The Flagellar Gene Regulates Biofilm Formation and Mussel Larval Settlement and Metamorphosis.

Biofilms are critical components of most marine systems and provide biochemical cues that can significantly impact overall community composition. Although progress has been made in the bacteria-animal interaction, the molecular basis of modulation of settlement and metamorphosis in most marine animals by bacteria is poorly understood. Here, Pseudoalteromonas marina showing inducing activity on mussel settlement and metamorphosis was chosen as a model to clarify the mechanism that regulates the bacteria-mussel interaction. We constructed a flagellin synthetic protein gene fliP deletion mutant of P. marina and checked whether deficiency of fliP gene will impact inducing activity, motility, and extracellular polymeric substances of biofilms. Furthermore, we examined the effect of flagellar proteins extracted from bacteria on larval settlement and metamorphosis. The deletion of the fliP gene caused the loss of the flagella structure and motility of the ∆fliP strain. Deficiency of the fliP gene promoted the biofilm formation and changed biofilm matrix by reducing ß-polysaccharides and increasing extracellular proteins and finally reduced biofilm-inducing activities. Flagellar protein extract promoted mussel metamorphosis, and ∆fliP biofilms combined with additional flagellar proteins induced similar settlement and metamorphosis rate compared to that of the wild-type strain. These findings provide novel insight on the molecular interactions between bacteria and mussels.

The Role of Epibionts of Bacteria of the Genus Pseudoalteromonas and Cellular Proteasomes in the Adaptive Plasticity of Marine Cold-Water Sponges.

It was found that cells of different color morphs of the cold-water marine sponges Halichondria panicea (Pallas, 1766) of the class Demospongiae differ in the content of epibionts of bacteria of the genus Pseudoalteromonas. The sponge cells with elevated levels of epibionts of bacteria of the genus Pseudoalteromonas showed an increased expression of Hsp70 proteins but had a reduced level of the proteasomal catalytic beta 5 subunit, which was accompanied by a change in their activity. Probably, epibionts of bacteria of the genus Pseudoalteromonas may affect the ubiquitin-proteasome system in the cells of cold-water marine sponges and, thereby, ensure their adaptive plasticity.

The lipopolysaccharide lipid A structure from the marine sponge-associated bacterium Pseudoalteromonas sp. 2A.

Suberites domuncula is a marine demosponge harbouring a large bacterioflora, including commensal, opportunistic and pathogenic bacteria, among which, species of the Gram-negative genus Pseudoalteromonas were identified. The sponge-bacteria interaction mechanisms are still not fully understood. As the main component of the Gram-negative bacterial outer membrane, the lipopolysaccharide (LPS) may play a role in such a crucial relationship. Moreover, the LPS is known to be the most versatile bioactive macromolecule of Gram-negative bacteria and its lipid A structure is responsible for the immunological activity of the whole LPS on eukaryotic host cells. Here it is reported the structural characterisation of the LPS lipid A moiety isolated from the S. domuncula-associated commensal bacterium, Pseudoalteromonas sp. 2A. Chemical and MALDI mass spectrometry analyses, performed on both the LPS and the isolated lipid A as well as on the intact bacterial cells, highlighted a complex family of penta-acylated lipid A species carrying two phosphate units on the disaccharide backbone.

The Pathogen of the Great Barrier Reef Sponge Rhopaloeides odorabile Is a New Strain of Pseudoalteromonas agarivorans Containing Abundant and Diverse Virulence-Related Genes.

Sponge diseases have increased dramatically, yet the causative agents of disease outbreaks have eluded identification. We undertook a polyphasic taxonomic analysis of the only confirmed sponge pathogen and identified it as a novel strain of Pseudoalteromonas agarivorans. 16S ribosomal RNA (rRNA) and gyraseB (gyrB) gene sequences along with phenotypic characteristics demonstrated that strain NW4327 was most closely related to P. agarivorans. DNA-DNA hybridization and in silico genome comparisons established NW4327 as a novel strain of P. agarivorans. Genes associated with type IV pili, mannose-sensitive hemagglutinin pili, and curli formation were identified in NW4327. One gene cluster encoding ATP-binding cassette (ABC) transporter, HlyD and TolC, and two clusters related to the general secretion pathway indicated the presence of type I secretion system (T1SS) and type II secretion system (T2SS), respectively. A contiguous gene cluster of at least 19 genes related to type VI secretion system (T6SS) which included all 13 core genes was found. The absence of T1SS and T6SS in nonpathogenic P. agarivorans S816 established NW4327 as the virulent strain. Serine proteases and metalloproteases of the classes S8, S9, M4, M6, M48, and U32 were identified in NW4327, many of which can degrade collagen. Collagenase activity in NW4327 and its absence in the nonpathogenic P. agarivorans KMM 255(T) reinforced the invasiveness of NW4327. This is the first report unambiguously identifying a sponge pathogen and providing the first insights into the virulence genes present in any pathogenic Pseudoalteromonas genome. The investigation supports a theoretical study predicting high abundance of terrestrial virulence gene homologues in marine bacteria.

