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Among various species of marine bacteria, those belonging to the genus Halomonas have several promising applications and have been studied well. However, not much information has been available on their antibiotic resistance. In our efforts to learn about the antibiotic resistance of strain Halomonas socia CKY01, which showed production of various hydrolases and growth promotion by osmolytes in previous study, we found that it exhibited resistance to multiple antibiotics including kanamycin, ampicillin, oxacillin, carbenicillin, gentamicin, apramycin, tetracycline, and spectinomycin. However, the H. socia CKY01 resistance pattern to kanamycin, gentamicin, apramycin, tetracycline, and spectinomycin differed in the presence of 10% NaCl and 1% NaCl in the culture medium. To determine the mechanism underlying this NaCl concentration-dependent antibiotic resistance, we compared four aminoglycoside resistance genes under different salt conditions while also performing time-dependent reverse transcription PCR. We found that the aph2 gene encoding aminoglycoside phosphotransferase showed increased expression under the 10% rather than 1% NaCl conditions. When these genes were overexpressed in an Escherichia coli strain, pETDuet-1:: aph2 showed a smaller inhibition zone in the presence of kanamycin, gentamicin, and apramycin than the respective control, suggesting aph2 was involved in aminoglycoside resistance. Our results demonstrated a more direct link between NaCl and aminoglycoside resistance exhibited by the H. socia CKY01 strain.

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

Halomonas TD01, which can grow under non-sterile and continuous processes at high pH and high salt concentrations, is a robust platform for PHA production from glucose. For extending other low-cost sustainable substrates and increasing the potential application in other value-added products, a better understanding of substrates utilization and chemicals tolerance is necessary. In this study, the substrate profiling of TD01 was analyzed via Biolog. Phenotype microarray results demonstrated that TD01 has a wide-ranging substrate spectrum and can utilize 140 of the 190 test compounds. Some cheap, abundant carbon sources, such as sodium acetate, glycerol, ethanol and lactate can well support the growth of TD01 in shake-flask, and are therefore suggested to be its alternative low-cost substrates for chemicals production in future. Furthermore, the tolerance of TD01 to various chemicals was tested. The results showed that the tolerability of TD01 to high concentrations of organic acid salts is prominent. When adding 75 g/L sodium acetate, 100 g/L succinic acid and 100 g/L itaconic acid in the medium, the growth rate reduced 56.14%, 52.63% and 47.37%, respectively. All these results highlight TD01 as a promising, next generation industrial workhorse in chemicals biomanufacturing from cheap organic acid salts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Imidazolium-based ionic liquids (IL) with short-alkyl side chain such as 1-ethyl-3-methyl-imidazolium chloride ([Emim]Cl) and 1-butyl-3-methyl-imidazolium chloride ([Bmim]Cl) has immense application potential including in lignocellulosic bioenergy production. But they are toxic to most microorganisms, and those isolated from different environments as IL-tolerant have salt tolerance capabilities. This study evaluates the relationship between salt and [Emim]Cl tolerance of microorganisms using different salinity sediments (2-19%) and brines (35%) of India's largest inland hypersaline lake, Sambhar in Rajasthan as the model system. While samples with 2% and 35% salinities do not yield any [Emim]Cl (100 mM) tolerant colonies, others have 6-50% colonies tolerant to the IL. Similar trend was observed with 50 mM [Bmim]Cl. Moderate halophilic isolates of genera Halomonas and Bacillus (growth in 0.7-3.0 M NaCl) isolated from the sediments could grow in as high as 375 mM [Emim]Cl, or 125 mM [Bmim]Cl facilitated by higher synthesis, and uptake of organic osmolytes; and up to 1.7-fold increased activity of active efflux pumps. [Bmim]Cl was more toxic than [Emim]Cl in all performed experiments. [Emim]Cl-adapted cells could trounce IL-induced stress. Interestingly, enrichment with 100 mM [Emim]Cl resulted in increase of IL-tolerant colonies in all sediments including the one with 2% salinity. However, the salt saturated brines (35%) do not yield any such colony even after repeated incubations. Extreme halophilic archaea, Natronomonas (growth in 3.0-4.0 M NaCl) isolated from such brines, were exceedingly sensitive to even 5 mM [Emim]Cl, or 1 mM [Bmim]Cl. Two additional extremophilic archaea, namely Haloferax and Haladaptatus were also sensitive to the tested ILs. Archaeal sensitivity is possibly due to the competitive interaction of [Emim] + with their acidic proteome (15.4-17.5% aspartic and glutamic acids, against 10.7-12.9% in bacteria) that they maintain to stabilize the high amount of K + ion accumulated by salt-in strategy. Thus, general salt adaptation strategies of moderate halophilic bacteria help them to restrain toxicity of these ILs, but extremophilic archaea are highly sensitive and demands meticulous use of these solvents to prevent environmental contamination., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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

