Halophilic bacteria in a Lake Michigan drainage basin as potential biological indicators of chloride-impacted freshwaters.

There are few biological indicators for freshwater systems subjected to high chloride levels. Freshwater systems receive many forms of chloride such as road salts (e.g., NaCl, CaCl2, MgCl2), fertilizers (e.g., KCl), and year-round water softener pollution. The goal our study was to investigate Halomonadaceae populations as prospective biological indicators of chloride-impacted freshwaters. The bacterial family Halomonadaceae are halophiles that generally require the presence of salt to survive, which make them an attractive candidate in determining chloride impaired areas. Field sediment surveys assessed how salt tolerant and halophilic bacteria abundance corresponded to chloride and conductivity measurements. Colony forming unit (CFU) counts on modified M9 6% NaCl plates (w/v) at urbanized sites compared to the rural sites had highest counts during winter and spring when chloride concentrations were also highest. Select isolates identified as Halomonadaceae through 16S rRNA sequencing were kept as active cultures to determine the NaCl concentration and temperature preference that resulted in the isolates optimal growth. Isolates tested under 5 °C (cold) grew optimally in 2 % NaCl (w/v), whereas under 18 °C (warm), isolates showed optimal growth at 6 % NaCl. The majority of isolates had maximum growth in the warmer temperature, however, select isolates grew better in the cold temperature. Culture-independent methods were used and identified Halomonadaceae were widespread and permeant members of the microbial community in a Lake Michigan drainage basin. Quantitative polymerase chain reaction (qPCR) targeting Halomonadaceae genera demonstrated that abundance varied by site, but overall were present throughout the year. However, community sequencing revealed there were a large relative proportion of specific Halomonadaceae populations present in winter versus summer. Methods targeting salt tolerant bacteria and specific members of Halomonadaceae appears to be a promising approach to assess chloride-impacted areas to better understand the long-term ecological impacts as we continue to salinize freshwater resources.

High natural PHA production from acetate in Cobetia sp. MC34 and Cobetia marina DSM 4741T and in silico analyses of the genus specific PhaC2 polymerase variant.

BACKGROUND: Several members of the bacterial Halomonadacea family are natural producers of polyhydroxyalkanoates (PHA), which are promising materials for use as biodegradable bioplastics. Type-strain species of Cobetia are designated PHA positive, and recent studies have demonstrated relatively high PHA production for a few strains within this genus. Industrially relevant PHA producers may therefore be present among uncharacterized or less explored members. In this study, we characterized PHA production in two marine Cobetia strains. We further analyzed their genomes to elucidate pha genes and metabolic pathways which may facilitate future optimization of PHA production in these strains. RESULTS: Cobetia sp. MC34 and Cobetia marina DSM 4741T were mesophilic, halotolerant, and produced PHA from four pure substrates. Sodium acetate with- and without co-supplementation of sodium valerate resulted in high PHA production titers, with production of up to 2.5 g poly(3-hydroxybutyrate) (PHB)/L and 2.1 g poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/L in Cobetia sp. MC34, while C. marina DSM 4741T produced 2.4 g PHB/L and 3.7 g PHBV/L. Cobetia marina DSM 4741T also showed production of 2.5 g PHB/L from glycerol. The genome of Cobetia sp. MC34 was sequenced and phylogenetic analyses revealed closest relationship to Cobetia amphilecti. PHA biosynthesis genes were located at separate loci similar to the arrangement in other Halomonadacea. Further genome analyses revealed some differences in acetate- and propanoate metabolism genes between the two strains. Interestingly, only a single PHA polymerase gene (phaC2) was found in Cobetia sp. MC34, in contrast to two copies (phaC1 and phaC2) in C. marina DSM 4741T. In silico analyses based on phaC genes show that the PhaC2 variant is conserved in Cobetia and contains an extended C-terminus with a high isoelectric point and putative DNA-binding domains. CONCLUSIONS: Cobetia sp. MC34 and C. marina DSM 4741T are natural producers of PHB and PHBV from industrially relevant pure substrates including acetate. However, further scale up, optimization of growth conditions, or use of metabolic engineering is required to obtain industrially relevant PHA production titers. The putative role of the Cobetia PhaC2 variant in DNA-binding and the potential implications remains to be addressed by in vitro- or in vivo methods.

Isolation and characterization of a halophilic Modicisalibacter sp. strain Wilcox from produced water.

