Monospecific Biofilms of Pseudoalteromonas Promote Larval Settlement and Metamorphosis of Mytilus coruscus.

As a stage of life cycle, larval settlement and metamorphosis are critical processes for persistence of many marine invertebrate populations. Bacterial biofilms (BFs) could induce larval settlement and metamorphosis. Pseudoalteromonas, a widely distributed genus of marine bacteria, showed inductive effects on several invertebrates. However, how Pseudoalteromonas BFs induce settlement and metamorphosis of Mytilus coruscus remains unclear. Pseudoalteromonas marina BFs with the highest inducing activity were further investigated to define inductive cues. Surface-bound products of P. marina BFs could induce larval settlement and metamorphosis. P. marina BFs treated with formalin, antibiotics, ultraviolet irradiation, heat and ethanol significantly reduced inductive effects and cell survival rates. The confocal laser scanning microscopy and the biovolume analysis showed the dominance of α-polysaccharides on P. marina BFs. Treatment of BFs with amylases, proteases and lipase led to the decrease of inducing activity, suggesting that inductive cues of P. marina BFs may comprise of molecular domains of polysaccharides, proteins, and lipids. Finding inductive cues of BFs could put forward further studies about the mechanism of larval settlement and metamorphosis of marine invertebrates.

Metabolomic and proteomic changes induced by growth inhibitory concentrations of copper in the biofilm-forming marine bacterium Pseudoalteromonas lipolytica.

Copper is an essential element for living cells but this metal is present in some marine environments at such high concentrations that it can be toxic for numerous organisms. In polluted areas, marine organisms may develop specific adaptive responses to prevent cell damage. To investigate the influence of copper on the metabolism of a single organism, a dual approach combining metabolomics and proteomics was undertaken on the biofilm-forming bacterial strain Pseudoalteromonas lipolytica TC8. In order to highlight differential adaptation according to the phenotype, the response of P. lipolytica TC8 to copper stress was studied in planktonic and biofilm culture modes under growth inhibitory copper concentrations. As expected, copper exposure led to the induction of defense and detoxification mechanisms. Specific metabolite and protein profiles were thus observed in each condition (planktonic vs. biofilm and control vs. copper-treated cultures). Copper exposure seems to induce drastic changes in the lipid composition of the bacterial cell membrane and to modulate the abundance of proteins functionally known to be involved in copper cell homeostasis in both planktonic and biofilm culture modes. Much more proteins differentially expressed after copper treatment were observed in biofilms than in planktonic cells, which could indicate a more heterogeneous response of biofilm cells to this metallic stress.

Functional exploration of Pseudoalteromonas atlantica as a source of hemicellulose-active enzymes: Evidence for a GH8 xylanase with unusual mode of action.

To address the need for efficient enzymes exhibiting novel activities towards cell wall polysaccharides, the bacterium Pseudoalteromonas atlantica was selected based on the presence of potential hemicellulases in its annotated genome. It was grown in the presence or not of hemicelluloses and the culture filtrates were screened towards 42 polysaccharides. P. atlantica showed appreciable diversity of enzymes active towards hemicelluloses from Monocot and Dicot origin, in agreement with its genome annotation. After growth on beechwood glucuronoxylan and fractionation of the secretome, a ß-xylosidase, a α-arabinofuranosidase and an acetylesterase activities were evidenced. A GH8 enzyme obtained in the same growth conditions was further cloned and heterologously overexpressed. It was shown to be a xylanase active on heteroxylans from various sources. The detailed study of its mode of action demonstrated that the oligosaccharides produced carried a long tail of un-substituted xylose residues on the reducing end.

Biofilm formation in Pseudoalteromonas lipolytica is related to IS5-like insertions in the capsular polysaccharide operon.

