Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents

Abstract

The newly established genus Pseudoalteromonas contains numerous marine species which synthesize biologically active molecules. The production of a range of compounds which are active against a variety of target organisms appears to be a unique characteristic for this genus and may greatly benefit Pseudoalteromonas cells in their competition for nutrients and colonization of surfaces. Species of Pseudoalteromonas are generally found in association with marine eukaryotes and display anti-bacterial, bacteriolytic, agarolytic and algicidal activities. Moreover, several Pseudoalteromonas isolates specifically prevent the settlement of common fouling organisms. While a wide range of inhibitory extracellular agents are produced, compounds promoting the survival of other marine organisms living in the vicinity of Pseudoalteromonas species have also been found.

Marine, Fouling, Pseudoalteromonas

1 Introduction

Bacteria readily isolated from marine waters are heterotrophic Gram-negative, flagellated bacteria [1] and can be divided into two subgroups depending on their capacity to ferment carbohydrates. Within the non-fermentative group, the genus designated Alteromonas was revised based on phylogenetic comparisons performed by Gauthier et al. in 1995. Their revision suggested that the genus Alteromonas should be divided into two genera, Alteromonas (which now includes one species only) and a new genus, Pseudoalteromonas[2] (Fig. 1 shows a phylogenetic tree of the different species currently assigned to the genus Pseudoalteromonas). This newly created genus has attracted significant interest for two reasons. First, Pseudoalteromonas species are frequently found in association with eukaryotic hosts in the marine environment and studies of such associations will elucidate the mechanisms important in microbe-host interactions. Second, many of the species produce biologically active metabolites which target a range of organisms. The different extracellular biological activities displayed by Pseudoalteromonas species are listed in Table 1. The aims of this review are to summarize some of the emerging information pertaining to the genus Pseudoalteromonas and to highlight the ecological relevance and range of biologically active compounds that are expressed by many of its member species.

1

A phylogenetic affiliation of the genus Pseudoalteromonas based on 16S rRNA gene sequence alignment. An alignment of 1157 characters was used to calculate genetic distances according to the method of Jukes and Cantor (1969). The phenogram was reconstructed from the pairwise distance matrix using the neighbor-joining method of Saitou and Nei (1996). The scale represents one base substitution per 10 nucleotide positions.

1

A summary of the biological activities displayed by Pseudoalteromonas species

Bacterium	Biological activity	References
P. aurantia	Anti-bacterial activity	[12]
P. luteoviolacea	Anti-bacterial activity	[13]
P. rubra	Anti-bacterial activity	[14]
P. citrea	Anti-bacterial, anti-fungal and agarolytic activities	[6,23]
Pseudoalteromonas sp. F-420	Anti-bacterial activity	[10]
P. agarolyticus	Agarolytic activity	[27]
P. antarctica strain N-1	Agarolytic activity	[27]
Pseudoalteromonas sp. strain C-1	Agarolytic activity	[27]
P. carrageenovora	Agarolytic activity	[9]
P. atlantica	Agarolytic activity	[9]
P. bacteriolytica	Bacteriolytic activity, believed to cause red spot disease of L. japonica	[28]
P. haloplanktis strain S5B	Produces trypsin-like proteases which are believed to cause fish spoilage	[47]
Pseudoalteromonas sp. strain Y	Algicidal activity	[30]
P. piscicidia	Produces a toxin which appears to cause fish mortality	[32]
P. tetraodonis	Produces a neurotoxin, tetradotoxin, which causes pufferfish poisoning	[7]
P. denitrificans	Produces autotoxic substances	[4]
Pseudoalteromonas sp. strain S9	Promotes the settlement of tunicate larvae	[39]
P. colwelliana	Promotes the settlement of oyster larvae	[35]
P. undina	Anti-bacterial and anti-viral activities	[48]
P. espejiana	Degrades polymers and also induces metamorphosis of hydroid larvae	[25,49]
P. tunicata	Anti-fouling and biocontrol activities against invertebrate larvae, algal spores, bacteria, fungi and diatoms	[8]

