Pseudoalteromonas Tunicata Research Articles

Crown ether dopant to reduce ion suppression and improve detection in the liquid microjunction surface sampling probe.

Sodium and potassium are required in agar media for the growth of some microorganisms (e.g., marine bacteria). However, alkali cations are a significant source of contamination for mass spectrometry causing ion suppression and adduct formation. Conventionally, salts can be removed before mass spectrometric analysis with appropriate and often lengthy sample preparation. The direct mass spectrometric sampling of bacterial colonies grown on agar media seeks to minimize or eliminate sample preparation to improve workflow. However, this may exacerbate ion suppression and contamination since these metal cations will degrade spectral quality and limit the rapid profiling of microbial metabolites. Different approaches are needed to sequester sodium and potassium ions to minimize unwanted background interferences. Herein, we use crown ethers (CEs) in combination with a liquid microjunction surface sampling probe (LMJ-SSP) to directly sample the surface of the bacterial colonies from two marine bacteria species (Pseudoalteromonas rubra DSM6842 and Pseudoalteromonas tunicata DSM 14096). CEs (e.g., 18-crown-6 or 15-crown-5) are added to the carrier solvent of the LMJ-SSP, the chemical noise is reduced, and spectra are easier to interpret. The liquid microjunction formed at the tip of LMJ-SSP was used to directly touch bacterial colonies on agar. The carrier solvent was either methanol (100%) or methanol: H2O (50:49.9%) with or without 0.01% CEs. Information-theoretic measures are employed to investigate qualitative changes between spectra before and after adding CEs. Our work demonstrates the capability of CEs to reduce background interferences within the direct profiling of bacterial colonies from agar plates. The data obtained from both P. rubra DSM6842 and P. tunicata DSM 14096 show that CEs can be used to mitigate the salty background and improve compound detection. Our approach can be implemented in natural product discovery using LMJ-SSP to allow fast and accurate detection of interesting/novel compounds.

The impact of interspecific competition on the genomic evolution of Phaeobacter inhibens and Pseudoalteromonas tunicata during biofilm growth.

Interspecific interactions in biofilms have been shown to cause the emergence of community-level properties. To understand the impact of interspecific competition on evolution, we deep-sequenced the dispersal population of mono- and co-culture biofilms of two antagonistic marine bacteria (Phaeobacter inhibens 2.10 and Pseudoalteromononas tunicata D2). Enhanced phenotypic and genomic diversification was observed in the P. tunicata D2 populations under both mono- and co-culture biofilms in comparison to P. inhibens 2.10. The genetic variation was exclusively due to single nucleotide variants and small deletions, and showed high variability between replicates, indicating their random emergence. Interspecific competition exerted an apparent strong positive selection on a subset of P. inhibens 2.10 genes (e.g., luxR, cobC, argH, and sinR) that could facilitate competition, while the P. tunicata D2 population was genetically constrained under competition conditions. In the absence of interspecific competition, the P. tunicata D2 replicate populations displayed high levels of mutations affecting the same genes involved in cell motility and biofilm formation. Our results show that interspecific biofilm competition has a complex impact on genomic diversification, which likely depends on the nature of the competing strains and their ability to generate genetic variants due to their genomic constraints.

Ecogenomics and cultivation reveal distinctive viral-bacterial communities in the surface microlayer of a Baltic Sea slick

Visible surface films, termed slicks, can extensively cover freshwater and marine ecosystems, with coastal regions being particularly susceptible to their presence. The sea-surface microlayer (SML), the upper 1-mm at the air-water interface in slicks (herein slick SML) harbors a distinctive bacterial community, but generally little is known about SML viruses. Using flow cytometry, metagenomics, and cultivation, we characterized viruses and bacteria in a brackish slick SML in comparison to non-slick SML as well as seawater below slick and non-slick areas (subsurface water = SSW). Size-fractionated filtration of all samples distinguished viral attachment to hosts and particles. The slick SML contained higher abundances of virus-like particles, prokaryotic cells, and dissolved organic carbon compared to non-slick SML and SSW. The community of 428 viral operational taxonomic units (vOTUs), 426 predicted as lytic, distinctly differed across all size fractions in the slick SML compared to non-slick SML and SSW. Specific metabolic profiles of bacterial metagenome-assembled genomes and isolates in the slick SML included a prevalence of genes encoding motility and carbohydrate-active enzymes (CAZymes). Several vOTUs were enriched in slick SML, and many virus variants were associated with particles. Nine vOTUs were only found in slick SML, six of them being targeted by slick SML-specific clustered-regularly interspaced short palindromic repeats (CRISPR) spacers likely originating from Gammaproteobacteria. Moreover, isolation of three previously unknown lytic phages for Alishewanella sp. and Pseudoalteromonas tunicata, abundant and actively replicating slick SML bacteria, suggests that viral activity in slicks contributes to biogeochemical cycling in coastal ecosystems.