Bacterial capsular polysaccharides (CPSs) participate in environmental adaptation in diverse bacteria species. However, the role and regulation of CPS production in marine bacteria have remained largely unexplored. We previously reported that both wrinkled and translucent Pseudoalteromonas lipolytica variants with altered polysaccharide production were generated in pellicle biofilm-associated cells. In this study, we observed that translucent variants were generated at a rate of ∼20% in colony biofilms of P. lipolytica cultured on HSLB agar plates for 12 days. The DNA sequencing results revealed that nearly 90% of these variants had an IS5-like element inserted within the coding or promoter regions of nine genes in the cps operon. In contrast, IS5 insertion into the cps operon was not detected in planktonic cells. Furthermore, we demonstrated that the IS5 insertion event inactivated CPS production, which leads to a translucent colony morphology. The CPS-deficient variants showed an increased ability to form attached biofilms but exhibited reduced resistance to sublethal concentrations of antibiotics. Moreover, deleting the DNA repair gene recA significantly decreased the frequency of occurrence of CPS-deficient variants during biofilm formation. Thus, IS insertion into the cps operon is an important mechanism for the production of genetic variants during biofilm formation of marine bacteria.

Optimized culturing conditions for an algicidal bacterium Pseudoalteromonas sp. SP48 on harmful algal blooms caused by Alexandrium tamarense.

Bacteria play an important role in preventing algal blooms and reducing their harm to the environment. To improve the algicidal activity of Pseudoalteromonas SP48 which had an inhibition effect on dinoflagellate Alexandrium tamarense, its growth medium and fermentation conditions were optimized for this bacterium. In this study, we used two steps to establish the optimum conditions. First, the proper proportion of medium was selected based on an orthogonal design. Then, the fermentation conditions were further optimized through uniform design in an enlarged 5L bioreactor. To test the algicidal ability of Pseudoalteromonas SP48 under the optimum conditions, algal cell morphology was observed by transmission electron microscopy (TEM). After the orthogonal design, we found that the optimum medium was [0.7% (m/v) tryptone, 0.2% (m/v) soluble starch, 0.2% (m/v) sucrose, 0.1% (m/v) FeSO4 , and 1.2% (m/v) K2 HPO4 ] for Pseudoalteromonas SP48 growth. Based on these results, optimum fermentation conditions were further explored in a 5L fermentation cylinder using a uniform design; the influence of variables such as incubation time, carbon type, and rotation speed were tested. The optimal fermentation conditions were fermentation time (42 hr), tryptone (1.1%), seeding volume (1.4 × 1013  cells), and rotation speed (250 r/min). Under these established optimum conditions, the biomass of strain SP48 increased by 79.2% and its lethal dose 50% (LD50 ) decreased by 54.0%, respectively. The TEM results showed that compared with the control group, the cell wall and cell membrane of A. tamarense were significantly damaged, and the structure and shape of the organelles were destroyed by algicidal bacteria of Pseudoalteromonas SP48. Overall, our results demonstrate that the optimized culture conditions could significantly enhance the algicidal activity of Pseudoalteromonas SP48 against a harmful dinoflagellate, such as A. tamarense. It will effectively provide a scientific foundation for both production of algicidal substances and HABs control.

Environmental conditions shape the biofilm of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Biofilms are the most widely distributed and successful microbial modes of life. The capacity of bacteria to colonize surfaces provides stability in the growth environment, allows the capturing of nutrients and affords protection from a range of environmental challenges and stress. Bacteria living in cold environments, like Antarctica, can be found as biofilms, even though the mechanisms of how this lifestyle is related to their environmental adaptation have been poorly investigated. In this paper, the biofilm of Pseudoalteromonas haloplanktis TAC125, one of the model organisms of cold-adapted bacteria, has been characterized in terms of biofilm typology and matrix composition. The characterization was performed on biofilms produced by the bacterium in response to different nutrient abundance and temperatures; in particular, this is the first report describing the structure of a biofilm formed at 0 °C. The results reported demonstrate that PhTAC125 produces biofilms in different amount and endowed with different physico-chemical properties, like hydrophobicity and roughness, by modulating the relative amount of the different macromolecules present in the biofilm matrix. The capability of PhTAC125 to adopt different biofilm structures in response to environment changes appears to be an interesting adaptation strategy and gives the first hints about the biofilm formation in cold environments.

Culturing marine bacteria from the genus Pseudoalteromonas on a cotton scaffold alters secondary metabolite production.