Production and characterization of a novel thermostable extracellular agarase from Pseudoalteromonas hodoensis newly isolated from the West Sea of South Korea.

A Gram-negative, aerobic, motile, rod-shaped, agarolytic bacterium, designated as H7, was isolated from a coastal seawater sample. This strain grows at pH 6.0-8.0, temperature of 15-40 °C, and at an NaCl concentration of 1-7% (w/v). Ubiquinone-8 was the predominant respiratory quinone, and the DNA G+C content was 45.82 mol%. Analysis of the 16S rRNA sequence suggests that strain H7 belongs to the genus Pseudoalteromonas. DNA-DNA hybridization analysis showed DNA relatedness of as low as 55.42 and 40.27% with its nearest phylogenetic neighbors Pseudoalteromonas atlantica IAM12927T and Pseudoalteromonas espejiana NCIMB2127T, respectively, which led us to name H7 Pseudoalteromonas hodoensis sp. nov. The type strain is H7T (=DSM25967T=KCTC23887T). An agarase (AgaA7) was purified to homogeneity from the cell-free culture broth of H7 through many steps of chromatography. Purified AgaA7 had an apparent molecular weight of 35 kDa, with a distinct NH2-terminal sequence of Ala-Asp-Ala-Thr-X-Pro (X, any amino acid) from the reported proteins, implying that it is a novel enzyme. The optimum pH and temperature for agarase activity were 7.0 and 45 °C, respectively. Thin-layer chromatography analysis, mass spectrometry, and enzyme assay using p-nitrophenyl-α/ß-D-galactopyranoside revealed that AgaA7 is both an exo- and endo-type ß-agarase that degrades agarose into neoagarotetraose, neoagarohexaose, and neoagarooctaose (minor).

Cytoplasmic and periplasmic proteomic signatures of exponentially growing cells of the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125.

The psychrophilic model bacterium Pseudoalteromonas haloplanktis is characterized by remarkably fast growth rates under low-temperature conditions in a range from 5°C to 20°C. In this study the proteome of cellular compartments, the cytoplasm and periplasm, of P. haloplanktis strain TAC125 was analyzed under exponential growth conditions at a permissive temperature of 16°C. By means of two-dimensional protein gel electrophoresis and mass spectrometry, a first inventory of the most abundant cytoplasmic and periplasmic proteins expressed in a peptone-supplemented minimal medium was established. By this approach major enzymes of the amino acid catabolism of this marine bacterium could be functionally deduced. The cytoplasmic proteome showed a predominance of amino acid degradation pathways and tricarboxylic acid (TCA) cycle enzymes but also the protein synthesis machinery. Furthermore, high levels of cold acclimation and oxidative stress proteins could be detected at this moderate growth temperature. The periplasmic proteome was characterized by a significant abundance of transporters, especially of highly expressed putative TonB-dependent receptors. This high capacity for protein synthesis, efficient amino acid utilization, and substrate transport may contribute to the fast growth rates of the copiotrophic bacterium P. haloplanktis in its natural environments.

Optimization of kappa-carrageenase production by Pseudoalteromonas sp. AJ5.

OBJECTIVES: To optimize the culture conditions of Pseudoalteromonas sp. AJ5 for a higher production of extracellular kappa-carrageenase. METHODS: A kappa-carrageenan-degrading bacterium AJ5, capable of utilizing kappa-carrageenan as sole source of carbon and energy, was isolated from the intestine of holothurian Apostichopus japonicus by enrichment culture technique. The strain was identified as the genus Pseudoalteromonas sp. according to its morphological and physiological characterization and 16S rRNA gene analysis. Culture conditions for the bacterium were standardized for the maximal productivity of the extracellular kappa-carrageenase by the single factor and orthogonal tests. RESULTS: According to the single factor test, the optimal culture conditions were: 75 mL medium in 250 mL Erlenmeyer flask, shaking speed of 150 r/min, inoculum's volume 7%, and pH 8.0. Based on the single factor and orthogonal tests the optimal medium components were: kappa-carrageenan (1 g/L), beef extract (2 g/L ), NaCl (20 g/L), K2HPO4.3H2O (1 g/L), MgSO4.7H2O (0.5 g/L), MnCl2.4H2O (0.2 g/L), FePO4.4H2O (0.01 g/L), with the incubation temperature and time of 28 degrees C and 28 h. CONCLUSION: Pseudoalteromonas sp. AJ5 secreted an extracellular kappa-carrageenase. Under the optimal culture conditions, four-fold increase in kappa-carrageenase activity was achieved as compared to the control.

