Description of Palleronia rufa sp. nov., a biofilm-forming and AHL-producing Rhodobacteraceae, reclassification of Hwanghaeicola aestuarii as Palleronia aestuarii comb. nov., Maribius pontilimi as Palleronia pontilimi comb. nov., Maribius salinus as Palleronia salina comb. nov., Maribius pelagius as Palleronia pelagia comb. nov. and emended description of the genus Palleronia.

Strain MOLA 401T was isolated from marine waters in the southwest lagoon of New Caledonia and was shown previously to produce an unusual diversity of quorum sensing signaling molecules. This strain was Gram-negative, formed non-motile cocci and colonies were caramel. Optimum growth conditions were 30°C, pH 8 and 3% NaCl (w/v). Based on 16S rRNA gene sequence analysis, this strain was found to be closely related to Pseudomaribius aestuariivivens NBRC 113039T (96.9% of similarity), Maribius pontilimi DSM 104950T (96.4% of similarity) and Palleronia marisminoris LMG 22959T (96.3% of similarity), belonging to the Roseobacter group within the family Rhodobacteraceae. As its closest relatives, strain MOLA 401T is able to form a biofilm on polystyrene, supporting the view of Roseobacter group strains as prolific surface colonizers. An in-depth genomic study allowed us to affiliate strain MOLA 401T as a new species of genus Palleronia and to reaffiliate some of its closest relatives in this genus. Consequently, we describe strain MOLA 401T (DSM 106827T=CIP 111607T=BBCC 401T) for which we propose the name Palleronia rufa sp. nov. We also propose to emend the description of the genus Palleronia and to reclassify Maribius and Hwanghaeicola species as Palleronia species.

Genomic reconstructions and potential metabolic strategies of generalist and specialist heterotrophic bacteria associated with an estuary Synechococcus culture.

Interactions between photoautotrophs and heterotrophs are central to marine microbial ecosystems. Synechococcus are dominant marine phototrophs, and they are frequently associated with heterotrophic bacteria. These co-cultures provide a useful research system to investigate photoautotroph-heterotroph interactions in marine systems. Bacteria within the Roseobacter clade and Flavobacteria are two of the main bacterial lineages that exhibit intimate associations with Synechococcus populations. We conducted metagenomic analyses of a Synechococcus culture, followed by genomic binning of metagenomic contigs, and recovered five nearly complete genomes, including members of the Roseobacter clade (i.e. Marivita sp. XM-24) and Flavobacteria (i.e. Fluviicola sp. XM-24). Marivita sp. XM-24 is an ecological generalist of the Roseobacter clade and displays diverse metabolic capacities for the acquisition of nutrients and energy sources. Specifically, the genome contained numerous gene complements involved in the uptake and metabolism of nitrogen- and phosphorus-containing inorganic and organic compounds, in addition to the potential for aerobic anoxygenic photosynthesis, oxidation of carbon monoxide, inorganic sulfur oxidation, DMSP demethylation and PHA metabolism. The genome of the Flavobacteria representative, Fluviicola sp. XM-24, contained numerous peptidases, glycoside hydrolases, adhesion-related proteins and genes involved in gliding motility. Fluviicola sp. XM-24 likely specialize in the degradation of high molecular weight compound exudates from Synechococcus cells, including polysaccharides and polypeptides via attachment to particles, surfaces or cells. The distinct metabolic strategies identified within several heterotrophic bacteria that are associated with Syneochococcus cells provide insights into their lifestyles and nutrient utilization patterns, in addition to their interactions with photoautotrophs. Biological interactions, including mutualism, competition and antagonism, shape the microbial community structure of marine environments and are critical for understanding biogeochemical cycling in the ocean. These results provide valuable insights into the nature of interactions between dominant marine photoautotrophs and associated bacterial heterotrophs.

Manganese Oxide Biomineralization Provides Protection against Nitrite Toxicity in a Cell-Density-Dependent Manner.