Hyperspectral Visualization-Based Mass Spectrometry Imaging by LMJ-SSP: A Novel Strategy for Rapid Natural Product Profiling in Bacteria.

Mass spectrometry imaging (MSI) has been widely used to discover natural products (NPs) from underexplored microbiological sources. However, the technique is limited by incompatibility with complicated/uneven surface topography and labor-intensive sample preparation, as well as lengthy compound profiling procedures. Here, liquid micro-junction surface sampling probe (LMJ-SSP)-based MSI is used for rapid profiling of natural products from Gram-negative marine bacteria Pseudoalteromonas on nutrient agar media without any sample preparation. A conductance-based autosampling platform with 1 mm spatial resolution and an innovative multivariant analysis-driven method was used to create one hyperspectral image for the sampling area. NP discovery requires general spatial correlation between m/z and colony location but not highly precise spatial resolution. The hyperspectral image was used to annotate different m/z by straightforward color differences without the need to directly interrogate the spectra. To demonstrate the utility of our approach, the rapid analysis of Pseudoalteromonas rubra DSM6842, Pseudoalteromonas tunicata DSM14096, Pseudoalteromonas piscicida JCM20779, and Pseudoalteromonas elyakovii ATCC700519 cultures was directly performed on Agar. Various natural products, including prodiginine and tambjamine analogues, were quickly identified from the hyperspectral image, and the dynamic extracellular environment was shown with compound heatmaps. Hyperspectral visualization-based MSI is an efficient and sensitive strategy for direct and rapid natural product profiling from different Pseudoalteromonas strains.

Discovery of a Tambjamine Gene Cluster in Streptomyces Suggests Convergent Evolution in Bipyrrole Natural Product Biosynthesis.

While bacterial natural products are a valuable source of therapeutics, the molecules produced by most biosynthetic gene clusters remain unknown. Tambjamine YP1, produced by Pseudoalteromonas tunicata, is partially derived from fatty acids siphoned from primary metabolism. A structurally similar tambjamine produced by Streptomyces, BE-18591, had not been linked to a gene cluster. Using enzymes putatively implicated in the construction of these two tambjamines, we used sequence similarity networks and gene knockout experiments to identify the biosynthetic gene cluster responsible for the production of tambjamine BE-18591 in Streptomyces albus. Despite the structural similarities between YP1 and BE-18591, the biosynthesis of the alkylamine tails of these molecules differs significantly, with the S. albus gene cluster putatively encoding a dedicated system for the construction of the fatty acid precursor to BE-18591. These different pathways in Pseudoalteromonas and Streptomyces suggest that evolutionary convergence is operative, with similar selective pressures leading to the emergence of structurally similar tambjamine natural products using different biosynthetic logic.

ChrII-Encoded DNA Helicase: A Preliminary Study.

