn(Ⅱ) levels were higher than in cultures without NaHS. Therefore, H2S improves Mn(Ⅱ) tolerance by eliminating intracellular reactive oxygen species rather than decreasing Mn(Ⅱ) concentration in solution. Our findings indicate that cysteine metabolism, particularly the intermediate H2S, plays a pivotal role in Mn(Ⅱ) resistance by mitigating the damage caused by reactive oxygen species. These findings provide new insights into the amino acid mechanisms associated with Mn(Ⅱ) resistance in bacteria.

Nonsterile microbial production of chemicals based on Halomonas spp.

The use of extremophile organisms such as Halomomas spp. can eliminate the need for fermentation sterilization, significantly reducing process costs. Microbial fermentation is considered a pivotal strategy to reduce reliance on fossil fuel resources; however, sustainable processes continue to incur higher costs than their chemical industry counterparts. Most organisms require equipment sterilization to prevent contamination, a practice that introduces complexity and financial strain. Fermentations involving extremophile organisms can eliminate the sterilization process, relying instead on conditions that are conductive solely to the growth of the desired organism. This review discusses current challenges in pilot- and industrial-scale bioproduction when using the extremophile bacteria Halomomas spp. under nonsterile conditions.

Halomonas rhizosphaerae sp. nov. and Halomonas kalidii sp. nov., two novel moderate halophilic phenolic acid-degrading species isolated from saline soil.

Four vanillic acid-degrading bacterial strains, named LR5S13T, LR5S20, and M4R5S39T and LN1S58, were isolated from Kalidium cuspidatum rhizosphere and bulk soils, respectively. Phylogenetic analysis based on 16S rRNA gene as well as core genome revealed that LR5S13T and LR5S20 clustered closely with each other and with Halomonas ventosae Al12T, and that the two strains shared the highest similarities (both 99.3 %) with H. ventosae Al12T, in contrast, M4R5S39T and LN1S58 clustered together and with Halomonas heilongjiangensis 9-2T, and the two strains shared the highest similarities (99.4 and 99.2 %, respectively) with H. heilongjiangensis 9-2T. The average nucleotides identities based on BLAST (ANIb) and digital DNA-DNA hybridization (dDDH) values of strains LR5S13T to LR5S20, and M4R5S39T to LN1S58, were both higher than the threshold values for delineation of a species. The ANIb and dDDH values of the four strains to their closely relatives were lower than the threshold values. All four strains take phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol as the major polar lipids, Summed Feature 8, Summed Feature 3, and C16:0 as the major fatty acids. Based on the phylogenetic and phenotypic results, the four strains should be classified as two novel Halomonas species. Therefore, Halomonas rhizosphaerae sp. nov. (type strain LR5S13T = KCTC 8016T = CGMCC 1.62049T) and Halomonas kalidii (type strain M4R5S39T = KCTC 8015T = CGMCC 1.62047T) are proposed. The geographical distribution analysis based on 16S rRNA gene revealed that the two novel species are widely distributed across the globe, specifically in highly saline habits, especially in Central and Eastern Asia.

Structure of the O-polysaccharide from the moderately halophilic bacterium Halomonas fontilapidosi KR26.

Lipopolysaccharide was obtained from the aerobic moderately halophilic bacterium Halomonas fontilapidosi KR26. The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide and was examined by chemical methods and by 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC, and HMBC experiments. The following structure of the linear tetrasaccharide repeating unit was deduced. →2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-ß-d-Galp-(1→.

Identification and analysis of a clinically isolated strain of Halomonas based on whole-genome sequencing and comparative genomics.

OBJECTIVE: The aim of this study was to identify the species of a Halomonas strain isolated from a neonatal blood sample and to understand the potential pathogenicity and characteristic genes of the strain. METHODS: The genomic DNA of strain 18071143 (identified as Halomonas by matrix-assisted laser desorption-ionization time of flight-mass spectrometry and the 16S ribosomal RNA (rRNA) gene sequence) was sequenced using Nanopore PromethION platforms. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were calculated using the complete genome sequences of the strain. Comparative genomic analyses were performed on strain 18071143 and 3 strains of Halomonas (Halomonas stevensii S18214, Halomonas hamiltonii KCTC 22154, and Halomonas johnsoniae KCTC 22157) that were associated with human infections and had high genomic similarity to strain 18071143. RESULTS: Phylogenetic, ANI, and dDDH similarity analyses based on genome sequence indicated that strain 18071143 belonged to the species H stevensii. Similarities exist between strain 18071143 and the other 3 Halomonas strains in terms of gene structure and protein function. Nonetheless, strain 18071143 has greater potential for DNA replication, recombination, repair, and horizontal transfer. CONCLUSION: Whole-genome sequencing holds great promise for accurate strain identification in clinical microbiology. In addition, the results of this study provide data for understanding Halomonas from the perspective of pathogenic bacteria.