Competitive interactions in mixed-species biofilms containing the marine bacterium Pseudoalteromonas tunicata

Pseudoalteromonas tunicata is a biofilm-forming marine bacterium that is often found in association with the surface of eukaryotic organisms. It produces a range of extracellular inhibitory compounds, including an antibacterial protein (AlpP) thought to be beneficial for P. tunicata during competition for space and nutrients on surfaces. As part of our studies on the interactions between P. tunicata and the epiphytic bacterial community on the marine plant Ulva lactuca, we investigated the hypothesis that P. tunicata is a superior competitor compared with other bacteria isolated from the plant. A number of U. lactuca bacterial isolates were (i) identified by 16S rRNA gene sequencing, (ii) characterized for the production of or sensitivity to extracellular antibacterial proteins, and (iii) labeled with a fluorescent color tag (either the red fluorescent protein DsRed or green fluorescent protein). We then grew single- and mixed-species bacterial biofilms containing P. tunicata in glass flow cell reactors. In pure culture, all the marine isolates formed biofilms containing microcolony structures within 72 h. However, in mixed-species biofilms, P. tunicata removed the competing strain unless its competitor was relatively insensitive to AlpP (Pseudoalteromonas gracilis) or produced strong inhibitory activity against P. tunicata (Roseobacter gallaeciensis). Moreover, biofilm studies conducted with an AlpP- mutant of P. tunicata indicated that the mutant was less competitive when it was introduced into preestablished biofilms, suggesting that AlpP has a role during competitive biofilm formation. When single-species biofilms were allowed to form microcolonies before the introduction of a competitor, these microcolonies coexisted with P. tunicata for extended periods of time before they were removed. Two marine bacteria (R. gallaeciensis and P. tunicata) were superior competitors in this study. Our data suggest that this dominance can be attributed to the ability of these organisms to rapidly form microcolonies and their ability to produce extracellular antibacterial compounds.     

Inhibition of algal spore germination by the marine bacterium Pseudoalteromonas tunicata

A collection of 56 bacteria isolated from different surfaces in the marine environment were assayed for their effects on the germination of spores from the common green alga Ulva lactuca. Thirteen bacterial isolates were shown to inhibit spore germination. Of these bacteria, Pseudoalteromonas tunicata displayed the most pronounced effects against algal spores. Further characterisation of the anti-algal activity of P. tunicata was performed and it was found that this bacterium produces an extracellular component with specific activity toward algal spores that is heat-sensitive, polar and between 3 and 10 kDa in size. This biologically active compound was also found to prevent the germination of spores from the red alga Polysiphonia sp. and, given the widespread occurrence of P. tunicata in a range of marine habitats, this may suggest that it is effective against a variety of marine algae.     

Biofilm Development and Cell Death in the Marine Bacterium Pseudoalteromonas tunicata

The newly described green-pigmented bacterium Pseudoalteromonas tunicata (D2) produces target-specific inhibitory compounds against bacteria, algae, fungi, and invertebrate larvae and is frequently found in association with living surfaces in the marine environment. As part of our studies on the ecology of P. tunicata and its interaction with marine surfaces, we examined the ability of P. tunicata to form biofilms under continuous culture conditions within the laboratory. P. tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water channels. Remarkably, we observed a repeatable pattern of cell death during biofilm development of P. tunicata, similar to that recently reported for biofilms of Pseudomonas aeruginosa (J. S. Webb et al., J. Bacteriol. 185:4585-4595, 2003). Killing and lysis occurred inside microcolonies, apparently resulting in the formation of voids within these structures. A subpopulation of viable cells was always observed within the regions of killing in the biofilm. Moreover, extensive killing in mature biofilms appeared to result in detachment of the biofilm from the substratum. A novel 190-kDa autotoxic protein produced by P. tunicata, designated AlpP, was found to be involved in this biofilm killing and detachment. A ΔalpP mutant derivative of P. tunicata was generated, and this mutant did not show cell death during biofilm development. We propose that AlpP-mediated cell death plays an important role in the multicellular biofilm development of P. tunicata and subsequent dispersal of surviving cells within the marine environment.     

Profiling the secretome of the marine bacterium Pseudoalteromonas tunicata using amine-specific isobaric tagging (iTRAQ)

The eukaryote-associated marine bacterium Pseudoalteromonas tunicata produces a range of target-specific compounds that inhibit different types of marine organisms including invertebrate larvae and algal spores, as well as a broad spectrum of fungi, protozoa, and bacteria. The ability to produce such bioactive compounds is correlated to the expression of a yellow and a purple pigment in P. tunicata. To investigate the regulation and biosynthesis of the pigments and bioactive compounds, the expressed secretome of the pigmented wild-type P. tunicata and a nonpigmented mutant (wmpD-) defective in the type-II secretion pathway were compared. Secreted proteins were digested with trypsin, labeled using amine-specific isobaric tagging reagents (iTRAQ), and identified using two-dimensional SCX and nano C18 RP liquid-chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS). The iTRAQ labeling experiments enabled accurate measurement of the proteins identified in this work. A sequence-base prediction of P. tunicata secretome was also obtained and compared to the expressed proteome to determine the role of the type-II secretion pathway in this bacterium. Our results suggest that this secretion pathway has a role in iron transport and acquisition in P. tunicata.     