DNA helicases are unwinding enzymes that are essential for many cellular processes. Research has suggested that both the model microorganisms of a single chromosome and the model microorganisms of multiple chromosomes adopt DNA helicases encoded by chromosome I. Therefore, studying DNA helicases encoded by chromosome II may lay some foundation for understanding nucleic acid metabolism processes. To prove the existence of DNA helicase encoded by chromosome II and to reveal its difference compared to DNA helicase encoded by chromosome I. The DNA helicases of Pseudoalteromonas spongiae JCM 12884T and Pseudoalteromonas tunicata DSM 14096T were analyzed by sequence alignment and phylogenetic relationships with other known DNA helicases. Then, proteins of P. spongiae JCM 12884T and P. tunicata DSM 14096T were obtained by heterologous expression. N-terminal sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were performed to confirm the form of proteins. A fluorescence resonance energy transfer (FRET) assay was used to measure the activity of helicases. DnaB-pspo and DnaB-ptun belong to the same family, the PRK08840 superfamily, and form a branch with helicases encoded by chromosome I. YwqA-pspo and YwqA-ptun have similar domains and form another branch with helicases encoded by chromosome II. All four helicases have DNA unwinding activity. YwqA is more efficient than DnaB for DNA unwinding, especially YwqA-pspo, which is encoded by bidirectional replication chromosome II. This is the first study to show that the existence of a DNA helicase encoded by chromosome II, and DNA helicase encoded by chromosome II is more efficient than chromosome I for DNA unwinding.

Coupled Natural Fusion Enzymes in a Novel Biocatalytic Cascade Convert Fatty Acids to Amines.

Tambjamine YP1 is a pyrrole-containing natural product. Analysis of the enzymes encoded in the Pseudoalteromonas tunicata “tam” biosynthetic gene cluster (BGC) identified a unique di-domain biocatalyst (PtTamH). Sequence and bioinformatic analysis predicts that PtTamH comprises an N-terminal, pyridoxal 5′-phosphate (PLP)-dependent transaminase (TA) domain fused to a NADH-dependent C-terminal thioester reductase (TR) domain. Spectroscopic and chemical analysis revealed that the TA domain binds PLP, utilizes l-Glu as an amine donor, accepts a range of fatty aldehydes (C7–C14 with a preference for C12), and produces the corresponding amines. The previously characterized PtTamA from the “tam” BGC is an ATP-dependent, di-domain enzyme comprising a class I adenylation domain fused to an acyl carrier protein (ACP). Since recombinant PtTamA catalyzes the activation and thioesterification of C12 acid to the holo-ACP domain, we hypothesized that C12 ACP is the natural substrate for PtTamH. PtTamA and PtTamH were successfully coupled together in a biocatalytic cascade that converts fatty acids (FAs) to amines in one pot. Moreover, a structural model of PtTamH provides insights into how the TA and TR domains are organized. This work not only characterizes the formation of the tambjamine YP1 tail but also suggests that PtTamA and PtTamH could be useful biocatalysts for FA to amine functional group conversion.

Antifouling Marine Coatings with a Potentially Safer and Sustainable Synthetic Polyphenolic Derivative.

The development of harmless substances to replace biocide-based coatings used to prevent or manage marine biofouling and its unwanted consequences is urgent. The formation of biofilms on submerged marine surfaces is one of the first steps in the marine biofouling process, which facilitates the further settlement of macrofoulers. Anti-biofilm properties of a synthetic polyphenolic compound, with previously described anti-settlement activity against macrofoulers, were explored in this work. In solution this new compound was able to prevent biofilm formation and reduce a pre-formed biofilm produced by the marine bacterium, Pseudoalteromonas tunicata. Then, this compound was applied to a marine coating and the formation of P. tunicata biofilms was assessed under hydrodynamic conditions to mimic the marine environment. For this purpose, polyurethane (PU)-based coating formulations containing 1 and 2 wt.% of the compound were prepared based on a prior developed methodology. The most effective formulation in reducing the biofilm cell number, biovolume, and thickness was the PU-based coating containing an aziridine-based crosslinker and 2 wt.% of the compound. To assess the marine ecotoxicity impact of this compound, its potential to disrupt endocrine processes was evaluated through the modulation of two nuclear receptors (NRs), peroxisome proliferator-activated receptor γ (PPARγ), and pregnane X receptor (PXR). Transcriptional activation of the selected NRs upon exposure to the polyphenolic compound (10 µM) was not observed, thus highlighting the eco-friendliness towards the addressed NRs of this new dual-acting anti-macro- and anti-microfouling agent towards the addressed NRs.

Differential priority effects impact taxonomy and functionality of host-associated microbiomes.