2 Isolation of Pseudoalteromonas species

In the classification of bacteria, the clear distinction that exists between marine and non-marine animals and plants is not applicable [3]. Currently, representatives of most culturable bacterial genera can be isolated both from terrestrial and marine environments. Yet, it is now established that the genus Pseudoalteromonas contains species that exclusively derive from marine waters and that members have been isolated from marine locations around the world [4]. Interestingly, a majority of the Pseudoalteromonas species seem to be associated with eukaryotic hosts. Species have been isolated from various animals, such as mussels [5,6], pufferfish [7], tunicates [8] and sponges [6], as well as from a range of marine plants [9,10]. Their existence in a variety of habitats and their world-wide spread suggest that the adaptive and survival strategies expressed by Pseudoalteromonas species are diverse, efficient and of great interest for both basic and applied research.

3 Biological activities expressed by Pseudoalteromonas and Alteromonas species

Recent years have witnessed the generation of considerable novel information on the production of biologically active metabolites by members of the genus Pseudoalteromonas and the interactions of these bacteria with different host organisms. In particular, many Pseudoalteromonas species have been demonstrated to produce anti-bacterial products which appear to aid them in the colonization of surfaces including those of their hosts. The production of agarases, toxins, bacteriolytic substances and other enzymes by many Pseudoalteromonas species may assist the bacterial cells in their competition for nutrients and space as well as in their protection against predators grazing at surfaces. The bacterial ecology at solid surfaces is complex and many additional factors besides the production of secondary metabolites and other secreted products affect bacterial responses. For example, Ivanova et al. [11] demonstrated that the degree of hydrophobicity of the substratum influences the production of anti-bacterial metabolites. The highest anti-microbial activity was found to occur on hydrophilic surfaces despite the fact that attached Pseudoalteromonas cells were more abundant on hydrophobic surfaces [11]. This finding may suggest that the expression of the anti-microbial activity may be switched on and off depending on fluctuations and stimuli present in the immediate environment of the cell.

3.1 Anti-bacterial activity

Pseudoalteromonas species display a broad range of antibiotic effects. Three species, P. aurantia[12], P. luteoviolacea[13] and P. rubra[14], have been demonstrated to produce high molecular mass anti-bacterial compounds [15]. The anti-bacterial activity displayed by the different strains of P. luteoviolacea is particularly interesting and has been suggested to be due to two classes of compounds. First, cell bound polyanionic macromolecules, which are partly diffusible in culture media, were thought to be acidic polysaccharides. A later study by McCarthy et al. demonstrated that these high molecular compounds are associated with proteins [16]. The second group of antibiotics produced by P. luteoviolaceus contains small brominated compounds [17] which are cell bound and not diffusible into the media. These brominated compounds are known to have a strong bactericidal effect [13]. In the production of the different antibiotics, heterogeneity among different P. luteoviolaceus strains has been demonstrated. Different strains have been found to synthesize either the polysaccharide molecule or the small brominated metabolites, or both. These variations in the production of anti-bacterial agents may suggest that P. luteoviolaceus strains originally selected different host organisms or habitats and with time evolved separately to produce divergent compounds. Self-inhibition has also been observed in P. luteoviolaceus cells and is suggested to be mediated by the macromolecular antibiotic compound [13]. These polyanionic carbohydrates have also been demonstrated to be important in the attachment of bacteria to solid surfaces [18], enabling the bacteria to be highly competitive in the colonization of host organisms [13]. Although auto-inhibition is observed for Pseudoalteromonas species, we demonstrated that it is not a widely occurring phenomenon in other marine bacteria [19]. The importance of this activity in the marine ecosystem has been questioned, given that the dilution of compounds in the aqueous phase probably keeps the concentration of extracellular compounds low in the vicinity of cells [17]. Yet, it is possible that the production of auto-inhibitory compounds in a bacterial population is important for maintaining the microbial diversity within a microhabitat [20]. Given the recent findings that extracellular auto-inducer compounds are important in many bacterial populations [21,22], a role for auto-inhibitory molecules can also be envisaged.