Manganese biomineralization is a widespread process among bacteria and fungi. To date, there is no conclusive experimental evidence for how and if this process impacts microbial fitness in the environment. Here, we show how a model organism for manganese oxidation is growth inhibited by nitrite, and that this inhibition is mitigated in the presence of manganese. We show that such manganese-mediated mitigation of nitrite inhibition is dependent on the culture inoculum size, and that manganese oxide (MnOX) forms granular precipitates in the culture, rather than sheaths around individual cells. We provide evidence that MnOX protection involves both its ability to catalyze nitrite oxidation into (nontoxic) nitrate under physiological conditions and its potential role in influencing processes involving reactive oxygen species (ROS). Taken together, these results demonstrate improved microbial fitness through MnOX deposition in an ecological setting, i.e., mitigation of nitrite toxicity, and point to a key role of MnOX in handling stresses arising from ROS.IMPORTANCE We present here a direct fitness benefit (i.e., growth advantage) for manganese oxide biomineralization activity in Roseobacter sp. strain AzwK-3b, a model organism used to study this process. We find that strain AzwK-3b in a laboratory culture experiment is growth inhibited by nitrite in manganese-free cultures, while the inhibition is considerably relieved by manganese supplementation and manganese oxide (MnOX) formation. We show that biogenic MnOX interacts directly with nitrite and possibly with reactive oxygen species and find that its beneficial effects are established through formation of dispersed MnOX granules in a manner dependent on the population size. These experiments raise the possibility that manganese biomineralization could confer protection against nitrite toxicity to a population of cells. They open up new avenues of interrogating this process in other species and provide possible routes to their biotechnological applications, including in metal recovery, biomaterials production, and synthetic community engineering.

Increased biofilm formation due to high-temperature adaptation in marine Roseobacter.

Ocean temperatures will increase significantly over the next 100 years due to global climate change1. As temperatures increase beyond current ranges, it is unclear how adaptation will impact the distribution and ecological role of marine microorganisms2. To address this major unknown, we imposed a stressful high-temperature regime for 500 generations on a strain from the abundant marine Roseobacter clade. High-temperature-adapted isolates significantly improved their fitness but also increased biofilm formation at the air-liquid interface. Furthermore, this altered lifestyle was coupled with genomic changes linked to biofilm formation in individual isolates, and was also dominant in evolved populations. We hypothesize that the increasing biofilm formation was driven by lower oxygen availability at elevated temperature, and we observe a relative fitness increase at lower oxygen. The response is uniquely different from that of Escherichia coli adapted to high temperature3 (only 3% of mutated genes were shared in both studies). Thus, future increased temperatures could have a direct effect on organismal physiology and an indirect effect via a decrease in ocean oxygen solubility, leading to an alteration in microbial lifestyle.

Aestuarium zhoushanense gen. nov., sp. nov., Isolated from the Tidal Flat.

A gram-stain-negative, aerobic, ovoid or short rod-shaped, and non-motile strain, designed G7T was isolated from a tidal flat sample collected from the coast of East Sea in Zhoushan, China. Strain G7T grew at 4-40 °C and pH 6.0-9.0 (optimum, 28 °C and pH 7.5) and with 0-7% (w/v) NaCl (optimum, 1%). The predominant respiratory quinone was Q-10 and the major fatty acids (>10%) identified were C18:1 ω7c, C16:0 and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The polar lipids of strain G7T consisted of phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, and four unidentified lipids. The genomic DNA G+C content was 56.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain G7T formed a distinct lineage belonging to the Roseobacter clade of the family Rhodobacteraceae. On the basis of morphological, physiological, and chemotaxonomic characteristics, together with the results of phylogenetic analysis, strain G7T is described as a novel species in a new genus, for which the name Aestuarium zhoushanense gen. nov., sp. nov. (type strain G7T = MCCC 1K03229T = KCTC 52584T) is proposed.

Bacterial transcriptome remodeling during sequential co-culture with a marine dinoflagellate and diatom.