Most multicellular eukaryotes host complex communities of microorganisms, but the factors that govern their assembly are poorly understood. The settlement of specific microorganisms may have a lasting impact on community composition, a phenomenon known as the priority effect. Priority effects of individual bacterial strains on a host's microbiome are, however, rarely studied and their impact on microbiome functionality remains unknown. We experimentally tested the effect of two bacterial strains (Pseudoalteromonas tunicata D2 and Pseudovibrio sp. D323) on the assembly and succession of the microbial communities associated with the green macroalga Ulva australis. Using 16S rRNA gene sequencing and qPCR, we found that both strains exert a priority effect, with strain D2 causing initially strong but temporary taxonomic changes and strain D323 causing weaker but consistent changes. Consistent changes were predominately facilitatory and included taxa that may benefit the algal host. Metagenome analyses revealed that the strains elicited both shared (e.g., depletion of type III secretion system genes) and unique (e.g., enrichment of antibiotic resistance genes) effects on the predicted microbiome functionality. These findings indicate strong idiosyncratic effects of colonizing bacteria on the structure and function of host-associated microbial communities. Understanding the idiosyncrasies in priority effects is key for the development of novel probiotics to improve host condition.

Novel Nematode-Killing Protein-1 (Nkp-1) from a Marine Epiphytic Bacterium Pseudoalteromonas tunicata.

Drug resistance among parasitic nematodes has resulted in an urgent need for the development of new therapies. However, the high re-discovery rate of anti-nematode compounds from terrestrial environments necessitates a new repository for future drug research. Marine epiphytes are hypothesised to produce nematicidal compounds as a defence against bacterivorous predators, thus representing a promising yet underexplored source for anti-nematode drug discovery. The marine epiphytic bacterium Pseudoalteromonas tunicata is known to produce several bioactive compounds. Screening heterologously expressed genomic libraries of P. tunicata against the nematode Caenorhabditis elegans, identified as an E. coli clone (HG8), shows fast-killing activity. Here we show that clone HG8 produces a novel nematode-killing protein-1 (Nkp-1) harbouring a predicted carbohydrate-binding domain with weak homology to known bacterial pore-forming toxins. We found bacteria expressing Nkp-1 were able to colonise the C. elegans intestine, with exposure to both live bacteria and protein extracts resulting in physical damage and necrosis, leading to nematode death within 24 h of exposure. Furthermore, this study revealed C. elegans dar (deformed anal region) and internal hatching may act as a nematode defence strategy against Nkp-1 toxicity. The characterisation of this novel protein and putative mode of action not only contributes to the development of novel anti-nematode applications in the future but reaffirms the potential of marine epiphytic bacteria as a new source of novel biomolecules.

Assessment of the environmental compatibility and antifouling performance of an innovative biocidal and foul-release multifunctional marine coating

The control of marine biofouling has raised serious environmental concerns, thus the continuous release of toxic and persistent biocidal agents applied as anti-biofouling coatings have triggered the search for non-toxic strategies. However, most of them still lack rigorous evaluation of their ecotoxicity and antifouling effects under real scenarios and their correlation with simulated assays. In this work, the biocide releasing risk and ecotoxicity of a biocidal and foul-release polydimethylsiloxane (PDMS)-based marine coating containing grafted Econea biocide (<0.6 wt.%) were evaluated under simulated real mechanical wear conditions at a pilot-scale system, and under extreme wear scenarios (washability settings). The coating system demonstrated low environmental impact against the model Vibrio fischeri bacterium and marine algae, associated with the effective biocide grafting in the coating matrix and subsequent biocide release minimization. This multifunctional coating system also showed auspicious antifouling (AF) effects, with an AF performance index significantly higher (API > 89) than a single foul-release system (AF < 40) after two and half years at a real immersion scenario in the Portuguese shore of the Atlantic Ocean. These field results corroborated the antibiofilm performance evaluated with Pseudoalteromonas tunicata at simulated dynamic marine conditions after seven-week assays. This eco-friendly multifunctional strategy, validated by both simulated testing conditions and real field tests, is believed to be a powerful tool for the development of AF technologies and a potential contribution to the quest for new environmentally friendly antifouling solutions.