We report the isolation a halophilic bacterium that degrades both aromatic and aliphatic hydrocarbons as the sole sources of carbon at high salinity from produced water. Phylogenetic analysis of 16S rRNA-gene sequences shows the isolate is a close relative of Modicisalibacter tunisiensis isolated from an oil-field water in Tunisia. We designate our isolate as Modicisalibacter sp. strain Wilcox. Genome analysis of strain Wilcox revealed the presence of a repertoire of genes involved in the metabolism of aliphatic and aromatic hydrocarbons. Laboratory culture studies corroborated the predicted hydrocarbon degradation potential. The strain degraded benzene, toluene, ethylbenzene, and xylenes at salinities ranging from 0.016 to 4.0 M NaCl, with optimal degradation at 1 M NaCl. Also, the strain degraded phenol, benzoate, biphenyl and phenylacetate as the sole sources of carbon at 2.5 M NaCl. Among aliphatic compounds, the strain degraded n-decane and n-hexadecane as the sole sources of carbon at 2.5 M NaCl. Genome analysis also predicted the presence of many heavy metal resistance genes including genes for metal efflux pumps, transport proteins, and enzymatic detoxification. Overall, due to its ability to degrade many hydrocarbons and withstand high salt and heavy metals, strain Wilcox may prove useful for remediation of produced waters.

Portiera Gets Wild: Genome Instability Provides Insights into the Evolution of Both Whiteflies and Their Endosymbionts.

Whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) are a superfamily of small phloem-feeding insects. They rely on their primary endosymbionts "Candidatus Portiera aleyrodidarum" to produce essential amino acids not present in their diet. Portiera has been codiverging with whiteflies since their origin and therefore reflects its host's evolutionary history. Like in most primary endosymbionts, the genome of Portiera stays stable across the Aleyrodidae superfamily after millions of years of codivergence. However, Portiera of the whitefly Bemisia tabaci has lost the ancestral genome order, reflecting a rare event in the endosymbiont evolution: the appearance of genome instability. To gain a better understanding of Portiera genome evolution, identify the time point in which genome instability appeared and contribute to the reconstruction of whitefly phylogeny, we developed a new phylogenetic framework. It targeted five Portiera genes and determined the presence of the DNA polymerase proofreading subunit (dnaQ) gene, previously associated with genome instability, and two alternative gene rearrangements. Our results indicated that Portiera gene sequences provide a robust tool for studying intergenera phylogenetic relationships in whiteflies. Using these new framework, we found that whitefly species from the Singhiella, Aleurolobus, and Bemisia genera form a monophyletic tribe, the Aleurolobini, and that their Portiera exhibit genome instability. This instability likely arose once in the common ancestor of the Aleurolobini tribe (at least 70 Ma), drawing a link between the appearance of genome instability in Portiera and the switch from multibacteriocyte to a single-bacteriocyte mode of inheritance in this tribe.

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.

Nature lessons: The whitefly bacterial endosymbiont is a minimal amino acid factory with unusual energetics.

Reductive genome evolution is a universal phenomenon observed in endosymbiotic bacteria in insects. As the genome reduces its size and irreversibly losses coding genes, the functionalities of the cell system, including the energetics processes, are more restricted. Several energetic pathways can also be lost. How do these reduced metabolic networks sustain the energy needs of the system? Among the bacteria with reduced genomes Candidatus Portiera aleyrodidarum, obligate endosymbiont of whiteflies, represents an extreme case since lacks several key mechanisms for ATP generation. Thus, to analyze the cell energetics in this system, a genome-scale metabolic model of this endosymbiont was constructed, and its energy production capabilities dissected using stoichiometric analysis. Our results suggest that energy generation is coupled to the synthesis of essential amino acids and carotenoids, crucial metabolites in the symbiotic association. A deeper insight showed that ATP production via carotenoid synthesis is also connected with amino acid production. This unusual association of energy production with anabolism suggests that, although minimized, endosymbiont metabolic networks maintain a remarkable biosynthetic potential.

Metabolic Coevolution in the Bacterial Symbiosis of Whiteflies and Related Plant Sap-Feeding Insects.

