Synthesis and Characterization of Dendritic and Linear Glycol Methacrylates and Their Performance as Marine Antifouling Coatings.

Dendritic polyglycerol (PG) was covalently coupled to 2-hydroxyethyl methacrylate (HEMA) by an anionically catalyzed ring-opening polymerization generating a dendritic PG-HEMA with four PG repetition units (PG4MA). Coatings of the methacrylate monomer were prepared by grafting-through and compared against commercially available hydrophilic monomers of HEMA, poly(ethylene) glycol methacrylate (PEGMA), and poly(propylene) glycol methacrylate (PPGMA). The obtained coatings were characterized by modern surface analytical techniques, including water contact angle goniometry (sessile and captive bubble), attenuated total internal reflection Fourier transform infrared spectroscopy, and atomic force microscopy. The antifouling (AF) and fouling-release (FR) properties of the coatings were tested against the model organisms Cobetia marina and Navicula perminuta in laboratory-scale dynamic accumulation assays as well as in a dynamic short-term field exposure (DSFE) in the marine environment. In addition, the hydration of the coatings and their susceptibility toward silt uptake were evaluated, revealing a strong correlation between water uptake, silt incorporation, and field assay performance. While all glycol derivatives showed good resistance in laboratory settlement experiments, PPGMA turned out to be less susceptible to silt incorporation and outperformed PEGMA and PG4MA in the DSFE assay.

Effect of ozone stress on the intracellular metabolites from Cobetia marina.

A GCxGC-MS system was employed with a non-polar × mid-polar column set for the metabolic non-target analysis of Cobetia marina, the model bacteria for marine biofouling. C. marina was treated with ozone to investigate the intracellular metabolic state change under oxidative stress. A minimal inhibitory concentration test was involved to guarantee that the applied ozone dosages were not lethal for the cells. In this study, non-target analyses were performed to identify the metabolites according to the NIST database. As a result, over 170 signals were detected under normal living conditions including 35 potential metabolites. By the comparison of ozone-treated and non-treated samples, five compounds were selected to describe observed trends of signals in the contour plots. Oleic acid exhibited a slight growth by increasing ozone dosage. In contrast, other metabolites such as the amino acid L-proline showed less abundance after ozone treatment, which was more evident once ozone dosage was raised. Thus, this work could provide a hint for searching for up/downregulating factors in such environmental stress conditions for C. marina. Graphical abstract.

Microfluidic accumulation assay to quantify the attachment of the marine bacterium Cobetia marina on fouling-release coatings.

Testing the adhesion of marine biofilm formers on bioresistant coatings is important to determine their fouling-release and antifouling properties. A dynamic attachment assay for the marine bacterium Cobetia marina (C. marina) was developed to test the adhesion on coatings and bioresistant surfaces. With well-defined culture conditions, the reproducibility of the microfluidic accumulation experiments with C. marina was verified using self-assembling monolayers as model surfaces. The assay discriminated the attachment of C. marina on four different surfaces with different wettability and protein resistances. In addition to these benchmark experiments on self-assembled monolayers, the adhesion of C. marina on polyglycerol coatings with different thicknesses was investigated.

Salinicola peritrichatus sp. nov., isolated from deep-sea sediment.

A Gram stain-negative, aerobic and rod-shaped bacterium, strain DY22(T), was isolated from a deep-sea sediment collected from the east Pacific Ocean. The isolate was found to grow in the presence of 0-20.0 % (w/v) NaCl and at pH 4.5-8.5; optimum growth was observed with 0.5-2.0 % (w/v) NaCl and at pH 5.0-7.0. Chemotaxonomic analysis showed the presence of ubiquinone-9 as predominant respiratory quinone and C16:0, C19:0 ω8c cyclo and C12:0 3-OH as major cellular fatty acids. The genomic DNA G+C content was determined to be 59.6 mol%. Comparative 16S rRNA gene sequence analysis revealed that the novel isolate belongs to the genus Salinicola. Strain DY22(T) exhibited the closest phylogenetic affinity to the type strain of Salinicola salarius with 97.2 % sequence similarity and less than 97 % sequence similarity with respect to other Salinicola species with validly published names. The DNA-DNA reassociation values between strain DY22(T) and S. salarius DSM 18044(T) was 52 ± 4 %. On the basis of phenotypic, chemotaxonomic and genotypic data, strain DY22(T) represents a novel species of the genus Salinicola, for which the name Salinicola peritrichatus sp. nov. (type strain DY22(T) = CGMCC 1.12381(T) = JCM 18795(T)) is proposed.