Experimental Assessment of the Performance of Two Marine Coatings to Curb Biofilm Formation of Microfoulers

Biofilms formed on submerged marine surfaces play a critical role in the fouling process, causing increased fuel consumption, corrosion, and high maintenance costs. Thus, marine biofouling is a major issue and motivates the development of antifouling coatings. In this study, the performance of two commercial marine coatings, a foul-release silicone-based paint (SilRef) and an epoxy resin (EpoRef), was evaluated regarding their abilities to prevent biofilm formation by Cyanobium sp. and Pseudoalteromonas tunicata (common microfoulers). Biofilms were developed under defined hydrodynamic conditions to simulate marine settings, and the number of biofilm cells, wet weight, and thickness were monitored for 7 weeks. The biofilm structure was analyzed by confocal laser scanning microscopy (CLSM) at the end-point. Results demonstrated that EpoRef surfaces were effective in inhibiting biofilm formation at initial stages (until day 28), while SilRef surfaces showed high efficacy in decreasing biofilm formation during maturation (from day 35 onwards). Wet weight and thickness analysis, as well as CLSM data, indicate that SilRef surfaces were less prone to biofilm formation than EpoRef surfaces. Furthermore, the efficacy of SilRef surfaces may be dependent on the fouling microorganism, while the performance of EpoRef was strongly influenced by a combined effect of surface and microorganism.

Slr4, a newly identified S-layer protein from marine Gammaproteobacteria, is a major biofilm matrix component.

S‐layers are paracrystalline proteinaceous lattices that surround prokaryotic cells, forming a critical interface between the cells and their extracellular environment. Here, we report the discovery of a novel S‐layer protein present in the Gram‐negative marine organism, Pseudoalteromonas tunicata D2. An uncharacterized protein (EAR28894) was identified as the most abundant protein in planktonic cultures and biofilms. Bioinformatic methods predicted a beta‐helical structure for EAR28894 similar to the Caulobacter S‐layer protein, RsaA, despite sharing less than 20% sequence identity. Transmission electron microscopy revealed that purified EAR28894 protein assembled into paracrystalline sheets with a unique square lattice symmetry and a unit cell spacing of ~9.1 nm. An S‐layer was found surrounding the outer membrane in wild‐type cells and completely removed from cells in an EAR28894 deletion mutant. S‐layer material also appeared to be “shed” from wild‐type cells and was highly abundant in the extracellular matrix where it is associated with outer membrane vesicles and other matrix components. EAR28894 and its homologs form a new family of S‐layer proteins that are widely distributed in Gammaproteobacteria including species of Pseudoalteromonas and Vibrio, and found exclusively in marine metagenomes. We propose the name Slr4 for this novel protein family.

Synthesis of N-acyl amide natural products using a versatile adenylating biocatalyst.

Natural products are secondary metabolites produced by many different organisms such as bacteria, fungi and plants. These biologically active molecules have been widely exploited for clinical application. Here we investigate TamA, a key enzyme from the biosynthetic pathway of tambjamine YP1, an acylated bipyrrole that is produced by the marine microorganism Pseudoalteromonas tunicata. TamA is a didomain enzyme composed of a catalytic adenylation (ANL) and an acyl carrier protein (ACP) domain that together control the fatty acid chain length of the YP1. Here we show that the TamA ANL domain alone can be used to generate a range of acyl adenylates that can be captured by a number of amines thus leading to the production of a series of fatty N-acyl amides. We exploit this biocatalytic promiscuity to produce the recently discovered class of N-acyl histidine amide natural products from Legionella pneumophila.

Causes and Consequences of a Variant Strain of Phaeobacter inhibens With Reduced Competition.

Phaeobacter inhibens 2.10 is an effective biofilm former and colonizer of marine surfaces and has the ability to outcompete other microbiota. During biofilm dispersal P. inhibens 2.10 produces heritable phenotypic variants, including those that have a reduced ability to inhibit the co-occurring bacterium Pseudoalteromonas tunicata. However, the genetic changes that underpin the phenotypic variation and what the ecological consequences are for variants within the population are unclear. To answer these questions we sequenced the genomes of strain NCV12a1, a biofilm variant of P. inhibens 2.10 with reduced inhibitory activity and the P. inhibens 2.10 WT parental strain. Genome wide analysis revealed point mutations in genes involved in synthesis of the antibacterial compound tropodithietic acid (TDA) and indirectly in extracellular polymeric substances (EPS) production. However, confocal laser scanning microscopy analyses found little differences in biofilm growth between P. inhibens 2.10 WT (parental) and NCV12a1. P. inhibens NCV12a1 was also not outcompeted in co-cultured biofilms with P. tunicata, despite its reduced inhibitory activity, rather these biofilms were thicker than those produced when the WT strain was co-cultured with P. tunicata. Notably, dispersal populations from biofilms of P. inhibens NCV12a1 had a higher proportion of WT-like morphotypes when co-cultured with P. tunicata. These observations may explain why the otherwise non-inhibiting variant persists in the presence of a natural competitor, adding to our understanding of the relative importance of genetic diversification in microbial biofilms.