Genomic decay is a common feature of intracellular bacteria that have entered into symbiosis with plant sap-feeding insects. This study of the whitefly Bemisia tabaci and two bacteria (Portiera aleyrodidarum and Hamiltonella defensa) cohoused in each host cell investigated whether the decay of Portiera metabolism genes is complemented by host and Hamiltonella genes, and compared the metabolic traits of the whitefly symbiosis with other sap-feeding insects (aphids, psyllids, and mealybugs). Parallel genomic and transcriptomic analysis revealed that the host genome contributes multiple metabolic reactions that complement or duplicate Portiera function, and that Hamiltonella may contribute multiple cofactors and one essential amino acid, lysine. Homologs of the Bemisia metabolism genes of insect origin have also been implicated in essential amino acid synthesis in other sap-feeding insect hosts, indicative of parallel coevolution of shared metabolic pathways across multiple symbioses. Further metabolism genes coded in the Bemisia genome are of bacterial origin, but phylogenetically distinct from Portiera, Hamiltonella and horizontally transferred genes identified in other sap-feeding insects. Overall, 75% of the metabolism genes of bacterial origin are functionally unique to one symbiosis, indicating that the evolutionary history of metabolic integration in these symbioses is strongly contingent on the pattern of horizontally acquired genes. Our analysis, further, shows that bacteria with genomic decay enable host acquisition of complex metabolic pathways by multiple independent horizontal gene transfers from exogenous bacteria. Specifically, each horizontally acquired gene can function with other genes in the pathway coded by the symbiont, while facilitating the decay of the symbiont gene coding the same reaction.

Genome evolution in the primary endosymbiont of whiteflies sheds light on their divergence.

Whiteflies are important agricultural insect pests, whose evolutionary success is related to a long-term association with a bacterial endosymbiont, Candidatus Portiera aleyrodidarum. To completely characterize this endosymbiont clade, we sequenced the genomes of three new Portiera strains covering the two extant whitefly subfamilies. Using endosymbiont and mitochondrial sequences we estimated the divergence dates in the clade and used these values to understand the molecular evolution of the endosymbiont coding sequences. Portiera genomes were maintained almost completely stable in gene order and gene content during more than 125 Myr of evolution, except in the Bemisia tabaci lineage. The ancestor had already lost the genetic information transfer autonomy but was able to participate in the synthesis of all essential amino acids and carotenoids. The time of divergence of the B. tabaci complex was much more recent than previous estimations. The recent divergence of biotypes B (MEAM1 species) and Q (MED species) suggests that they still could be considered strains of the same species. We have estimated the rates of evolution of Portiera genes, synonymous and nonsynonymous, and have detected significant differences among-lineages, with most Portiera lineages evolving very slowly. Although the nonsynonymous rates were much smaller than the synonymous, the genomic dN/dS ratios were similar, discarding selection as the driver of among-lineage variation. We suggest variation in mutation rate and generation time as the responsible factors. In conclusion, the slow evolutionary rates of Portiera may have contributed to its long-term association with whiteflies, avoiding its replacement by a novel and more efficient endosymbiont.

Direct ethanol production from cellulosic materials by Zymobacter palmae carrying Cellulomonas endoglucanase and Ruminococcus ß-glucosidase genes.

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we conferred the ability to ferment cellulosic materials directly on Zymobacter palmae by co-expressing foreign endoglucanase and ß-glucosidase genes. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, the six genes encoding the cellulolytic enzymes (CenA, CenB, CenD, CbhA, CbhB, and Cex) from Cellulomonas fimi were introduced and expressed in Z. palmae. Of these cellulolytic enzyme genes cloned, CenA degraded carboxymethylcellulose and phosphoric acid-swollen cellulose (PASC) efficiently. The extracellular CenA catalyzed the hydrolysis of barley ß-glucan and PASC to liberate soluble cello-oligosaccharides, indicating that CenA is the most suitable enzyme for cellulose degradation among those cellulolytic enzymes expressed in Z. palmae. Furthermore, the cenA gene and ß-glucosidase gene (bgl) from Ruminococcus albus were co-expressed in Z. palmae. Of the total endoglucanase and ß-glucosidase activities, 57.1 and 18.1 % were localized in the culture medium of the strain. The genetically engineered strain completely saccharified and fermented 20 g/l barley ß-glucan to ethanol within 84 h, producing 79.5 % of the theoretical yield. Thus, the production and secretion of CenA and BGL enabled Z. palmae to efficiently ferment a water-soluble cellulosic polysaccharide to ethanol.

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.

The hydrocarbon-degrading marine bacterium Cobetia sp. strain MM1IDA2H-1 produces a biosurfactant that interferes with quorum sensing of fish pathogens by signal hijacking.