Cyanobactins are a family of linear and cyclic peptides produced through the post-translational modification of short precursor peptides. A mass spectrometry-based screening of potential cyanobactin producers led to the discovery of a new prenylated member of this family of compounds, sphaerocyclamide (1), from Sphaerospermopsis sp. LEGE 00249. The sphaerocyclamide biosynthetic gene cluster (sph) encoding the novel macrocyclic prenylated cyanobactin, was sequenced. Heterologous expression of the sph gene cluster in Escherichia coli confirmed the connection between genomic and mass spectrometric data. Unambiguous establishment of the orientation and site of prenylation required the full structural elucidation of 1 using Nuclear Magnetic Resonance (NMR), which demonstrated that a forward prenylation occurred on the tyrosine residue. Compound 1 was tested in pharmacologically or ecologically relevant biological assays and revealed moderate antimicrobial activity towards the fouling bacterium Halomonas aquamarina CECT 5000.

In living systems, environmental stress due to biotic and abiotic factors triggers the production of myriad metabolites as a potential mechanism for combating stress. Among these metabolites are the small polycationic aliphatic amine molecules - polyamines, which are ubiquitous in all living organisms. In this work, we demonstrate a correlation between cellular concentration of three major polyamines (putrescine, spermidine and spermine) with lead exposure on bacteria for a period of 6-24 h. We report that indigenously isolated Halomonas sp. strain BVR 1 exhibits lead induced fluctuations in their cellular polyamine concentration. This response to lead occurs within 6 h post metal treatment. During the same time interval there was a surge in the growth of bacteria along with an enhancement in the putrescine levels. We conclude that in Halomonas sp. strain BVR 1, an early response is seen with respect to modulation of polyamines as a result of lead treatment and hypothesize that endogenous polyamines contribute towards scavenging lead in these bacteria.

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

Levan is a bioactive fructan polymer that is mainly associated with high-value applications where exceptionally high purity requirements call for well-defined cultivation conditions. In this study, microbial levan production by the halophilic extremophile Halomonas smyrnensis AAD6(T) was investigated systematically. For this, different feeding strategies in fed-batch cultures were employed and fermentation profiles of both shaking and bioreactor cultures were analyzed. Initial carbon and nitrogen source concentrations, production pH, NaCl and nitrogen pulses, nitrogen and phosphorous limitations, trace elements and thiamine contents of the basal production medium were found to affect the levan yields at different extends. Boric acid was found to be the most effective stimulator of levan production by increasing the sucrose utilization three-fold and levan production up to five-fold. This significant improvement implied the important role of quorum sensing phenomenon and its regulatory impact on levan production mechanism. Levan produced by bioreactor cultures under conditions optimized within this study was found to retain its chemical structure. Moreover, its biocompatibility was assessed for a broad concentration range. Hence H. smyrnensis AAD6(T) has been firmly established as an industrially important resource microorganism for high-quality levan production., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The limits to biological processes on Earth are determined by physicochemical parameters, such as extremes of temperature and low water availability. Research into microbial extremophiles has enhanced our understanding of the biophysical boundaries which define the biosphere. However, there remains a paucity of information on the degree to which rates of microbial multiplication within extreme environments are determined by the availability of specific chemical elements. Here, we show that iron availability and the composition of the gaseous phase (aerobic versus microaerobic) determine the susceptibility of a marine bacterium, Halomonas hydrothermalis, to suboptimal and elevated temperature and salinity by impacting rates of cell division (but not viability). In particular, iron starvation combined with microaerobic conditions (5% [vol/vol] O2, 10% [vol/vol] CO2, reduced pH) reduced sensitivity to temperature across the 13°C range tested. These data demonstrate that nutrient limitation interacts with physicochemical parameters to determine biological permissiveness for extreme environments. The interplay between resource availability and stress tolerance, therefore, may shape the distribution and ecology of microorganisms within Earth's biosphere., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The leaf surfaces of the salt-excreting tree Tamarix aphylla harbor a wide diversity of halophilic microorganisms, including Halomonas sp., but little is known of the factors that shape community composition in this extreme habitat. We isolated a strain of Halomonas variabilis from the leaf surface of T. aphylla and used it to determine the heterogeneity of salt concentrations experienced by bacteria in this environment. This halophilic strain was transformed with a proU::gfp reporter gene fusion, the fluorescence of which was responsive to NaCl concentrations up to 200 g liter(-1). These bioreporting cells were applied to T. aphylla leaves and were subsequently recovered from dew droplets adhering to the leaf surface. Although cells from within a given dew droplet exhibited similar green fluorescent protein fluorescence, the fluorescence intensity varied between droplets and was correlated with the salt concentration measured in each drop. Growth of H. variabilis was observed in all droplets, regardless of the salt concentration. However, cells found in desiccated microniches between dew drops were low in abundance and generally dead. Other bacteria recovered from T. aphylla displayed higher desiccation tolerance than H. variabilis, both in culture and on inoculated plants, despite having lower osmotic tolerance. Thus, the Tamarix leaf surface can be described as a salty desert with occasional oases where water droplets form under humid conditions. While halotolerant bacteria such as Halomonas grow in high concentrations of salt in such wet microniches, other organisms are better suited to survive desiccation in sites that are not wetted.

