NaCl Concentration-Dependent Aminoglycoside Resistance of Halomonas socia CKY01 and Identification of Related Genes.

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.

Effects of osmolytes on salt resistance of Halomonas socia CKY01 and identification of osmolytes-related genes by genome sequencing.

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.

Substrate profiling and tolerance testing of Halomonas TD01 suggest its potential application in sustainable manufacturing of chemicals.

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.

Efficacy of alkyltrimethylammonium bromide for decontaminating salt-cured hides from the red heat causing moderately halophilic bacteria.

Conventionally, animal hide and skin necessitates 95% saturated brine solution (SBS) for its preservation. This salt is primarily derived from different sources including solar-saltern, evaporation ponds, etc., which are laden with different types of halophilic micro-organisms. Previous studies confirmed that the presence of moderately halophilic bacteria caused red heat on cured hide, which adversely affects the leather quality and causes substantial economic losses for leather industries. Thus, this investigation was carried out to examine the effects of different concentrations of alkyltrimethylammonium bromide (ATMB) on selected halophilic-bacteria attributed to the deterioration of hide quality. In nutrient broth solution (NBS), ATMB at 250 and 500 ppm reduced individual halo-bacteria, that is, Halomonas halodenitrificans, Halomonas eurihalina, Alkalibacillus haloalkaliphilus and Salimicrobium album, by averages of 0·64 and 1·90, 1·5 and 2·61, 0·90 and 2·27, 1·65 and 3·36 log CFU per ml respectively in 5 min. ATMB treatment in SBS at 500 ppm for 18 h resulted in a reduction of H. halodenitrificans, H. eurihalina, A. haloalkaliphilus and S. album by averages of 1·9, 1·25, 0·96 and 1·34 log CFU per ml respectively, when compared with the controls. Likewise, 5000 ppm ATMB reduced the cocktail population nearly to zero from that cultivated in SBS for 18 h. SIGNIFICANCE AND IMPACT OF THE STUDY: In this investigation, the inhibition of different halophilic bacteria that causes red heat in salt-preserved hides is described for the first time. The antimicrobial susceptibility test executed via solution procedures for selected halophilic bacterial strains (i.e. resistant to the salt environment) revealed significant efficacy of alkyltrimethylammonium bromide (ATMB). The current study suggests that, chemical compound like ATMB could be utilized to prevent red heat-related damage on salt-cured hides caused by halophilic bacteria, which is a persisting concern of the leather industry.

Moderate halophilic bacteria, but not extreme halophilic archaea can alleviate the toxicity of short-alkyl side chain imidazolium-based ionic liquids.

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.

Potassium resistance of halotolerant and alkaliphilic Halomonas sp. Y2 by a Na+-induced K+ extrusion mechanism.

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.

PHA granules help bacterial cells to preserve cell integrity when exposed to sudden osmotic imbalances.

Polyhydroxyalkanoates (PHA) are microbial polyesters which accumulate as intracellular granules in numerous prokaryotes and mainly serve as storage materials; beyond this primary function, PHA also enhance the robustness of bacteria against various stress factors. We have observed that the presence of PHA in bacterial cells substantially enhances their ability to maintain cell integrity when suddenly exposed to osmotic imbalances. In the case of the non-halophilic bacterium Cupriavidus necator, the presence of PHA decreased plasmolysis-induced cytoplasmic membrane damage during osmotic up-shock, which subsequently enabled the cells to withstand subsequent osmotic downshock. In contrast, sudden induction of osmotic up- and subsequent down-shock resulted in massive hypotonic lysis of non-PHA containing cells as determined by Transmission Electron Microscopy and Thermogravimetrical Analysis. Furthermore, a protective effect of PHA against hypotonic lysis was also observed in the case of the halophilic bacterium Halomonas halophila; here, challenged PHA-rich cells were capable of retaining cell integrity more effectively than their PHA-poor counterparts. Hence, it appears that the fact that PHA granules, as an added value to their primary storage function, protect halophiles from the harmful effect of osmotic down-shock might explain why PHA accumulation is such a common feature among halophilic prokaryotes. The results of this study, apart from their fundamental importance, are also of practical biotechnological significance: because PHA-rich bacterial cells are resistant to osmotic imbalances, they could be utilized in in-situ bioremediation technologies or during enrichment of mixed microbial consortia in PHA producers under conditions of fluctuating salinity.

Isolation, cloning and characterization of an azoreductase and the effect of salinity on its expression in a halophilic bacterium.