Ecological advantages of autolysis during the development and dispersal of Pseudoalteromonas tunicata biofilms

In the ubiquitous marine bacterium Pseudoalteromonas tunicata, subpopulations of cells are killed by the production of an autocidal protein, AlpP, during biofilm development. Our data demonstrate an involvement of this process in two parameters, dispersal and phenotypic diversification, which are of importance for the ecology of this organism and for its survival within the environment. Cell death in P. tunicata wild-type biofilms led to a major reproducible dispersal event after 192 h of biofilm development. The dispersal was not observed with a DeltaAlpP mutant strain. Using flow cytometry and the fluorescent dye DiBAC4(3), we also show that P. tunicata wild-type cells that disperse from biofilms have enhanced metabolic activity compared to those cells that disperse from DeltaAlpP mutant biofilms, possibly due to nutrients released from dead cells. Furthermore, we report that there was considerable phenotypic variation among cells dispersing from wild-type biofilms but not from the DeltaAlpP mutant. Wild-type cells that dispersed from biofilms showed significantly increased variations in growth, motility, and biofilm formation, which may be important for successful colonization of new surfaces. These findings suggest for the first time that the autocidal events mediated by an antibacterial protein can confer ecological advantages to the species by generating a metabolically active and phenotypically diverse subpopulation of dispersal cells.     

Analysis of the Pseudoalteromonas tunicata genome reveals properties of a surface-associated life style in the marine environment

Background

Colonisation of sessile eukaryotic host surfaces (e.g. invertebrates and seaweeds) by bacteria is common in the marine environment and is expected to create significant inter-species competition and other interactions. The bacterium Pseudoalteromonas tunicata is a successful competitor on marine surfaces owing primarily to its ability to produce a number of inhibitory molecules. As such P. tunicata has become a model organism for the studies into processes of surface colonisation and eukaryotic host-bacteria interactions.

Methodology/Principal Findings

To gain a broader understanding into the adaptation to a surface-associated life-style, we have sequenced and analysed the genome of P. tunicata and compared it to the genomes of closely related strains. We found that the P. tunicata genome contains several genes and gene clusters that are involved in the production of inhibitory compounds against surface competitors and secondary colonisers. Features of P. tunicata''s oxidative stress response, iron scavenging and nutrient acquisition show that the organism is well adapted to high-density communities on surfaces. Variation of the P. tunicata genome is suggested by several landmarks of genetic rearrangements and mobile genetic elements (e.g. transposons, CRISPRs, phage). Surface attachment is likely to be mediated by curli, novel pili, a number of extracellular polymers and potentially other unexpected cell surface proteins. The P. tunicata genome also shows a utilisation pattern of extracellular polymers that would avoid a degradation of its recognised hosts, while potentially causing detrimental effects on other host types. In addition, the prevalence of recognised virulence genes suggests that P. tunicata has the potential for pathogenic interactions.

Conclusions/Significance

The genome analysis has revealed several physiological features that would provide P. tunciata with competitive advantage against other members of the surface-associated community. We have also identified properties that could mediate interactions with surfaces other than its currently recognised hosts. This together with the detection of known virulence genes leads to the hypothesis that P. tunicata maintains a carefully regulated balance between beneficial and detrimental interactions with a range of host surfaces.     

The use of functional genomics for the identification of a gene cluster encoding for the biosynthesis of an antifungal tambjamine in the marine bacterium Pseudoalteromonas tunicata

A large insert library was created in Escherichia coli from the DNA of the surface-associated marine bacterium Pseudoalteromonas tunicata. Screening of the library for antifungal activity resulted in the detection and identification of a large gene cluster encoding for the biosynthesis of an antifungal tambjamine. A biosynthetic pathway has been proposed based on analysis and annotation of the gene cluster.     

