Ability of Pseudoalteromonas tunicata to colonize natural biofilms and its effect on microbial community structure.

We investigated the effectiveness of surface colonization by the epiphytic marine bacterium Pseudoalteromonas tunicata firstly on a complex biofilm community on glass slides, and secondly, on the epiphytic community of Ulva australis. The effectiveness of P. tunicata was compared with the performance of Phaeobacter sp. 2.10, also a marine epiphytic isolate in the U. australis colonization experiments. Pseudoalteromonas tunicata cells were able to colonize the glass slide community at densities found naturally in the water column (9.7 x 10(4) cells mL(-1)). However, P. tunicata was a poor invader of the epiphytic community on U. australis at densities of 10(6) cells mL(-1). At densities of 10(8) cells mL(-1), P. tunicata again exerted little impact on the epiphytic community. Phaeobacter sp. 2.10 was also a poor invader at lower densities, but was able to invade and become dominant at densities of 10(8) cells mL(-1). Differences in the ability of P. tunicata and Phaeobacter sp. 2.10 to invade natural communities may be due to differences in the antibacterial compounds produced by the two species. These experiments suggest that epiphytic communities may have protective effects compared with inanimate surfaces.

Variability and abundance of the epiphytic bacterial community associated with a green marine Ulvacean alga.

Marine Ulvacean algae are colonized by dense microbial communities predicted to have an important role in the development, defense and metabolic activities of the plant. Here we assess the diversity and seasonal dynamics of the bacterial community of the model alga Ulva australis to identify key groups within this epiphytic community. A total of 48 algal samples of U. australis that were collected as 12 individuals at 3 monthly intervals, were processed by applying denaturing gradient gel electrophoresis (DGGE), and three samples from each season were subjected to catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). CARD-FISH revealed that the epiphytic microbial community was comprised mainly of bacterial cells (90%) and was dominated by the groups Alphaproteobacteria (70%) and Bacteroidetes (13%). A large portion (47%) of sequences from the Alphaproteobacteria fall within the Roseobacter clade throughout the different seasons, and an average relative proportion of 19% was observed using CARD-FISH. DGGE based spatial (between tidal pools) and temporal (between season) comparisons of bacterial community composition demonstrated that variation occurs. Between individuals from both the same and different tidal pools, the variation was highest during winter (30%) and between seasons a 40% variation was observed. The community also includes a sub-population of bacteria that is consistently present. Sequences from excised DGGE bands indicate that members of the Alphaproteobacteria and the Bacteroidetes are part of this stable sub-population, and are likely to have an important role in the function of this marine epiphytic microbial community.

Antimicrobial activity observed among cultured marine epiphytic bacteria reflects their potential as a source of new drugs.

The surfaces of marine eukaryotes provide a unique habitat for colonizing microorganisms where competition between members of these communities and chemically mediated interactions with their host are thought to influence both microbial diversity and function. For example, it is believed that marine eukaryotes may use their surface-associated bacteria to produce bioactive compounds in defence against competition and to protect the host against further colonization. With the increasing need for novel drug discovery, marine epibiotic bacteria may thus represent a largely underexplored source of new antimicrobial compounds. In the current study, 325 bacterial isolates were obtained from the surfaces of marine algae Delisea pulchra and Ulva australis. Thirty-nine showed to have antimicrobial activity and were identified via 16S rRNA gene sequencing. The majority of those isolates belonged to Alpha- and Gammaproteobacteria. Interestingly, the most commonly isolated bacterial strain, Microbulbifer sp., from the surface of D. pulchra has previously been described as an ecologically significant epibiont of different marine eukaryotes. Other antimicrobial isolates obtained in this study belonged to the phyla Actinobacteria, Firmicutes and Bacteroidetes. Phylogenetically, little overlap was observed among the bacteria obtained from surfaces of D. pulchra and U. australis. The high abundance of cultured isolates that produce antimicrobials suggest that culturing remains a powerful resource for exploring novel bioactives of bacterial origin.

Low densities of epiphytic bacteria from the marine alga Ulva australis inhibit settlement of fouling organisms.