Eco-friendly non-biocide-release coatings for marine biofouling prevention

Environmental concerns have been changing the way of looking for solutions to problems. The hydrosphere, together with its biosphere, has been feeling the impact of many pollutants, used for instance in the marine industry for economic reasons or lack of knowledge of their effects. In particular biocides, applied as coatings in paints, are released into the waters becoming toxic and persistent extending their action to an area far beyond the initial coated surface they should protect. In order to minimize these side effects, two biocides, Irgarol (I) and Econea (E), were covalently attached to polyurethane (PU) and foul-release silicone based (PDMS) marine paints through an isocyanate linker. Their antifouling bioactivity was better in PDMS coatings, both for single (Econea) and combined biocides (E/I ratio = 1.5) with contents lower than 0.6 wt%. The treated samples remained almost clean after more than one year immersion in the Portuguese shore of the Atlantic Ocean, and after about 24 weeks under the tropical conditions of Singapore (Fouling rate < 1%). Complementary biofilm adhesion susceptibility tests against Pseudoalteromonas tunicata D2 showed adhesion reduction higher than 90% for PU formulations containing single biocides and close to 100% for PDMS with combined biocides. The eco-toxicity assessment evidenced a low environmental impact, in accordance with the European standards. In addition, shipping field trial tests showed the best antifouling performance for the Econea-based PDMS formulations (E = 0.6 wt%), which remained clean for about nine months in open seawaters, proving the efficacy of this non-release strategy, when applied under dynamic conditions.

The carbon chain-selective adenylation enzyme TamA: the missing link between fatty acid and pyrrole natural product biosynthesis.

The marine bacterium Pseudoalteromonas tunicata produces the bipyrrole antibiotic tambjamine YP1. This natural product is built from common amino acid and fatty acid building blocks in a biosynthetic pathway that is encoded in the tam operon which contains 19 genes. The exact role that each of these Tam proteins plays in tambjamine biosynthesis is not known. Here, we provide evidence that TamA initiates the synthesis and controls the chain length of the essential tambjamine fatty amine tail. Sequence analysis suggests the unusual TamA is comprised of an N-terminal adenylation (ANL) domain fused to a C-terminal acyl carrier protein (ACP). Mass spectrometry analysis of recombinant TamA revealed the surprising presence of bound C11 and C12 acyl-adenylate intermediates. Acylation of the ACP domain was observed upon attachment of the phosphopantetheine (4'-PP) arm to the ACP. We also show that TamA can transfer fatty acids ranging in chain length from C6-C13 to an isolated ACP domain. Thus TamA bridges the gap between primary and secondary metabolism by linking fatty acid and pyrrole biosynthetic pathways.

Phaeobacter porticola sp. nov., an antibiotic-producing bacterium isolated from a sea harbour.