Biosurfactants are produced by hydrocarbon-degrading marine bacteria in response to the presence of water-insoluble hydrocarbons. This is believed to facilitate the uptake of hydrocarbons by bacteria. However, these diffusible amphiphilic surface-active molecules are involved in several other biological functions such as microbial competition and intra- or inter-species communication. We report the isolation and characterization of a marine bacterial strain identified as Cobetia sp. MM1IDA2H-1, which can grow using the sulfur-containing heterocyclic aromatic hydrocarbon dibenzothiophene (DBT). As with DBT, when the isolated strain is grown in the presence of a microbial competitor, it produces a biosurfactant. Because the obtained biosurfactant was formed by hydroxy fatty acids and extracellular lipidic structures were observed during bacterial growth, we investigated whether the biosurfactant at its critical micelle concentration can interfere with bacterial communication systems such as quorum sensing. We focused on Aeromonas salmonicida subsp. salmonicida, a fish pathogen whose virulence relies on quorum sensing signals. Using biosensors for quorum sensing based on Chromobacterium violaceum and Vibrio anguillarum, we showed that when the purified biosurfactant was mixed with N-acyl homoserine lactones produced by A. salmonicida, quorum sensing was inhibited, although bacterial growth was not affected. In addition, the transcriptional activities of A. salmonicida virulence genes that are controlled by quorum sensing were repressed by both the purified biosurfactant and the growth in the presence of Cobetia sp. MM1IDA2H-1. We propose that the biosurfactant, or the lipid structures interact with the N-acyl homoserine lactones, inhibiting their function. This could be used as a strategy to interfere with the quorum sensing systems of bacterial fish pathogens, which represents an attractive alternative to classical antimicrobial therapies in fish aquaculture.

[Taxonomical status of the psychrotolerant Antarctic microorganisms].

The aerobic chemoorganotrophic bacteria, dominating in soils and phytocenosis of the Antarctic Region, on combination of morphological and biochemical properties belong to several taxons of Bacteria domain. Gram-negative strains 3189, 3415 (fam. Halomonadaceae, Halomonas sp.) and 3088, 3468, 3469 (fam. Moraxellaceae, Psychrobacter sp.) belong to phylum Proteobacteria, to class Gammaproteobacteria. Gram-negative strains 3294 3392 (Rhizobiales, fam. Methylobacteriaceae, Methylobacterium sp.) relate to class Alphaproteobacteria of this phylum. Gram-positive strains 3179, 3275, 3470, 3471 (fam. Microbacteriaceae, Cryobacterium sp.), 3054, 3058, 3411 (fam. Corynebacteriaceae, Corynebacterium sp.) and 3194, 3398 (fam. Micrococcaceae, Micrococcus sp.) relate to phylum Actinobacteria, class Actinobacteria. Thus, the psychrophilic and psychrotolerant Antarctic bacteria (aerobic chemoorganotrophic) isolated from phytocenosis and soils of polar region are characterized by wide taxonomic variety.

Complete genome sequence of "Candidatus Portiera aleyrodidarum" BT-QVLC, an obligate symbiont that supplies amino acids and carotenoids to Bemisia tabaci.

The genome of "Candidatus Portiera aleyrodidarum," the primary endosymbiont of the whitefly Bemisia tabaci (Mediterranean species), is reported. It presents a reduced genome (357 kb) encoding the capability to synthetize, or participate in the synthesis of, several amino acids and carotenoids, being the first insect endosymbiont capable of supplying carotenoids.

Expression and surface display of Cellulomonas endoglucanase in the ethanologenic bacterium Zymobacter palmae.

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we developed a tool for cell surface display of cellulolytic enzymes on the ethanologenic bacterium Zymobacter palmae. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, to express and display heterologous cellulolytic enzymes on the Z. palmae cell surface, we utilized the cell-surface display motif of the Pseudomonas ice nucleation protein Ina. The gene encoding Ina from Pseudomonas syringae IFO3310 was cloned, and its product was comprised of three functional domains: an N-terminal domain, a central domain with repeated amino acid residues, and a C-terminal domain. The N-terminal domain of Ina was shown to function as the anchoring motif for a green fluorescence protein fusion protein in Escherichia coli. To express a heterologous cellulolytic enzyme extracellularly in Z. palmae, we fused the N-terminal coding sequence of Ina to the coding sequence of an N-terminal-truncated Cellulomonas endoglucanase. Z. palmae cells carrying the fusion endoglucanase gene were shown to degrade carboxymethyl cellulose. Although a portion of the expressed fusion endoglucanase was released from Z. palmae cells into the culture broth, we confirmed the display of the protein on the cell surface by immunofluorescence microscopy. The results indicate that the N-terminal anchoring motif of Ina from P. syringae enabled the translocation and display of the heterologous cellulase on the cell surface of Z. palmae.