Bacteria that produce inhibitory compounds on the surface of marine algae are thought to contribute to the defense of the host plant against colonization of fouling organisms. However, the number of bacterial cells necessary to defend against fouling on the plant surface is not known. Pseudoalteromonas tunicata and Phaeobacter sp. strain 2.10 (formerly Roseobacter gallaeciensis) are marine bacteria often found in association with the alga Ulva australis and produce a range of extracellular inhibitory compounds against common fouling organisms. P. tunicata and Phaeobacter sp. strain 2.10 biofilms with cell densities ranging from 10(2) to 10(8) cells cm(-2) were established on polystyrene petri dishes. Attachment and settlement assays were performed with marine fungi (uncharacterized isolates from U. australis), marine bacteria (Pseudoalteromonas gracilis, Alteromonas sp., and Cellulophaga fucicola), invertebrate larvae (Bugula neritina), and algal spores (Polysiphonia sp.) and gametes (U. australis). Remarkably low cell densities (10(2) to 10(3) cells cm(-2)) of P. tunicata were effective in preventing settlement of algal spores and marine fungi in petri dishes. P. tunicata also prevented settlement of invertebrate larvae at densities of 10(4) to 10(5) cells cm(-2). Similarly, low cell densities (10(3) to 10(4)cells cm(-2)) of Phaeobacter sp. strain 2.10 had antilarval and antibacterial activity. Previously, it has been shown that abundance of P. tunicata on marine eukaryotic hosts is low (<1 x 10(3) cells cm(-2)) (T. L. Skovhus et al., Appl. Environ. Microbiol. 70:2373-2382, 2004). Despite such low numbers of P. tunicata on U. australis in situ, our data suggest that P. tunicata and Phaeobacter sp. strain 2.10 are present in sufficient quantities on the plant to inhibit fouling organisms. This strongly supports the hypothesis that P. tunicata and Phaeobacter sp. strain 2.10 can play a role in defense against fouling on U. australis at cell densities that commonly occur in situ.

Inhibition of fouling by marine bacteria immobilised in kappa-carrageenan beads.

Antifouling solutions that leave little or no impact in the world's oceans are constantly being sought. This study employed the immobilisation of the antifouling bacterium Pseudoalteromonas tunicata in kappa-carrageenan to demonstrate how a surface may be protected from fouling by bacteria, i.e. a 'living paint'. Attempts so far to produce a 'living paint' have been limited in both longevity of effectiveness and demonstration of applicability, most noticeably regarding the lack of any field data. Here survival of bacteria immobilised in kappa-carrageenan for 12 months in the laboratory is demonstrated and evidence presented for inhibition of fouling for up to 7 weeks in the field (Sydney Harbour, NSW, Australia).

Molecular investigation of the distribution, abundance and diversity of the genus Pseudoalteromonas in marine samples.

The genus Pseudoalteromonas has attracted interest because it has frequently been found in association with eukaryotic hosts, and because many Pseudoalteromonas species produce biologically active compounds. One distinct group of Pseudoalteromonas species is the antifouling subgroup containing Pseudoalteromonas tunicata and Ps. ulvae, which both produce extracellular compounds that inhibit growth and colonization by different marine organisms. PCR primers targeting the 16S rRNA gene of the genus Pseudoalteromonas and the antifouling subgroup were developed and applied in this study. Real-time quantitative PCR (qPCR) was applied to determine the relative bacterial abundance of the genus and the antifouling subgroup, and denaturing gradient gel electrophoresis (DGGE) was applied to study the diversity of the genus in 11 different types of marine samples from Danish coastal waters. The detection of Ps. tunicata that contain the antifouling subgroup was achieved through specific PCR amplification of the antibacterial protein gene (alpP). The Pseudoalteromonas species accounted for 1.6% of the total bacterial abundance across all samples. The Pseudoalteromonas diversity on the three unfouled marine organisms Ciona intestinalis, Ulva lactuca and Ulvaria fusca was found to be low, and Ps. tunicata was only detected on these three hosts, which all contain accessible cellulose polymers in their cell walls.

Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies.

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such "heterogeneity hot spots" occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.