Phenol degradation by halophilic bacteria isolated from hypersaline environments.

Phenol is a toxic aromatic compound used or produced in many industries and as a result a common component of industrial wastewaters. Phenol containing waste streams are frequently hypersaline and therefore require halophilic microorganisms for efficient biotreatment without dilution. In this study three halophilic bacteria isolated from different saline environments and identified as Halomonas organivorans, Arhodomonas aquaeolei and Modicisalibacter tunisiensis were shown to be able to grow on phenol in hypersaline media containing 100 g/L of total salts at a concentration of 3 mM (280 mg/L), well above the concentration found in most waste streams. Genes encoding the aromatic dioxygenase enzymes catechol 1,2 dioxygenase and protocatechuate 3,4-dioxygenase were present in all strains as determined by PCR amplification using primers specific for highly conserved regions of the genes. The gene for protocatechuate 3,4-dioxygenase was cloned from the isolated H. organivorans and the translated protein was evaluated by comparative protein sequence analysis with protocatechuate 3,4-dioxygenase proteins from other microorganisms. Although the analysis revealed a wide range of sequence divergence among the protocatechuate 3,4-dioxygenase family, all of the conserved domain amino acid structures identified for this enzyme family are identical or conservatively substituted in the H. organivorans enzyme.

Interfacial tension analysis of oligo(ethylene glycol)-terminated self-assembled monolayers and their resistance to bacterial attachment.

The fouling resistance of oligo(ethylene glycol) (OEG)-terminated self-assembled monolayers (SAMs) of alkanethiolates on gold has been well established. Although hydration of the OEG chains seems key to OEG-SAM resistance to macromolecular adsorption and cellular attachment, the details of how hydration prevents biofouling have been inferred largely through computational methods. Because OEG-SAMs of different lengths exhibit differing degrees of fouling resistance, the interactions between water and OEG-SAMs leading to fouling resistance can be deduced by comparing the properties of fouling and nonfouling OEG-SAMs. While all OEG-SAMs had similar water contact angles, contact angles taken with glycerol were able to individuate between different OEG-SAMs and between fouling and nonfouling OEG-SAMs. Subsequent estimation of surface and interfacial tension using a colloidal model showed that nonfouling surfaces are associated with an increased negative interfacial tension between those OEG-SAMs that resisted attachment and water. Further analysis of this interfacial tension experimentally confirmed current mathematical models that cite OEG-water hydrogen-bond formation as a driving force behind short-term fouling resistance. Finally, we found a correlation between solid-water interfacial tension and packing density and molecular density of ethylene glycol.

GFAJ-1 is an arsenate-resistant, phosphate-dependent organism.

The bacterial isolate GFAJ-1 has been proposed to substitute arsenic for phosphorus to sustain growth. We have shown that GFAJ-1 is able to grow at low phosphate concentrations (1.7 µM), even in the presence of high concentrations of arsenate (40 mM), but lacks the ability to grow in phosphorus-depleted (<0.3 µM), arsenate-containing medium. High-resolution mass spectrometry analyses revealed that phosphorylated central metabolites and phosphorylated nucleic acids predominated. A few arsenylated compounds, including C6 sugar arsenates, were detected in extracts of GFAJ-1, when GFAJ-1 was incubated with arsenate, but further experiments showed they formed abiotically. Inductively coupled plasma mass spectrometry confirmed the presence of phosphorus in nucleic acid extracts, while arsenic could not be detected and was below 1 per mil relative to phosphorus. Taken together, we conclude that GFAJ-1 is an arsenate-resistant, but still a phosphate-dependent, bacterium.

An extended suite of genetic tools for use in bacteria of the Halomonadaceae: an overview.