Endosymbiotic bacteria as a source of carotenoids in whiteflies.

Although carotenoids serve important biological functions, animals are generally unable to synthesize these pigments and instead obtain them from food. However, many animals, such as sap-feeding insects, may have limited access to carotenoids in their diet, and it was recently shown that aphids have acquired the ability to produce carotenoids by lateral transfer of fungal genes. Whiteflies also contain carotenoids but show no evidence of the fungus-derived genes found in aphids. Because many sap-feeding insects harbour intracellular bacteria, it has long been hypothesized that these endosymbionts could serve as an alternative source of carotenoid biosynthesis. We sequenced the genome of the obligate bacterial endosymbiont Portiera from the whitefly Bemisia tabaci. The genome exhibits typical signatures of obligate endosymbionts in sap-feeding insects, including extensive size reduction (358.2 kb) and enrichment for genes involved in essential amino acid biosynthesis. Unlike other sequenced insect endosymbionts, however, Portiera has bacterial homologues of the fungal carotenoid biosynthesis genes in aphids. Therefore, related lineages of sap-feeding insects appear to have convergently acquired the same functional trait by distinct evolutionary mechanisms-bacterial endosymbiosis versus fungal lateral gene transfer.

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.

Absence of detectable arsenate in DNA from arsenate-grown GFAJ-1 cells.

A strain of Halomonas bacteria, GFAJ-1, has been claimed to be able to use arsenate as a nutrient when phosphate is limiting and to specifically incorporate arsenic into its DNA in place of phosphorus. However, we have found that arsenate does not contribute to growth of GFAJ-1 when phosphate is limiting and that DNA purified from cells grown with limiting phosphate and abundant arsenate does not exhibit the spontaneous hydrolysis expected of arsenate ester bonds. Furthermore, mass spectrometry showed that this DNA contains only trace amounts of free arsenate and no detectable covalently bound arsenate.

"Artifactual" arsenate DNA.

The recent claim by Wolfe-Simon et al. that the Halomonas bacterial strain GFAJ-1 when grown in arsenate-containing medium with limiting phosphate is able to substitute phosphate with arsenate in biomolecules including nucleic acids and in particular DNA(1) arose much skepticism, primarily due to the very limited chemical stability of arsenate esters (see ref. 2 and references therein). A major part of the criticisms was concerned with the insufficient (bio)chemical evidence in the Wolfe-Simon study for the actual chemical incorporation of arsenate in DNA (and/or RNA). Redfield et al. now present evidence that the identification of arsenate DNA was artifactual.

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.

Biofilm formation in moderately halophilic bacteria is influenced by varying salinity levels.

Bacteria in a biofilm have a co-dependent lifestyle resulting in a harmonized and complex coordination of the bacterial cells within an exopolysaccharide (EPS) matrix. We hypothesized that biofilm formation and EPS production in salt-tolerant bacteria are helpful for plant growth improvement in saline soil, but that they are influenced differently. To investigate this hypothesis, we tested the effect of different salinity levels on the biofilm formation of the bacterial strains PAa6 (Halomonas meridiana), HT2 (Kushneria indalinina) and ST2 (Halomonas aquamarina) on different abiotic and biotic surfaces. Maximum biofilm formation was established at 1 M salt concentration. However, EPS production was maximal at 0-1 M NaCl stress. We also studied the effect of salt stress on EPS produced by the bacterial strains and confirmed the presence of EPS on Cicer arietinum var. CM 98 roots and in soil at different salinity levels, using Alcian blue staining. Overall, the strain PAa6 was more effective in biofilm formation and EPS production. Under saline and non-saline conditions, this strain also colonized the plant roots more efficiently as compared to the other two strains. We conclude that the strain PAa6 has the potential of biofilm formation and EPS production at different salinity levels. The presence of EPS in the biofilm helped the bacterial strains to better colonize the roots.