Antibiotic activity mediated by a polyanionic antibiotic molecule has also been demonstrated for the species P. citrea[23]. These polyvalent anions, which also mediate the antibiotic activity of P. rubra and P. luteoviolacea, have been found to inhibit bacterial respiration [13]. Interestingly, it was demonstrated that the production of such compounds in several Pseudoalteromonas species was media dependent. The cells did not express any anti-bacterial activity when grown on blood-containing media and the expression of the active compounds was very low when grown on nutrient agar and tryptic soy agar media containing salt [13,14,24]. Given that specific nutrient conditions may be needed for bacteria to express their extracellular biologically active compounds, it is suggested that some bacteria-host associations in the marine environments are controlled by available food sources.

3.2 Extracellular enzymes and toxins

Agar is a polysaccharide present in the cell walls of some red algae. It appears that the bacterial degradation of agar occurs by two mechanisms based on the specificity of the enzymes β-agarase and α-agarase. Cleavage of the polysaccharide chains causes agar softening so that agarase activity expressed by bacteria living in association with red algae may help the bacteria to easily acquire nutrients from the algae, as is the case for the bacterial degradation of the fronds of green [25] and brown algae [26]. Vera et al. identified an agarolytic isolate, P. antarctica strain N-1, and characterized its extracellular produced agarase to be an endo β-agarase I [27]. Other agarase decomposing strains within the genus Pseudoalteromonas are P. agarolyticus, P. sp. strain C-1, P. carrageenovora, P. atlantica[9,27] and P. citrea[6]. Most of the reported agarolytic Pseudoalteromonas strains have been found to produce extracellular β-agarases while the P. agarolyticus strain was reported to produce both an α- and a β-agarase.

Other bacteria expressing biological activity against marine plants include P. bacteriolytica strains which were isolated from the brown alga Laminaria japonica and are believed to be the causative agent of red spot disease in L. japonica[28]. These bacterial isolates have also been found to have bacteriolytic activity against both Gram-positive and Gram-negative bacteria [28]. Given the advantages that bacteriolytic activity provide for the producer strains by the release of nutritive compounds, this is most likely a significant trait for bacteria living in oligotrophic environments. Bacteriolytic activity may also benefit the producer strain in the competition for space in the marine environment.

Competitive advantages for Pseudoalteromonas and Alteromonas bacterial strains in nutritrient aquisition and colonization have been proposed for their algicidal activities against phytoplankton [29]. In this case, the ecological relevance of algicidal activity may also be to control phytoplankton succession in the marine environment as was shown for Pseudoalteromonas sp. strain Y against blooms of harmful micro algae [30]. This bacterium was demonstrated to cause rapid cell lysis and death of species within the genera Chatonella, Gymnodinium and Heterosigma. A bacterium Pseudoalteromonas sp. A28 was also demonstrated to lyse marine algae [31]. The active components were proteases and it was suggested that the protease expression is regulated by the acylated homoserine lactone regulatory system [31].

Several other Pseudoalteromonas species have also been shown to produce extracellular toxins (see Table 1). These include P. tetraodonis, which produces the neurotoxin, tetradotoxin, the causative agent of pufferfish poisoning [7], and P. piscicidia, which releases a toxin suggested to cause fish mortality [32]. Furthermore, Pseudoalteromonas tunicata cells have been demonstrated to be toxic against invertebrate larvae [33] and algal spores [34] and P. denitrificans produces autotoxic substances which kill the bacterial cells and inhibit further growth in dense culture [4]. Production of toxic compounds may allow for the bacteria to control large scale processes, in contrast to the more restricted modification of their microhabitats caused by specific non-toxic extracellular agents.