Halophilic gammaproteobacteria of the family Halomonadaceae (including the genera Aidingimonas, Carnimonas, Chromohalobacter, Cobetia, Halomonas, Halotalea, Kushneria, Modicisalibacter, Salinicola, and Zymobacter) have current and promising applications in biotechnology mainly as a source of compatible solutes (powerful stabilizers of biomolecules and cells, with exciting potentialities in biomedicine), salt-tolerant enzymes, biosurfactants, and extracellular polysaccharides, among other products. In addition, they display a number of advantages to be used as cell factories, alternative to conventional prokaryotic hosts like Escherichia coli or Bacillus, for the production of recombinant proteins: (1) their high salt tolerance decreases to a minimum the necessity for aseptic conditions, resulting in cost-reducing conditions, (2) they are very easy to grow and maintain in the laboratory, and their nutritional requirements are simple, and (3) the majority can use a large range of compounds as a sole carbon and energy source. In the last 15 years, the efforts of our group and others have made possible the genetic manipulation of this bacterial group. In this review, the most relevant and recent tools for their genetic manipulation are described, with emphasis on nucleic acid isolation procedures, cloning and expression vectors, genetic exchange mechanisms, mutagenesis approaches, reporter genes, and genetic expression analyses. Complementary sections describing the influence of salinity on the susceptibility of these bacteria to antimicrobials, as well as the growth media most routinely used and culture conditions, for these microorganisms, are also included.

Resistance of galactoside-terminated alkanethiol self-assembled monolayers to marine fouling organisms.

Self-assembled monolayers (SAMs) of galactoside-terminated alkanethiols have protein-resistance properties which can be tuned via the degree of methylation [Langmuir 2005, 21, 2971-2980]. Specifically, a partially methylated compound was more resistant to nonspecific protein adsorption than the hydroxylated or fully methylated counterparts. We investigate whether this also holds true for resistance to the attachment and adhesion of a range of marine species, in order to clarify to what extent resistance to protein adsorption correlates with the more complex adhesion of fouling organisms. The partially methylated galactoside-terminated SAM was further compared to a mixed monolayer of ω-substituted methyl- and hydroxyl-terminated alkanethiols with wetting properties and surface ratio of hydroxyl to methyl groups matching that of the galactoside. The settlement (initial attachment) and adhesion strength of four model marine fouling organisms were investigated, representing both micro- and macrofoulers; two bacteria (Cobetia marina and Marinobacter hydrocarbonoclasticus), barnacle cypris larvae (Balanus amphitrite), and algal zoospores (Ulva linza). The minimum in protein adsorption onto the partially methylated galactoside surface was partly reproduced in the marine fouling assays, providing some support for a relationship between protein resistance and adhesion of marine fouling organisms. The mixed alkanethiol SAM, which was matched in wettability to the partially methylated galactoside SAM, consistently showed higher settlement (initial attachment) of test organisms than the galactoside, implying that both wettability and surface chemistry are insufficient to explain differences in fouling resistance. We suggest that differences in the structure of interfacial water may explain the variation in adhesion to these SAMs.

Isolation and characterization of halophilic bacteria from Urmia Lake in Iran.

Urmia Lake is one of the most permanent hypersaline lakes in the world which is threatened by hypersalinity and serious dryness. In spite of its importance no paper has been published regarding bacterial community of this lake. Accordingly, the present study aimed to investigate the halophilic bacteria in the aforementioned lake. In so doing, thirty seven strains were isolated on six different culture media. The isolated strains were characterized using phenotypic and genotypic methods. Growth of the strains occurred at 2535 degrees C, pH 6-9 and 7 to 20% (w/v) NaCl indicating that most of the isolates were moderately halophiles. Catalase, oxidase and urease activities were found to be positive for the majority of the isolates. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolated bacteria belonged to two major taxa: Gammaproteobacteria (92%, including Salicola [46%], Pseudomonas [13.5%], Marinobacter [ 11%], Idiomarina [11%], and Halomonas [8%]) and Firmicutes (8%, including Bacillus [5%] and Halobacillus [3%]). In addition, a novel bacterium whose 16S rRNA gene sequence showed almost 98% sequence identity with the taxonomically troubled DSM 3050T, Halovibrio denitrificans HGD 3T and Halospina denitrificans HGD 1-3T, each, was isolated. 16S rRNA gene similarity levels along with phenotypic characteristics suggest that some of the isolated strains could be regarded as potential type strain for novel species, on which further studies are recommended.

Prevalence of endosymbionts in Bemisia tabaci populations and their in vivo sensitivity to antibiotics.