A mannose-sensitive haemagglutinin (MSHA)-like pilus promotes attachment of Pseudoalteromonas tunicata cells to the surface of the green alga Ulva australis.

This study demonstrates that attachment of the marine bacterium Pseudoalteromonas tunicata to the cellulose-containing surface of the green alga Ulva australis is mediated by a mannose-sensitive haemagglutinin (MSHA-like) pilus. We have identified an MSHA pilus biogenesis gene locus in P. tunicata, termed msh/1/2JKLMNEGFBACDOPQ, which shows significant homology, with respect to its genetic characteristics and organization, to the MSHA pilus biogenesis gene locus of Vibrio cholerae. Electron microscopy studies revealed that P. tunicata wild-type cells express flexible pili peritrichously arranged on the cell surface. A P. tunicata mutant (SM5) with a transposon insertion in the mshJ region displayed a non-piliated phenotype. Using SM5, it has been demonstrated that the MSHA pilus promotes attachment of P. tunicata wild-type cells in polystyrene microtitre plates, as well as to microcrystalline cellulose and to the living surface of U. australis. P. tunicata also demonstrated increased pilus production in response to cellulose and its monomer constituent cellobiose. The MSHA pilus thus functions as a determinant of attachment in P. tunicata, and it is proposed that an understanding of surface sensing mechanisms displayed by P. tunicata will provide insight into specific ecological interactions that occur between this bacterium and higher marine organisms.

A CARD-FISH protocol for the identification and enumeration of epiphytic bacteria on marine algae.

A CARD-FISH protocol was developed and applied to analyse surface-associated bacteria on the marine algae Ulva lactuca, Delisea pulchra, Corallina officinalis, Amphiroa anceps, Porphyra sp. and Sargassum linearifolium. The combination of Alexa(546)-labelled tyramide as the reporter molecule with SYBR Green II counterstain allowed for superior detection of the hybridised probe fluorescence against plant tissue from which pigment autofluorescence has been reduced.

Isolation and structure elucidation of a novel yellow pigment from the marine bacterium Pseudoalteromonas tunicata.

The marine environment is a major source for many novel natural compounds. A new yellow pigment has been isolated from the marine bacterium P. tunicata and identified as a new member of the tambjamine class of compounds. The structural identification was achieved by a combination of 1D and 2D-NMR spectroscopy and high resolution mass spectrometry data.

Real-time quantitative PCR for assessment of abundance of Pseudoalteromonas species in marine samples.

A real-time quantitative PCR (RTQ-PCR) method for measuring the abundance of Pseudoalteromonas species in marine samples is presented. PCR primers targeting a Pseudoalteromonas-specific region of the 16S rRNA gene were tested at three different levels using database searches (in silico), a selection of pure cultures (in vitro), and a combined denaturing gradient gel electrophoresis and cloning approach on environmental DNA (in situ). The RTQ-PCR method allowed for the detection of SYBR Green fluorescence from double-stranded DNA over a linear range spanning six orders of magnitude. The detection limit was determined as 1.4 fg of target DNA (1,000 gene copies) measured in the presence of 20 ng of nontarget DNA from salmon testes. In this study, we discuss the importance of robust post-PCR analyses to overcome pitfalls in RTQ-PCR when samples from different complex marine habitats are analyzed and compared on a nonroutine basis. Representatives of the genus Pseudoalteromonas were detected in samples from all investigated habitats, suggesting a widespread distribution of this genus across many marine habitats (e.g., seawater, rocks, macroalgae, and marine animals). Three sample types were analyzed by RTQ-PCR to determine the relative abundance of Pseudoalteromonas ribosomal DNA (rDNA) compared to the total abundance of eubacterial rDNA. The rDNA fractions of Pseudoalteromonas compared to all Eubacteria were 1.55% on the green alga Ulva lactuca, 0.10% on the tunicate Ciona intestinalis, and 0.06% on the green alga Ulvaria fusca.

Impact of oil contamination and biostimulation on the diversity of indigenous bacterial communities in soil microcosms.