In their role as primary producers, marine phytoplankton modulate heterotrophic bacterial activities through differences in the types and amounts of organic matter they release. This study investigates the transcriptional response of bacterium Ruegeria pomeroyi, a member of the Roseobacter clade known to affiliate with diverse phytoplankton groups in the ocean, during a shift in phytoplankton taxonomy. The bacterium was initially introduced into a culture of the dinoflagellate Alexandrium tamarense, and then experienced a change in phytoplankton community composition as the diatom Thalassiosira pseudonana gradually outcompeted the dinoflagellate. Samples were taken throughout the 30-day experiment to track shifts in bacterial gene expression informative of metabolic and ecological interactions. Transcriptome data indicate fundamental differences in the exometabolites released by the two phytoplankton. During growth with the dinoflagellate, gene expression patterns indicated that the main sources of carbon and energy for R. pomeroyi were dimethysulfoniopropionate (DMSP), taurine, methylated amines, and polyamines. During growth with the diatom, dihydroxypropanesulfonate (DHPS), xylose, ectoine, and glycolate instead appeared to fuel the bulk of bacterial metabolism. Expression patterns of genes for quorum sensing, gene transfer agent, and motility suggest that bacterial processes related to cell communication and signaling differed depending on which phytoplankton species dominated the co-culture. A remodeling of the R. pomeroyi transcriptome implicating more than a quarter of the genome occurred through the change in phytoplankton regime.

Nutrient recycling facilitates long-term stability of marine microbial phototroph-heterotroph interactions.

Biological interactions underpin the functioning of marine ecosystems, be it via competition, predation, mutualism or symbiosis processes. Microbial phototroph-heterotroph interactions propel the engine that results in the biogeochemical cycling of individual elements, and they are critical for understanding and modelling global ocean processes. Unfortunately, studies thus far have focused on exponentially growing cultures in nutrient-rich media, meaning knowledge of such interactions under in situ conditions is rudimentary at best. Here, we have performed long-term phototroph-heterotroph co-culture experiments under nutrient-amended and natural seawater conditions, and show that it is not the concentration of nutrients but rather their circulation that maintains a stable interaction and a dynamic system. Using the Synechococcus-Roseobacter interaction as a model phototroph-heterotroph case study, we show that although Synechococcus is highly specialized for carrying out photosynthesis and carbon fixation, it relies on the heterotroph to remineralize the inevitably leaked organic matter, making nutrients circulate in a mutualistic system. In this sense we challenge the general belief that marine phototrophs and heterotrophs compete for the same scarce nutrients and niche space, and instead suggest that these organisms more probably benefit from each other because of their different levels of specialization and complementarity within long-term stable-state systems.

Genomic, physiologic, and proteomic insights into metabolic versatility in Roseobacter clade bacteria isolated from deep-sea water.

Roseobacter clade bacteria are ubiquitous in marine environments and now thought to be significant contributors to carbon and sulfur cycling. However, only a few strains of roseobacters have been isolated from the deep-sea water column and have not been thoroughly investigated. Here, we present the complete genomes of phylogentically closed related Thiobacimonas profunda JLT2016 and Pelagibaca abyssi JLT2014 isolated from deep-sea water of the Southeastern Pacific. The genome sequences showed that the two deep-sea roseobacters carry genes for versatile metabolisms with functional capabilities such as ribulose bisphosphate carboxylase-mediated carbon fixation and inorganic sulfur oxidation. Physiological and biochemical analysis showed that T. profunda JLT2016 was capable of autotrophy, heterotrophy, and mixotrophy accompanied by the production of exopolysaccharide. Heterotrophic carbon fixation via anaplerotic reactions contributed minimally to bacterial biomass. Comparative proteomics experiments showed a significantly up-regulated carbon fixation and inorganic sulfur oxidation associated proteins under chemolithotrophic conditions compared to heterotrophic conditions. Collectively, rosebacters show a high metabolic flexibility, suggesting a considerable capacity for adaptation to the marine environment.

Biogeography and environmental genomics of the Roseobacter-affiliated pelagic CHAB-I-5 lineage.