Bemisia tabaci can harbor both primary and secondary endosymbionts, and the specific endosymbionts can differ among different B. tabaci biotypes. This study determined (1) the prevalence of the primary endosymbiont Portiera aleyrodidarum and secondary endosymbionts Arsenophonus and Wolbachia in two invasive biotypes (B and Q) and one indigenous biotype (Cv) in China and (2) the in vivo effect of three antibiotics (tetracycline, ampicillin trihydrate, and rifampicin) against the endosymbionts; if an antibiotic substantially inhibits an endosymbiont, it could be used to determine the effect of that endosymbiont on B. tabaci. P. aleyrodidarum and Wolbachia were detected in all the three biotypes, while Arsenophonus was found only in the Q and Cv biotypes. P. aleyrodidarum was found in all tested individuals of the three biotypes. Infection rates of Wolbachia in the B, Cv, and Q biotypes were 58, 68, and 48%, respectively. The infection rate of Arsenophonus was 44% in the Q biotype but only 22% in the Cv biotype. The antibiotics failed to eliminate P. aleyrodidarum from any individual of the B, Cv, and Q biotypes but eliminated the secondary endosymbionts, Arsenophonus and Wolbachia, from 50 to 80% of the adult B. tabaci. The effect of the antibiotics depended on the species of endosymbiont, the antibiotic, the B. tabaci biotype, and various interactions between these factors. When used against Arsenophonus, the efficiency of rifampicin was better than ampicillin and tetracycline, regardless of B. tabaci biotype. When inactivating Wolbachia in Cv and Q biotypes, the efficiency tetracycline was better than ampicillin and rifampicin, and while the efficiency of tetracycline was better than rifampicin and ampicillin when they were used against Wolbachia in B biotype.

Bacterial assay for the rapid assessment of antifouling and fouling release properties of coatings and materials.

An assay has been developed to accurately quantify the growth and release behaviour of bacterial biofilms on several test reference materials and coatings, using the marine bacterium Cobetia marina as a model organism. The assay can be used to investigate the inhibition of bacterial growth and release properties of many surfaces when compared to a reference. The method is based upon the staining of attached bacterial cells with the nucleic acid-binding, green fluorescent SYTO 13 stain. A strong linear correlation exists between the fluorescence of the bacterial suspension measured (RFU) using a plate reader and the total bacterial count measured with epifluorescence microscopy. This relationship allows the fluorescent technique to be used for the quantification of bacterial cells attached to surfaces. As the bacteria proliferate on the surface over a period of time, the relative fluorescence unit (RFU) measured using the plate reader also shows an increase with time. This was observed on all three test surfaces (glass, Epikote and Silastic T2) over a period of 4 h of bacterial growth, followed by a release assay, which was carried out by the application of hydrodynamic shear forces using a custom-made rotary device. Different fixed rotor speeds were tested, and based on the release analysis, 12 knots was used to provide standard shear force. The assay developed was then applied for assessing three different antifouling coatings of different surface roughness. The novel assay allows the rapid and sensitive enumeration of attached bacteria directly on the coated surface. This is the first plate reader assay technique that allows estimation of irreversibly attached bacterial cells directly on the coated surface without their removal from the surface or extraction of a stain into solution.

The potential of nano-structured silicon oxide type coatings deposited by PACVD for control of aquatic biofouling.

SiO(x)-like coatings were deposited on glass slides from a hexamethylsiloxane precursor by plasma-assisted CVD (PACVD). Surface energies (23.1-45.7 mJ m(-1)) were correlated with the degree of surface oxidation and hydrocarbon contents. Tapping mode AFM revealed a range of surface topologies with Ra values 1.55-3.16 nm and RMS roughness 1.96-4.11 nm. Settlement of spores of the green alga Ulva was significantly less, and detachment under shear significantly more on the lowest surface energy coatings. Removal of young plants (sporelings) of Ulva under shear was positively correlated with reducing the surface energy of the coatings. The most hydrophobic coatings also showed good performance against a freshwater bacterium, Pseudomonas fluorescens, significantly reducing initial attachment and biofilm formation, and reducing the adhesion strength of attached bacterial cells under shear. Taken together the results indicate potential for further investigation of these coatings for applications such as heat exchangers and optical instruments.

[Resistance of the petroleum-oxidizing microorganism Dietzia sp. to hyperosmotic shock in reconstituted biofilms].