Understanding the molecular mechanisms of azo dye decolorization is important for the development of effective bioremediation for textile-colored wastewater. A halophilic bacterium Halomonas sp. strain GT was isolated, which could degrade the azo dye Acid Brilliant Scarlet GR at 10% NaCl. The complete genome sequence of this strain was obtained using the PacBio RS II platform. Genome annotation revealed that four proteins are related to decolorization of azo dyes, such as azoreductase, laccases, benzene 1,2-dioxygenase, and catechol 1,2-dioxygenase. The putative azoreductase gene of Halomonas sp. strain GT responsible for the decolorization of azo dye in high salt environment was isolated. Phylogenetic tree analysis showed that the azoG (azoreductase gene of Halomonas sp. strain GT) and its homologs constituted a new branch of the NADH depending azoreductases, with all the homologous sequence of the protein from halophilic bacteria. At high NaCl concentrations, azoreductase gene expression and azoreductase activity were restrained in Halomonas sp. strain GT, which resulted in low a decolorization rate.

Sphaerocyclamide, a prenylated cyanobactin from the cyanobacterium Sphaerospermopsis sp. LEGE 00249.

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.

Toxicities of polystyrene nano- and microplastics toward marine bacterium Halomonas alkaliphila.

Nano- and microplastics have been shown to cause negative effects on marine organisms. However, the toxicities of nano- and microplastics toward marine bacteria are poorly understood. In this study, we investigated the toxic effects of polystyrene nano- and microplastics on the marine bacterium Halomonas alkaliphila by determining growth inhibition, chemical composition, inorganic nitrogen conversion efficiencies and reactive oxygen species (ROS) generation. The results showed that both nano- and microplastics inhibited the growth of H. alkaliphila in high concentrations, while nanoplastics rather than microplastics influenced the growth inhibition, chemical composition and ammonia conversion efficiencies of H. alkaliphila at concentration of 80 mg/L. The ROS generation indicated oxidative stress induced by nano- but not microplastics, and the oxidative stress induced by nanoplastics may provide a significant effect on bacteria. Furthermore, the positively charged nanoplastics (amine-modified 50 nm) induced higher oxidative stress toward bacteria than that induced by negatively charged nanoplastics (non-modified 55 nm). The increased extracellular polymeric substances as evidenced by transmission electron microscope (TEM) observation suggested the possible bacterial protective mechanisms. The present study illustrates for the first time the impact of plastics debris on the inorganic nitrogen conversion efficiencies of marine bacteria. Our findings highlight the effects of microplastics on the ecological function of marine organisms.

Impact of heavy metal lead stress on polyamine levels in Halomonas BVR 1 isolated from an industry effluent.

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.

Green synthesis of graphene-silver nanocomposites and its application as a potent marine antifouling agent.

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.

Isolation of an exopolysaccharide-producing heavy metal-resistant Halomonas sp. MG.

An exopolysaccharide (EPS)-producing heavy metal-resistant Gram-negative bacterium was isolated from ore-contaminated soil. The selected strain was identified by 16S rDNA sequencing and designated as Halomonas sp. MG. Phylogenetic analysis of the gene sequence showed its close similarity with Halomonas sp. Field emission scanning electron microscopy analysis revealed that the EPS had a porous structure with small pores. X-ray diffractograms showed the non-crystalline nature of the EPS. Further, FTIR spectroscopic analysis revealed the presence of carboxyl, hydroxyl and amide groups corresponding to a typical EPS.

Isolation and Characterization of Multi-Metal-Resistant Halomonas sp. MG from Tamil Nadu Magnesite Ore Soil in India.

The aim of the study was to isolate and characterize potential multi-metal-resistant bacteria from ore soils. A total of three bacteria were isolated and assayed for resistance to arsenic (As), copper (Cu), and lead (Pb). Isolate Halomonas sp. MG exhibited maximum resistance to 1000 mg Pb/L, 800 mg As/L, and 500 mg Cu/L and it was identified as Halomonas sp. based on the partial 16S rDNA sequences. The metal(loid)s resistance mechanisms were further confirmed by amplification of arsC (As) copAU (Cu), and pbrT (Pb) genes. Biological transmission electron micrographs and XRD studies showed that the isolate Halomonas sp. MG transformed and/or biomineralized the metals either intracellularly or extracellularly. These results suggest that the isolate could be used as a potential candidate for the bioremediation of As, Cu, and Pb.

Combinatorial materials research applied to the development of new surface coatings XVI: fouling-release properties of amphiphilic polysiloxane coatings.