The aim of this study was to analyse the effect of oil contamination and biostimulation (soil pH raise, and nitrogen, phosphate and sulphur addition) on the diversity of a bacterial community of an acidic Cambisol under Atlantic Forest. The experiment was based on the enumeration of bacterial populations and hydrocarbon degraders in microcosms through the use of conventional plating techniques and molecular fingerprinting of samples directly from the environment. PCR followed by denaturing gradient gel electrophoresis (DGGE) was used to generate microbial community fingerprints employing 16S rRNA gene as molecular marker. Biostimulation led to increases of soil pH (to 7.0) and of the levels of phosphorus and K, Ca, and Mg. Oil contamination caused an increase in soil organic carbon (170-190% higher than control soil). Total bacterial counts were stable throughout the experiment, while MPN counts of hydrocarbon degraders showed an increase in the biostimulated and oil-contaminated soil samples. Molecular fingerprinting performed with 16S rRNA gene PCR and DGGE analysis revealed stable patterns along the 360 days of experiment, showing little change in oil-contaminated microcosms after 90 days. The DGGE patterns of the biostimulated samples showed severe changes due to decreases in the number of bands as compared to the control samples as from 15 days after addition of nutrients to the soil. Results obtained in the present study indicate that the addition of inorganic compounds to soil in conjunction with oil contamination has a greater impact on the bacterial community than oil contamination only.

Correlation between pigmentation and antifouling compounds produced by Pseudoalteromonas tunicata.

Pseudoalteromonas tunicata is a marine bacterium with the ability to prevent biofouling by the production of at least four target-specific compounds. In addition to these antifouling compounds, P. tunicata produces at least two pigments. These include a yellow and a purple pigment which, when combined, give the bacterium a dark green appearance. Transposon mutagenesis was used in this study to investigate the correlation between pigment production and the expression of specific antifouling phenotypes in P. tunicata. Four different categories of pigmentation mutants were isolated including yellow, dark-purple, light-purple and white mutants. The mutants were tested for their ability to inhibit the settlement of invertebrate larvae, algal spore germination, fungal growth and bacterial growth. The results showed that the yellow-pigmented mutants retained full antifouling activity, whereas the purple and white mutant strains had lost some, or all, of their ability to inhibit target organisms. This demonstrates that the loss of antifouling capabilities correlates with the loss of yellow pigment and not purple pigment. Sequencing and analysis of the genes disrupted by the transposons in these mutants identified a number of potential biosynthetic enzymes and transport systems involved in the synthesis and regulation of pigmentation and fouling inhibitors in this organism.

Antifouling activities expressed by marine surface associated Pseudoalteromonas species.

Abstract Members of the marine bacterial genus Pseudoalteromonas have been found in association with living surfaces and are suggested to produce bioactive compounds against settlement of algal spores, invertebrate larvae, bacteria and fungi. To determine the extent by which these antifouling activities and the production of bioactive compounds are distributed amongst the members of the genus Pseudoalteromonas, 10 different Pseudoalteromonas species mostly derived from different host organisms were tested in a broad range of biofouling bioassays. These assays included the settlement of larvae of two ubiquitous invertebrates Hydroides elegans and Balanus amphitrite as well as the settlement of spores of the common fouling algae Ulva lactuca and Polysiphonia sp. The growth of bacteria and fungi, which are the initial fouling organisms on marine surfaces, was also assayed in the presence of each of the 10 Pseudoalteromonas species. It was found that most members of this genus produced a variety of bioactive compounds. The broadest range of inhibitory activities was expressed by Pseudoalteromonas tunicata which inhibited all target fouling organisms. Only two species, Pseudoalteromonas haloplanktis and Pseudoalteromonas nigrifaciens, displayed negligible activity in the bioassays. These were also the only two non-pigmented species tested in this study which indicates a correlation between production of bioactive compounds and expression of pigment. Three members, P. tunicata, Pseudoalteromonas citrea and Pseudoalteromonas rubra, were demonstrated to express autoinhibitory activity. It is suggested that most Pseudoalteromonas species are efficient producers of antifouling agents and that the production of inhibitory compounds by surface associated Pseudoalteromonas species may aid the host against colonisation of its surface.