The identification and functional characterization of microbial communities remains a prevailing topic in microbial oceanography as information on environmentally relevant pelagic prokaryotes is still limited. The Roseobacter group, an abundant lineage of marine Alphaproteobacteria, can constitute large proportions of the bacterioplankton. Roseobacters also occur associated with eukaryotic organisms and possess streamlined as well as larger genomes from 2.2 to >5 Mpb. Here, we show that one pelagic cluster of this group, CHAB-I-5, occurs globally from tropical to polar regions and accounts for up to 22% of the active North Sea bacterioplankton in the summer. The first sequenced genome of a CHAB-I-5 organism comprises 3.6 Mbp and exhibits features of an oligotrophic lifestyle. In a metatranscriptome of North Sea surface waters, 98% of the encoded genes were present, and genes encoding various ABC transporters, glutamate synthase and CO oxidation were particularly upregulated. Phylogenetic gene content analyses of 41 genomes of the Roseobacter group revealed a unique cluster of pelagic organisms distinct from other lineages of this group, highlighting the adaptation to life in nutrient-depleted environments.

CO2 leaking from sub-seabed storage: Responses of two marine bacteria strains.

Carbon capture and storage (CCS) in stable geological locations is one of the options to mitigate the negative effects of global warming produced by the increase in CO2 concentrations in the atmosphere. A CO2 leak is one of the risks associated with this strategy. Marine bacteria attached to the sediment may be affected by an acidification event. Responses of two marine strains (Roseobacter sp. CECT 7117 and Pseudomonas litoralis CECT 7670) were assessed under different scenarios using a range of pH values (7.8, 7, 6.5, 6, and 5.5) to mimic a CO2 leak. A CO2 injection system was used to simulate an escape from a stable sub-seabed. Growth rate (µ), cell number, inhibition of Relative Inhibitory Effect (RI CO2) and inhibited population were analysed as endpoints. P. litoralis showed more sensitivity to high CO2 concentrations than Roseobacter sp. Our results highlight the diversity and resistance in marine bacteria and their capacity to adapt under a stressful CO2 leakage.

Multiple opportunistic pathogens can cause a bleaching disease in the red seaweed Delisea pulchra.

While macroalgae (or seaweeds) are increasingly recognized to suffer from disease, in most cases the causative agents are unknown. The model macroalga Delisea pulchra is susceptible to a bleaching disease and previous work has identified two epiphytic bacteria, belonging to the Roseobacter clade, that cause bleaching under laboratory conditions. However, recent environmental surveys have shown that these in vitro pathogens are not abundant in naturally bleached D. pulchra, suggesting the presence of other pathogens capable of causing this algal disease. To test this hypothesis, we cultured bacteria that were abundant on bleached tissue across multiple disease events and assessed their ability to cause bleaching disease. We identified the new pathogens Alteromonas sp. BL110, Aquimarina sp. AD1 and BL5 and Agarivorans sp BL7 that are phylogenetically diverse, distinct from the previous two pathogens and can also be found in low abundance in healthy individuals. Moreover, we found that bacterial communities of diseased individuals that were infected with these pathogens were less diverse and more divergent from each other than those of healthy algae. This study demonstrates that multiple and opportunistic pathogens can cause the same disease outcome for D. pulchra and we postulate that such pathogens are more common in marine systems than previously anticipated.

Biofilm plasmids with a rhamnose operon are widely distributed determinants of the 'swim-or-stick' lifestyle in roseobacters.

Alphaproteobacteria of the metabolically versatile Roseobacter group (Rhodobacteraceae) are abundant in marine ecosystems and represent dominant primary colonizers of submerged surfaces. Motility and attachment are the prerequisite for the characteristic 'swim-or-stick' lifestyle of many representatives such as Phaeobacter inhibens DSM 17395. It has recently been shown that plasmid curing of its 65-kb RepA-I-type replicon with >20 genes for exopolysaccharide biosynthesis including a rhamnose operon results in nearly complete loss of motility and biofilm formation. The current study is based on the assumption that homologous biofilm plasmids are widely distributed. We analyzed 33 roseobacters that represent the phylogenetic diversity of this lineage and documented attachment as well as swimming motility for 60% of the strains. All strong biofilm formers were also motile, which is in agreement with the proposed mechanism of surface attachment. We established transposon mutants for the four genes of the rhamnose operon from P. inhibens and proved its crucial role in biofilm formation. In the Roseobacter group, two-thirds of the predicted biofilm plasmids represent the RepA-I type and their physiological role was experimentally validated via plasmid curing for four additional strains. Horizontal transfer of these replicons was documented by a comparison of the RepA-I phylogeny with the species tree. A gene content analysis of 35 RepA-I plasmids revealed a core set of genes, including the rhamnose operon and a specific ABC transporter for polysaccharide export. Taken together, our data show that RepA-I-type biofilm plasmids are essential for the sessile mode of life in the majority of cultivated roseobacters.