The discovery of secondary metabolites from marine microorganisms is beset by numerous challenges including difficulties cultivating and subsequently eliciting expression of biosynthetic genes from marine microbes in the laboratory. In this paper, we describe a method of culturing three species from the marine bacterial genus Pseudoalteromonas using cotton scaffold supplemented liquid media. This simple cultivation method was designed to mimic the natural behavior of some members of the genus wherein they form epibiotic/symbiotic associations with higher organisms such as sponges and corals or attach to solid structures as a biofilm. Our scaffolded cultivation is highly effective at stimulating an attachment/biofilm phenotype and causes large changes to metabolite profiles for the microbes investigated. Metabolite changes include alteration to the production levels of known molecules such as violacein, thiomarinol A, and the alterochromide and prodiginine families of molecules. Finally and critically, our technique stimulates the production of unknown compounds that will serve as leads for future natural product discovery. These results suggest our cultivation approach could potentially be used as a general strategy for the activation of silent gene clusters in marine microbes to facilitate access to their full natural product biosynthetic capacity.

Dissemination of Genetic Acquisition/Loss Provides a Variety of Quorum Sensing Regulatory Properties in Pseudoalteromonas.

A bstract: Quorum sensing (QS) enables single-celled bacteria to communicate with chemical signals in order to synchronize group-level bacterial behavior. Pseudoalteromonas are marine bacteria found in versatile environments, of which QS regulation for their habitat adaptation is extremely fragmentary. To distinguish genes required for QS regulation in Pseudoalteromonas, comparative genomics was deployed to define the pan-genomics for twelve isolates and previously-sequenced genomes, of which acyl-homoserine lactone (AHL)-based QS traits were characterized. Additionally, transposon mutagenesis was used to identify the essential QS regulatory genes in the selected Pseudoalteromonas isolate. A remarkable feature showed that AHL-based colorization intensity of biosensors induced by Pseudoalteromonas most likely correlates with QS regulators genetic heterogeneity within the genus. This is supported by the relative expression levels of two of the main QS regulatory genes (luxO and rpoN) analyzed in representative Pseudoalteromonas isolates. Notably, comprehensive QS regulatory schema and the working model proposed in Pseudoalteromonas seem to phylogenetically include the network architectures derived from Escherichia coli, Pseudomonas, and Vibrio. Several associated genes were mapped by transposon mutagenesis. Among them, a right origin-binding protein-encoding gene (robp) was functionally identified as a positive QS regulatory gene. This gene lies on a genomic instable region and exists in the aforementioned bioinformatically recruited QS regulatory schema. The obtained data emphasize that the distinctly- and hierarchically-organized mechanisms probably target QS association in Pseudoalteromonas dynamic genomes, thus leading to bacterial ability to accommodate their adaption fitness and survival advantages.

Metabolome and proteome changes between biofilm and planktonic phenotypes of the marine bacterium Pseudoalteromonas lipolytica TC8.

A number of bacteria adopt various lifestyles such as planktonic free-living or sessile biofilm stages. This enables their survival and development in a wide range of contrasting environments. With the aim of highlighting specific metabolic shifts between these phenotypes and to improve the overall understanding of marine bacterial adhesion, a dual metabolomics/proteomics approach was applied to planktonic and biofilm cultures of the marine bacterium Pseudoalteromonas lipolytica TC8. The liquid chromatography mass spectrometry (LC-MS) based metabolomics study indicated that membrane lipid composition was highly affected by the culture mode: phosphatidylethanolamine (PEs) derivatives were over-produced in sessile cultures while ornithine lipids (OLs) were more specifically synthesized in planktonic samples. In parallel, differences between proteomes revealed that peptidases, oxidases, transcription factors, membrane proteins and the enzymes involved in histidine biosynthesis were over-expressed in biofilms while proteins involved in heme production, nutrient assimilation, cell division and arginine/ornithine biosynthesis were specifically up-regulated in free-living cells.

Towards smart biocide-free anti-biofilm strategies: Click-based synthesis of cinnamide analogues as anti-biofilm compounds against marine bacteria.

A set of triazole-based analogues of N-coumaroyltyramine was designed to discover potential leads that may help in the control of bacterial biofilms. the most potent compounds act as inhibitors of biofilm development with EC50 closed to ampicillin (EC50 = 11 µM) without toxic effect on bacterial growth even at high concentrations(100 µM).