High-throughput methods were used to prepare and characterize the fouling-release (FR) properties of an array of amphiphilic polysiloxane-based coatings possessing systematic variations in composition. The coatings were derived from a silanol-terminated polydimethylsiloxane, a silanol-terminated polytrifluorpropylmethylsiloxane (CF3-PDMS), 2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane (TMS-PEG), methyltriacetoxysilane and hexamethyldisilazane-treated fumed silica. The variables investigated were the concentration of TMS-PEG and the concentration of CF3-PDMS. In general, it was found that the TMS-PEG and the CF3-PDMS had a synergist effect on FR properties with these properties being enhanced by combining both compounds into the coating formulations. In addition, reattached adult barnacles removed from coatings possessing both TMS-PEG and relatively high levels of CF3-PDMS displayed atypical base-plate morphologies. The majority of the barnacles removed from these coatings exhibited a cupped or domed base-plate as compared to the flat base-plate observed for the control coating that did not contain TMS-PEG or CF3-PDMS. Coating surface analysis using water contact angle measurements indicated that the presence of CF3-PDMS facilitated migration of TMS-PEG to the coating/air interface during the film formation/curing process. In general, coatings containing both TMS-PEG and relatively high levels of CF3-PDMS possessed excellent FR properties.

Reduction of the temperature sensitivity of Halomonas hydrothermalis by iron starvation combined with microaerobic conditions.

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.

Effective stimulating factors for microbial levan production by Halomonas smyrnensis AAD6T.

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.

Investigation of mercury tolerance in Chromohalobacter israelensis DSM 6768T and Halomonas zincidurans B6T by comparative genomics with Halomonas xinjiangensis TRM 0175T.

Chromohalobacter israelensis DSM 6768(T), Halomonas zincidurans B6(T), and Halomonas xinjiangensis TRM 0175(T) are three phylogenetically close strains belonging to the class Gammaproteobacteria. Both strains DSM 6768(T) and B6(T) can grow on plate containing 0.5mM HgCl2. Strain TRM 0175(T) could not grow on plates containing 0.1mM or more HgCl2. Here we report the draft genomes of strains DSM 6768(T) and TRM 0175(T) for comparative genomic analysis. Gene cluster with putative function in mercury resistance in strain DSM 6768(T) includes a mercuric ion reductase, whose homologues distribute among several marine microbes. Strain B6(T), which was isolated from the Atlantic Ocean, has one more gene cluster with putative function in mercury resistance than strain DSM 6768(T). This study will enhance our understanding of the mercury tolerance and further investigation in marine microbes.

Efficient secretion of (R)-3-hydroxybutyric acid from Halomonas sp. KM-1 by nitrate fed-batch cultivation with glucose under microaerobic conditions.

To establish a sustainable society, commodity chemicals need to be developed from biomass resources. Recently, (R)-3-hydroxybutyric acid ((R)-3-HB), a monomer of bioplastic poly-(R)-3-hydroxybutyric acid (PHB), has attracted attention for its possible use in the chemical industry. Halophilic bacteria have been considered for bioprocess applications due to certain characteristics such as the ability to grow in media containing high levels of the starting carbon source and the ability to be rarely contaminated. A halophilic bacterium Halomonas sp. KM-1 stores PHB intracellularly under aerobic conditions and secretes (R)-3-HB under microaerobic conditions. In this study, we optimized culture conditions to maximize (R)-3-HB secretion by KM-1 cells. By a simple nitrate fed-batch cultivation, Halomonas sp. KM-1 secreted 40.3g/L (R)-3-HB with a productivity of 0.48g L(-1)h(-1) with 20% (w/v) glucose. This level is one of the highest recorded productivity of (R)-3-HB to date.

Type 2 quorum sensing monitoring, inhibition and biofilm formation in marine microrganisms.

The quorum sensing (QS) dependent behaviour of micro-organisms, in particular expression of virulence genes, biofilm formation and dispersal, have provided impetus for investigating practical approaches to interfere with microbial QS. This study tests Halomonas pacifica and Marinobacter hydrocarbonoclasticus, two halophilic marine micro-organism, for their AI-2 dependent QS signalling and the effect of two well-known quorum-sensing inhibitors (QSIs), patulin and penicillic acid, on biofilm formation. We report, for the first time, the successful amplification of a putative luxS gene in H. pacifica using degenerated primers and AI-2 dependent QS as well as inhibition using QSIs. Penicillic acid had a strong inhibitory effect on AI-2 induction of H. pacifica at non-growth inhibitory concentrations, while patulin has an adverse effect only at the highest concentration (25 µM). QSIs effect on biofilm forming capability was isolate specific, with maximum inhibition at 25 µM of patulin in H. pacifica. In M. hydrocarbonoclasticus, no adverse effects were noted at any tested concentration of either QSIs. Detection of bioluminescence and the presence of a putative luxS gene provide biochemical and genetic evidence for the production of a signalling molecule(s) which is the essential first step in characterizing H. pacifica QS. This study highlights the importance of AI-2 dependent QS in a marine setting, not previously reported. It further suggests that QSI compounds must be selected in the specific system in which they are to function, and they cannot easily be transferred from one QS system to another.