3.3 Extracellular polysaccharides

The range of biological activities discussed above suggests that the expression of bacterial extracellular compounds allows for the producer to successfully compete with other organisms. However, bacteria can also produce compounds which aid in the survival of other marine organisms. For example, the production of exopolysaccharides (EPS) has been demonstrated to enhance the chances for other organisms to survive in specific marine habitats. Despite this fact, EPS effects have only been examined in detail in a few instances.

EPS producing bacterial strains are common within the genera Pseudoalteromonas and Alteromonas[35,36]. Interestingly, Alteromonas sp. strain HYD-1545, isolated from tube worms, produces an EPS containing acidic sugars [37] which were demonstrated to have heavy metal binding properties. The bacterium is suggested to be important for the survival of its host organism which lives in an environment where the exposure to chemicals (e.g. metallic sulfides) is high [37].

Further beneficial effects of EPS production by bacteria on organisms have been demonstrated for the settlement of invertebrate larvae [35,36]. For example, Pseudoalteromonas sp. strain S9 produces an EPS both in liquid culture and on surfaces during the stationary phase of growth [38]. The wild-type and a transposon-generated mutant deficient in the release of EPS were tested against the settlement of tunicate larvae [39] and it was demonstrated that the wild-type resulted in a higher degree of larval settlement and subsequent metamorphosis and development compared to the mutant strain.

The role of EPS production by Pseudoalteromonas sp. strain S9 in the bacterial cell attachment process was studied in some detail [38,40,41]. Wrangstadh et al. demonstrated that an increase in the production of EPS during starvation conditions correlated with a decrease in both cell surface hydrophobicity and adhesion of cells to inanimate surfaces. The increase in the amount of EPS at the bacterial cell surface further correlated with an increase in cell detachment [41]. These responses were triggered by starvation and were postulated to help the cells to escape nutrient-depleted environments. Following detachment, the free-living bacterial cells may ‘search’ for other surfaces to colonize that may provide more suitable conditions for their proliferation.

It would appear that bacterial EPSs can serve as anti-bacterial components [13], control bacterial attachment [18,40] and benefit the survival of both the host and other organisms that live in the vicinity of the producer strain [37,39]. Additionally EPSs can act as protective barriers against antibiotics, against predation by protozoa [42], function as enhancers for nutrient uptake and reduce the diffusion of some substances to and from the cells [43]. Many Pseudoalteromonas species employ EPS production and it is likely that this characteristic provides a range of survival strategies for the cells.

3.4 Biological activities expressed by P. tunicata

A well studied bacterium within the genus Pseudoalteromonas is P. tunicata (previously designated D2). This strain was isolated in coastal waters of Sweden from the surface of an adult tunicate collected at a depth of 10 m [33]. Subsequent studies demonstrated that P. tunicata-like strains exist on the green alga Ulva lactuca in Australian waters [34]. P. tunicata is a dark green-pigmented bacterium and has been found to produce at least five extracellular compounds which inhibit other organisms from establishing themselves in a biofouling community (Fig. 2). The compounds inhibit settlement of invertebrate larvae and algal spores, growth of bacteria and fungi and surface colonization by diatoms [34]. The anti-larval component is a heat stable polar molecule less than 500 Da in size [33]. The anti-bacterial molecule is a novel large protein (190 kDa) which consists of at least two subunits (80 and 60 kDa in size). The protein inhibits the growth of most Gram-negative and Gram-positive bacteria isolated from both marine and terrestrial environments [19]. P. tunicata cells also express auto-inhibition and cells in the exponential phase of growth are sensitive to this protein. However, as the cells reach the stationary phase, which is the physiological state in which the protein is produced, they become resistant. The ecological role of this anti-bacterial protein in P. tunicata colonization of surfaces is currently being studied using strains mutated in the subunit genes. The anti-spore component is a peptide of around 3 kDa in size [34]. Recent studies indicate that the anti-fungal molecule may be a cell bound long chain fatty acid derivative (unpublished data). The anti-diatom compound has not yet been characterized.