A number of halotolerant and halophilic bacterial strains were isolated from the Romashkinskoe oil field (Tatarstan) stratal waters having a salinity of up to 100 g/l. The isolation of pure cultures involved biofilm reconstitution on M9 medium with paraffins. The associations obtained were dispersed and reinoculated onto solid media that contained either peptone and yeast extract (PY) or paraffins. It was shown that such associations included both oil-oxidizing bacteria and accompanying chemoheterotrophic bacteria incapable of oil oxidation. The pure cultures that were isolated were used for creating binary biofilms. In these biofilms, interactions between halophilic and nonhalophilic bacteria under hypo- and hyperosmotic shocks were investigated. We conducted a detailed study of a biofilm obtained from an oil-oxidizing halotolerant species (with an upper growth limit of 10-12% NaCl) identified as Dietzia sp. and an extremely halophilic gram-negative bacterium (growing within the 5-20% NaCl concentration range) of the genus Chromohalobacter that did not oxidize paraffins. If these microorganisms were grown in a mixed suspension (planktonic) culture that was not supplemented with an additional amount of NaCl, no viable cells of the halophilic microorganism were detected after reinoculation. In contrast, only halophilic cells were detected at a NaCl concentration of 15%. Thus, no mutual protective influence of the microorganisms manifested itself in suspension culture, either under hypo- or under hyperosmotic shock. Neither could the halophile cells be detected after reinoculating a biofilm obtained on a peptone medium without addition of NaCl. However, biofilms produced at a NaCl concentration of 15% contained approximately equal numbers of cells of the halophilic and halotolerant organisms. Thus, the halophile in biofilms sustaining a hyperosmotic shock exerts a protective influence on the halotolerant microorganism. Preliminary data suggest that this effect is due to release by the halophile of osmoprotective substances (ectoine and glutamate), which are taken up by the halotolerant species. Such substances are diluted by a large medium volume in suspension cultures, whereas, in biofilms, their diffusion into the medium is apparently hampered by their interaction with the intercellular polymer matrix.

How to be moderately halophilic with broad salt tolerance: clues from the genome of Chromohalobacter salexigens.

We analyzed the amino acid composition of different categories of proteins of the moderately halophilic bacterium Chromohalobacter salexigens, as deduced from its genome sequence. Comparison with non-halophilic representatives of the gamma-Proteobacteria (Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae) shows only a slight excess of acidic residues in the cytoplasmic proteins, and no significant differences were found in the acidity of membrane-bound proteins. In contrast, a very pronounced difference in mean pI value was observed for the periplasmic binding proteins of the ABC transport systems of C. salexigens and the non-halophiles E. coli and P. aeruginosa. V. cholerae, which is adapted to life in brackish water, showed intermediate values. The findings suggest that there is a major difference between the proteins of the moderate halophile C. salexigens and non-halophilic bacteria in their periplasmic proteins, exemplified by the substrate binding proteins of transport systems. The highly acidic nature of these proteins may enable them to function at high salt concentrations. The evolution of highly salt-tolerant prokaryotes may have depended on an increase in acidity of the proteins located external to the cytoplasmic membrane, enabling effective transport of nutrients into the cell.

Salt-inducible multidrug efflux pump protein in the moderately halophilic bacterium Chromohalobacter sp.

It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained. We have addressed this question by identifying the gene responsible for multidrug resistance. A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp. strain 160 showed a two- to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane. The mutant produced an elevated level of the 58-kDa outer membrane protein. This mutant (160R) accumulated about one-third the level of EtBr that the parent cells did. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, caused a severalfold increase in the intracellular accumulation of EtBr, with the wild-type and mutant cells accumulating nearly equal amounts. The hrdC gene encoding the 58-kDa outer membrane protein has been cloned. Disruption of this gene rendered the cells hypersusceptible to antibiotics and EtBr and led to a high level of accumulation of intracellular EtBr. The primary structure of HrdC has a weak similarity to that of Escherichia coli TolC. Interestingly, both drug resistance and the expression of HrdC were markedly increased in the presence of a high salt concentration in the growth medium, but this was not observed in hrdC-disrupted cells. These results indicate that HrdC is the outer membrane component of the putative efflux pump assembly and that it plays a major role in the observed induction of drug resistance by salt in this bacterium.