[Antibacterial activity of sponge associated marine bacterium Pseudoalteromonas sp. NJ6-3-1 regulated by quorum sensing].

OBJECTIVE: We chose a sponge-associated marine-bacterium Pseudoalteromonas piscicida NJ6-3-1 to study whether its antibacterial activity were regulated by quorum sensing. METHODS: We studied the relationship between the antibacterial activity and bacterial density under various growth conditions. To simulate the natural competitive environment, we monitored the antibacterial activity at low cell density when the species were co-cultured with Staphylococcus aureus. RESULTS: Antibacterial activity correlated closely with cell density. Marine bacterium NJ6-3-1 started producing antibacterial compounds when cell density reached the threshold value of OD(630) = 0.4. Some signal molecules existing in the metabolites of S. aureus could induce the production of antibacterial substance by marine-bacteria NJ6-3-1 even cell density below the required threshold. CONCLUSION: The results provide preliminary evidence to support the hypothesis that the antibacterial activity of NJ6-3-1 was regulated by the quorum sensing system of intraspecies and interspecies.

Rapid chemotactic response enables marine bacteria to exploit ephemeral microscale nutrient patches.

Because ocean water is typically resource-poor, bacteria may gain significant growth advantages if they can exploit the ephemeral nutrient patches originating from numerous, small sources. Although this interaction has been proposed to enhance biogeochemical transformation rates in the ocean, it remains questionable whether bacteria are able to efficiently use patches before physical mechanisms dissipate them. Here we show that the rapid chemotactic response of the marine bacterium Pseudoalteromonas haloplanktis substantially enhances its ability to exploit nutrient patches before they dissipate. We investigated two types of patches important in the ocean: nutrient pulses and nutrient plumes, generated for example from lysed algae and sinking organic particles, respectively. We used microfluidic devices to create patches with environmentally realistic dimensions and dynamics. The accumulation of P. haloplanktis in response to a nutrient pulse led to formation of bacterial hot spots within tens of seconds, resulting in a 10-fold higher nutrient exposure for the fastest 20% of the population compared with nonmotile cells. Moreover, the chemotactic response of P. haloplanktis was >10 times faster than the classic chemotaxis model Escherichia coli, leading to twice the nutrient exposure. We demonstrate that such rapid response allows P. haloplanktis to colonize nutrient plumes for realistic particle sinking speeds, with up to a 4-fold nutrient exposure compared with nonmotile cells. These results suggest that chemotactic swimming strategies of marine bacteria in patchy nutrient seascapes exert strong influence on carbon turnover rates by triggering the formation of microscale hot spots of bacterial productivity.

Polymyxin B induces lysis of marine pseudoalteromonads.

Polymyxin B (PMB) is a cationic antibiotic that interacts with the envelopes of gram-negative bacterial cells. The therapeutic use of PMB was abandoned for a long time due to its undesirable side effects; however, the spread of resistance to currently used antibiotics has forced the reevaluation of PMB for clinical use. Previous studies have used enteric bacteria to examine the mode of PMB action, resulting in a somewhat limited understanding of this process. This study examined the effects of PMB on marine pseudoalteromonads and demonstrates that the frequently accepted view that "what is true for Escherichia coli is true for all bacteria" does not hold true. We show here that in contrast to the growth inhibition observed for enteric bacteria, PMB induces lysis of pseudoalteromonads, which is not prevented by high concentrations of divalent cations. Furthermore, we demonstrate that a high membrane voltage is required for the interaction of PMB with the cytoplasmic membranes of pseudoalteromonads, further elucidating the mechanisms by which PMB interacts with the cell envelopes of those gram-negative bacteria.

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).

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.

Laser impact assessment in a biofilm-forming bacterium Pseudoalteromonas carrageenovora using a flow cytometric system.

Impact by pulsed laser irradiations from an Nd:YAG laser on the marine biofilm-forming bacterium Pseudoalteromonas carrageenovora has been studied using a flow cytometric system. The biofilm-forming bacteria in the planktonic state have been irradiated while flowing, and the mortality and bacterial attachment have been determined by exposing TiN coupons in the system. Coupons suspended in the non-irradiated bacterial flow were treated as the control. The fluence used in the study was 0.1 J/cm(2). Three flow rates (14, 28, and 42 cm/min) and two exposure durations (15 and 30 min) were tested. The results showed the increase in bacterial mortality with the decrease in flow rate. The maximum mortality of 27.5% was observed when the flow rate was 14 cm/min. The bacterial attachment increased with the increase in flow rate and exposure duration. The area of bacterial attachment on the experimental coupons exposed to the irradiated sample was significantly lesser than that for the nonirradiated sample. The results thus show in a flowing system, low power pulsed laser irradiations could reduce the bacterial attachment even though it did not cause significant mortality.