2

The anti-fouling activities expressed by P. tunicata. The different extracellular compounds are active against at least five different groups of organisms. These include invertebrate larvae, algal spores, bacteria, fungi and diatoms. Each compound targets a specific group of organisms.

The unique features of P. tunicata relate to the diversity of anti-fouling and inhibitory compounds that are produced and the fact that each metabolite has been demonstrated to target specific groups of organisms (Fig. 2). This phenomenon has not been reported for any other bacteria and may suggest that bacteria displaying such features are not common in the marine environment, although P. tunicata strains have been isolated from surfaces of higher organisms in both waters of the Swedish west coast and around Sydney. However, the paucity of reports on such bacteria and characteristics may reflect that laboratories generally do not have access to broad range bioassays.

4 Biological control and commercial use of Pseudoalteromonas species

Biocontrol is based on antagonistic interactions between microorganisms and between bacteria and higher organisms. The mode of action may be non-toxic and specific, as displayed by biofilm bacteria that repel fouling macroorganisms. More often however, the microbial biocontrol is based on toxicity as is generally the case for the control of microbial disease causing organisms. Given that Pseudoalteromonas species express a wide range of biological activities, it has been proposed that this genus includes valuable biocontrol strains for use in, aquaculture, anti-fouling technologies and the control of toxic algal blooms. Indeed, these proposals have been explored in some research programs. The authors of this minireview developed a method to immobilize P. tunicata cells into polyacrylamide gels [44] and polyvinylalcohol gels [45]. The method was employed to keep the cells entrapped and alive in a gel matrix surface coating which allowed for an outflux of anti-fouling components. Viable immobilized cells were demonstrated after more than 2 months in marine waters in the laboratory [45]. Such a ‘living paint’ concept may offer a novel environmentally friendly alternative to current toxic marine anti-foulants.

The methods employed for reducing numbers of pathogenic microorganisms when culturing fish, abalone, oyster and other organisms include filtration of the water, ozonation, ultraviolet light exposure and the use of artificial food containing antibiotics. However, none of these methods has proven effective in controlling microbial diseases and there is an obvious need to introduce alternative methods. Maeda et al. [48] investigated the use of an anti-microbial producing Pseudoalteromonas undina strain as a biocontrol agent and demonstrated that this organism successfully represses the growth of deleterious bacteria and viruses and improves the growth of farmed fish and crustaceans. Furthermore, the ability of many Pseudoalteromonas species to degrade polymers and to attach to surfaces was applied in the degradation of freeze-dried Ulva fronds for a hatchery diet of Artemia nauplii larvae [25]. It was demonstrated that the addition of Pseudoalteromonas espejiana to the fronds enhanced their conversion into microalgae-like forms. These particles also contained double the amount of protein, as a result of the bacterial biofilm, in comparison with the particles that were treated under sterile conditions [25]. This technology may be applicable in other processes for the generation of animal food.

5 Fouling control by the Pseudoalteromonas species in the marine environment

In response to various metabolites or other environmental stimuli, bacterial cells can produce chemical compounds which benefit the producer strain and/or the host organism in their establishment in suitable marine habitats. Examples include where host organisms may employ bacterially produced compounds for their own chemical defence against fouling organisms as for the green algae Ulva lactuca and Enteromorpha intestinalis and the tunicate Ciona intestinalis. These organisms are known not to produce any secondary metabolites for their protection against fouling but have been reported to carry anti-fouling producing Pseudoalteromonas species [34,46]. We hypothesize that their success in remaining unfouled in the field is due to the associated anti-fouling bacteria.

Acknowledgments

Research in the author's laboratory was funded by the Australian Research Council, a Vice Chancellor's post doctoral research fellowship at the University of New South Wales to C.H. and by the Centre for Marine Biofouling and Bio-Innovation at UNSW. We would like to thank Sally James, Suhelen Egan, Ashley Franks, Torsten Thomas and Harriet Baillie for assistance and valuable discussions.