Three heterotrophic, aerobic, brown-pigmented strains, designated P97T, P100 and P104, were isolated from a harbour in the southern North Sea. Phylogenetic analysis of 16S rRNA gene sequences revealed that the isolates are affiliated to the genus Phaeobacter. In silico DNA-DNA hybridization of the genome of strain P97T against those of existing type species indicated that P97T represents a novel species within the genus Phaeobacter, with Phaeobacter inhibens T5T as the closest described organism (29.6 % DNA-DNA relatedness) followed by P. gallaeciensis CIP 105210T (26.4 %). DNA-DNA hybridization demonstrated that the three new strains belong to the same species. The new isolates inhibited Pseudoalteromonas tunicata DSM 14096T, and were Gram-stain-negative, catalase- and oxidase-positive, chemo-organoheterotrophic and motile. Growth occurred at pH 6.5-9.5 (optimum 7.0-8.0) and at 4-30 °C (optimum 20-28 °C). The strains required NaCl for growth. The salinity range was 0.5-6.0 % (w/v) NaCl for P97T and P100, and 0.5-5.0 % for P104, lower than values described for Phaeobacter gallaeciensis and Phaeobacter inhibens. The optimum NaCl concentration for strains P97T and P104 was 2.0-4.0 %, and for P100 was 2.0-3.0 %. Fatty acids (>1 %) comprised 18 : 1ω7c, 16 : 0, 18 : 1 ω7c 11-methyl, 18 : 0, 12 :1, 18 : 2ω7c,12, 10 : 0 3-OH and 12 : 0 3-OH. Polar lipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an aminolipid, one unknown lipid and one additional unknown lipid in strain P97T. The major respiratory quinone was Q10. Based on phylogenetic and phenotypic differences, the strains represent a novel species in the genus Phaeobacter, for which the name Phaeobacter porticola sp. nov. is proposed. The type strain is P97T (=DSM 103148T=LMG 29594T).

Effect of interspecific competition on trait variation in Phaeobacter inhibens biofilms.

Interspecific competition between bacteria shapes community dynamics, causing evolutionary changes that affect life history traits. Here, we studied the role of interspecific competition on the generation of trait diversity using a two-species model system of marine, surface-associated bacteria. Bacterial biofilms of Phaeobacter inhibens were established alone or in competition with Pseudoalteromonas tunicata and phenotypic traits of dispersal cells were assessed during biofilm development. P. inhibens dispersal isolates from competition biofilms displayed less phenotypic variation, were superior competitors, were less susceptible to predation, and reached higher planktonic biomass than isolates from noncompetition biofilms. Moreover, the maintenance of competitive ability exhibited by individual dispersal isolates from competition biofilms did not result in an obvious reduction (measured as a negative trait correlation) in other traits, but was rather positively correlated with planktonic growth. However, where negative correlations between traits were found, they were exhibited by individuals derived from noncompetitive biofilms, whose populations also had a higher degree of trait variation than those from biofilms experiencing competition. Our observations indicate that interspecific competition during biofilm formation is important for maintaining both competitive ability and affects variation in ecologically relevant traits. Given that most bacteria in biofilms exist in diverse, multispecies communities, an understanding of how bacteria respond to biotic factors such as interspecific competition is critical for understanding the dynamics of bacterial populations in both ecological and evolutionary time.

MICROBIAL ECOLOGY AND BIOFILMS IN THE TAPHONOMY OF SOFT TISSUES

Authigenic mineralization of embryos (and potentially other soft-bodied organisms) requires first stabilization of cells against rapid self-autolytic destruction, and secondly a role for bacterial biofilms that preserve rather than destructively consume tissue. We predict that the ecology of the second stage in preservation will depend on environmental effects on the bacterial species present, coupled with mutual interactions between the bacteria themselves. We have created a simple experimental model made up of two antagonistic marine bacterial species, tested on a taphonomic target of autolysis-inhibited killed marine embryos. Pseudoalteromonas tunicata forms a three-dimensional preserving biofilm with killed embryos, whereas P. luteoviolacea destroys embryo tissue. Our model system allows controlled laboratory tests of microbial interactions under taphonomic conditions selected to test inferred paleoenvironments present in Lagerstatten. We varied environmental conditions one at a time, and observed the taphonomic outcome for killed embryos in the presence of each species alone, and with both species present in direct competition. Parameters tested include temperature, pH, oxygen level, salinity, and nutrient state. Pseudoalteromonas tunicata was robust in generating preserving biofilm pseudomorphs over a wide range of conditions. In competition, P. luteoviolacea destruction dominated in most conditions. However, we identified conditions of temperature, pH, and salinity where P. luteoviolacea grows poorly and preservation by P. tunicata dominates. Elevated external nutrients reduced the fidelity of P. tunicata pseudomorphs. In low oxygen, P. tunicata physiology was altered and it switched to become a destroyer, dramatically showing the extent to which environment can determine taphonomic outcomes.