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

A bioflocculant-producing bacterium isolated from seawater was identified based on 16S rRNA gene nucleotide sequence to have 99% similarity to that of Halomonas sp. Au160H and the nucleotide sequence was deposited as Halomonas sp. OKOH (Genbank accession number is HQ875722). Influences of carbon source, nitrogen source, salt ions and pH on flocculating activity were investigated. The bioflocculant was optimally produced when glucose (87% flocculating activity) and urea (88% flocculating activity) were used as sources of carbon and nitrogen, respectively. Also, initial pH of 7.0 and Ca²⁺ supported optimal production of the bioflocculant with flocculating activities of 87% respectively. Chemical analyses revealed the bioflocculant to be a polysaccharide.

A process comprising two-step fed-batch cultivation has been investigated for the production of ectoines using the halophilic bacterium Halomonas boliviensis DSM 15516(T). The first cultivation was performed under optimal conditions for cell growth and resulted in cell mass concentration of about 41 g l(-1) after 24 h of cultivation. During the second cultivation at higher salt concentration, accumulation of ectoines increased while cell mass decreased with increasing salt concentration. Maximum productivity of total ectoines reached was 10 g l(-1) d(-1) with ectoine concentration of 6 g l(-1) and hydroxyectoine concentration of 8 g l(-1) after 9 h of cultivation at 18.5% NaCl, which is among the highest reported so far. H. boliviensis cells were further recycled for the production process after releasing the ectoines. About 75% of the accumulated ectoines were released by subjecting the cells to hypoosmotic shock. On subsequent reincubation in a medium containing higher salt concentration the cells were able to re-synthesize the ectoines resulting in a global productivity of 11.1 g l(-1) d(-1), and ectoine and hydroxyectoine productivities of 9.1 g l(-1) d(-1) and 2.0 g l(-1) d(-1), respectively., (2010 Elsevier B.V. All rights reserved.)

Biodiesel fuel is favored as a type of carbon neutral energy. To popularize its usage, by-product waste glycerol utilization is a critical problem. We tried to isolate waste glycerol utilizing bacteria, and obtained the alkalo- and halophile bacteria Halomonas sp. KM-1. This strain produced bioplastic poly(3-hydroxybutyrate) (PHB) in a simple medium and diluted waste glycerol as a sole carbon source.

Marine biofilms are a virtually untapped source of bioactive molecules that may find application as novel antifoulants in the marine paint industry. This study aimed at determining the potential of marine biofilm bacteria to produce novel biomolecules with potential application as natural antifoulants. Nine representative strains were isolated from a range of surfaces and were grown in YEB medium and harvested during the late exponential growth phase. Bacterial biomass and spent culture medium were extracted with ethanol and ethyl acetate, respectively. Extracts were assayed for their antifouling activity using two tests: (1) antimicrobial well diffusion test against a common fouling bacterium, Halomonas marina, and (2) anti-crustacean activity test using Artemia salina. Our results showed that none of the ethanolic extracts (bacterial biomass) were active in either test. In contrast, most of the organic extracts had antimicrobial activity (88%) and were toxic towards A. salina (67%). Sequencing of full 16 S ribosomal DNA analysis showed that the isolates were related to Bacillus mojavensis and Bacillus firmus. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) profiling of ethyl acetate extracts of culture supernatants showed that these species produce the bioactive lipopeptides surfactin A, mycosubtilin and bacillomycin D.