Competitive Interactions in Mixed-Species Biofilms Containing the Marine Bacterium Pseudoalteromonas tunicata

Pseudoalteromonas tunicata is a biofilm-forming marine bacterium that is often found in association with the surface of eukaryotic organisms. It produces a range of extracellular inhibitory compounds, including an antibacterial protein (AlpP) thought to be beneficial for P. tunicata during competition for space and nutrients on surfaces. As part of our studies on the interactions between P. tunicata and the epiphytic bacterial community on the marine plant Ulva lactuca, we investigated the hypothesis that P. tunicata is a superior competitor compared with other bacteria isolated from the plant. A number of U. lactuca bacterial isolates were (i) identified by 16S rRNA gene sequencing, (ii) characterized for the production of or sensitivity to extracellular antibacterial proteins, and (iii) labeled with a fluorescent color tag (either the red fluorescent protein DsRed or green fluorescent protein). We then grew single- and mixed-species bacterial biofilms containing P. tunicata in glass flow cell reactors. In pure culture, all the marine isolates formed biofilms containing microcolony structures within 72 h. However, in mixed-species biofilms, P. tunicata removed the competing strain unless its competitor was relatively insensitive to AlpP (Pseudoalteromonas gracilis) or produced strong inhibitory activity against P. tunicata (Roseobacter gallaeciensis). Moreover, biofilm studies conducted with an AlpP⁻ mutant of P. tunicata indicated that the mutant was less competitive when it was introduced into preestablished biofilms, suggesting that AlpP has a role during competitive biofilm formation. When single-species biofilms were allowed to form microcolonies before the introduction of a competitor, these microcolonies coexisted with P. tunicata for extended periods of time before they were removed. Two marine bacteria (R. gallaeciensis and P. tunicata) were superior competitors in this study. Our data suggest that this dominance can be attributed to the ability of these organisms to rapidly form microcolonies and their ability to produce extracellular antibacterial compounds.     

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.     

Biofilm development of the polyethylene-degrading bacterium Rhodococcus ruber

We have recently isolated a biofilm-producing strain (C208) of Rhodococcus ruber that degraded polyethylene at a rate of 0.86% per week (r ²=0.98). Strain C208 adheres to polyethylene immediately upon exposure to the polyolefin. This initial biofilm differentiates (in a stepwise process that lasts about 20 h) into cell-aggregation-forming microcolonies. Further organization yields “mushroom-like” three-dimensional structures on the mature biofilm. The ratio between the population densities of the biofilm and the planktonic C208 cells after 10 days of incubation was about 60:1, indicating a high preference for the biofilm mode of growth. Analysis of extracellular polymeric substances (EPS) in the biofilm of C208 revealed that the polysaccharides level was up to 2.5 folds higher than that of the protein. The biofilm showed a high viability even after 60 days of incubation, apparently due to polyethylene biodegradation.     

Genetic examination of the marine bacterium Pseudoalteromonas luteoviolacea and effects of its metamorphosis-inducing factors

Pseudoalteromonas luteoviolacea is a globally distributed marine bacterium that stimulates the metamorphosis of marine animal larvae, an important bacteria–animal interaction that can promote the recruitment of animals to benthic ecosystems. Recently, different P. luteoviolacea isolates have been shown to produce two stimulatory factors that can induce tubeworm and coral metamorphosis; Metamorphosis-Associated Contractile structures (MACs) and tetrabromopyrrole (TBP) respectively. However, it remains unclear what proportion of P. luteoviolacea isolates possess the genes encoding MACs, and what phenotypic effect MACs and TBP have on other larval species. Here, we show that 9 of 19 sequenced P. luteoviolacea genomes genetically encode both MACs and TBP. While P. luteoviolacea biofilms producing MACs stimulate the metamorphosis of the tubeworm Hydroides elegans, TBP biosynthesis genes had no effect under the conditions tested. Although MACs are lethal to larvae of the cnidarian Hydractinia symbiologicarpus, P. luteoviolacea mutants unable to produce MACs are capable of stimulating metamorphosis. Our findings reveal a hidden complexity of interactions between a single bacterial species, the factors it produces and two species of larvae belonging to different phyla.     

Genome sequences of type strains of seven species of the marine bacterium Pseudoalteromonas

There are over 30 species in the marine bacterial genus Pseudoalteromonas. However, our knowledge about this genus is still limited. We sequenced the genomes of type strains of seven species in the genus, facilitating the study of the physiology, adaptation, and evolution of this genus.     

Ecological Advantages of Autolysis during the Development and Dispersal of Pseudoalteromonas tunicata Biofilms

In the ubiquitous marine bacterium Pseudoalteromonas tunicata, subpopulations of cells are killed by the production of an autocidal protein, AlpP, during biofilm development. Our data demonstrate an involvement of this process in two parameters, dispersal and phenotypic diversification, which are of importance for the ecology of this organism and for its survival within the environment. Cell death in P. tunicata wild-type biofilms led to a major reproducible dispersal event after 192 h of biofilm development. The dispersal was not observed with a ΔAlpP mutant strain. Using flow cytometry and the fluorescent dye DiBAC₄(3), we also show that P. tunicata wild-type cells that disperse from biofilms have enhanced metabolic activity compared to those cells that disperse from ΔAlpP mutant biofilms, possibly due to nutrients released from dead cells. Furthermore, we report that there was considerable phenotypic variation among cells dispersing from wild-type biofilms but not from the ΔAlpP mutant. Wild-type cells that dispersed from biofilms showed significantly increased variations in growth, motility, and biofilm formation, which may be important for successful colonization of new surfaces. These findings suggest for the first time that the autocidal events mediated by an antibacterial protein can confer ecological advantages to the species by generating a metabolically active and phenotypically diverse subpopulation of dispersal cells.     

Proteomic studies highlight outer‐membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer‐membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer‐membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB‐dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT‐PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.     

Purification and characterization of antibacterial substances produced by a marine bacterium Pseudoalteromonas haloplanktis strain

Aims: To purify and characterize compounds with antimicrobial activity from Pseudoalteromonas haloplanktis inhibition (INH) strain. Methods and Results: The P. haloplanktis isolated from a scallop hatchery was used to analyse antibacterial activities. Crude extracts were obtained with ethyl acetate of the cultured broth, after separation of bacterial cells, and assays against six strains of marine bacteria and nine clinically important pathogenic bacteria. The active compounds were purified from ethyl acetate extracts, by a combination of SiO₂ column and thin layer chromatography. Two active fractions were isolated, and chemical structures of two products from the major one were unambiguously identified as isovaleric acid (3-methylbutanoic acid) and 2-methylbutyric acid (2-methylbutanoic acid), by comparing their mass spectra and ¹H- and ¹³C-nuclear magnetic resonance spectra to those of authentic compounds. Conclusions: In the antibacterial activity of P. haloplanktis INH strain, extra cell compounds are involucred, mainly isovaleric and 2-methylbutyric acids. Significance and Impact of the Study: Production of antimicrobial compounds by marine micro-organisms has been widely reported; however, the efforts not always are conducted to purification and applications of these active compounds. This study is a significant contribution to the knowledge of compounds unique from marine bacteria as potential sources of new drugs in the pharmacological industry.     

Impact of phosphate concentration on the metabolome of biofilms of the marine bacterium Pseudoalteromonas lipolytica

Metabolomics - Recent advances in high-throughput methodologies in the ‘omics’ and synthetic biology fields call for rapid and sensitive workflows in the metabolic phenotyping of...     

Structure of an acidic O-specific polysaccharide of the marine bacterium Pseudoalteromonas sp. KMM 634

An acidic O-specific polysaccharide containing D-glucuronic acid (D-GlcA), 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (D-GlcNAc3NAcA), 2,3-diacetamido-2,3-dideoxy-D-mannuronoyl-L-alanine (D-ManNAc3NAcA6Ala), and 2-acetamido-2,4, 6-trideoxy-4-[(S)-3-hydroxybutyramido]-D-glucose (D-QuiNAc4NAcyl) was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Pseudoalteromonas sp. KMM 634 followed by gel-permeation chromatography. The polysaccharide was cleaved selectively with a new solvolytic agent, trifluoromethanesulfonic acid, to give a disaccharide and a trisaccharide with D-GlcNAc3NAcA at the reducing end. The borohydride-reduced oligosaccharides and the initial polysaccharide were studied by GLC-MS and 1H- and 13C-NMR spectroscopy, and the following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established: -->3)-alpha-D-QuipNAc4Ac4NAcyl-(1-->4)-beta-D-ManpNAc3NAcA6Ala+ ++-(1-->4)-b eta-D-GlcpNAc3NAc3NAcA-(1-->4)-beta-D-GlcpA-(1-->.     

Molecular level damages of low power pulsed laser radiation in a marine bacterium Pseudoalteromonas carrageenovora

AIM: To study the molecular level damages in a marine bacterium, Pseudoalteromonas carrageenovora, exposed to low power pulsed laser radiation from an Nd:YAG laser. METHODS AND RESULTS: The laser damages in bacterial DNA were monitored by studying the formation of apurinic/apyrimidinic (AP) sites. Molecular probe kits were used for this purpose. Occurrence of lesions in the cell walls was monitored under a transmission electron microscope (TEM). The results showed that laser radiation significantly increased the number of AP sites in the bacterial DNA. This increase corresponded to the laser fluence (J cm(-2)) and to the duration of laser irradiation. TEM observation showed the occurrence of lesions in bacterial cell walls upon laser irradiation. CONCLUSIONS: It is concluded that bacteria exposed to laser irradiation suffers DNA damages and resulted in broken cell walls. These events led to bacterial mortality. These are in addition to the mechanisms reported earlier such as the photochemical reactions occurring inside the cells upon exposure to low power laser. SIGNIFICANCE AND IMPACT OF THE STUDY: These results help us to understand the mechanisms of bacterial mortality on exposure to low power pulsed laser irradiation and are useful in formulating a laser treatment strategy to kill bacteria.