Bacteria may induce the secretion of mucin-like proteins by the diatom Phaeodactylum tricornutum.

Benthic diatoms live in photoautotrophic/heterotrophic biofilm communities embedded in a matrix of secreted extracellular polymeric substances. Closely associated bacteria influence their growth, aggregation, and secretion of exopolymers. We have studied a diatom/bacteria model community, in which a marine Roseobacter strain is able to grow with secreted diatom exopolymers as a sole source of carbon. The strain influences the aggregation of Phaeodactylum tricornutum by inducing a morphotypic transition from planktonic, fusiform cells to benthic, oval cells. Analysis of the extracellular soluble proteome of P. tricornutum in the presence and absence of bacteria revealed constitutively expressed newly identified proteins with mucin-like domains that appear to be typical for extracellular diatom proteins. In contrast to mucins, the proline-, serine-, threonine-rich (PST) domains in these proteins were also found in combination with protease-, glucosidase- and leucine-rich repeat-domains. Bioinformatic functional predictions indicate that several of these newly identified diatom-specific proteins may be involved in algal defense, intercellular signaling, and aggregation.

Potential DMSP-degrading Roseobacter clade dominates endosymbiotic microflora of Pyrodinium bahamense var. compressum (Dinophyceae) in vitro.

Many aspects of the biology and ecology of the toxic dinoflagellate Pyrodinium bahamense var. compressum are still poorly understood. In this brief note, we present identification of its associated intracellular bacteria or endosymbionts via PCR cloning and 16s rRNA gene sequencing and their localization by confocal microscopy, a first for Pyrodinium. The most frequently observed species in the endosymbiotic microflora were from Roseobacter clade (Alphaproteobacteria, 68%) and Gilvibacter sediminis (Flavobacteriaceae, 20%). Roseobacter lineage, the most abundant taxa in this study, is known to be involved in dimethylsulfoniopropionate metabolism which is highly produced in dinoflagellates-a possible strong factor shaping the structure of the associated bacterial community.

Homoserine Lactones, Methyl Oligohydroxybutyrates, and Other Extracellular Metabolites of Macroalgae-Associated Bacteria of the Roseobacter Clade: Identification and Functions.

Twenty-four strains of marine Roseobacter clade bacteria were isolated from macroalgae and investigated for the production of quorum-sensing autoinducers, N-acylhomoserine lactones (AHLs). GC/MS analysis of the extracellular metabolites allowed us to evaluate the release of other small molecules as well. Nineteen strains produced AHLs, ranging from 3-OH-C10:0-HSL (homoserine lactone) to (2E,11Z)-C18:2-HSL, but no specific phylogenetic or ecological pattern of individual AHL occurrence was observed when cluster analysis was performed. Other identified compounds included indole, tropone, methyl esters of oligomers of 3-hydroxybutyric acid, and various amides, such as N-9-hexadecenoylalanine methyl ester (9-C16:1-NAME), a structural analogue of AHLs. Several compounds were tested for their antibacterial and antialgal activity on marine isolates likely to occur in the habitat of the macroalgae. Both AHLs and 9-C16:1-NAME showed high antialgal activity against Skeletonema costatum, whereas their antibacterial activity was low.

Induction of Larval Settlement in the Reef Coral Porites astreoides by a Cultivated Marine Roseobacter Strain.

Successful larval settlement and recruitment by corals is critical for the survival of coral reef ecosystems. Several closely related strains of γ-proteobacteria have been identified as cues for coral larval settlement, but the inductive properties of other bacterial taxa naturally occurring in reef ecosystems have not yet been explored. In this study, we assayed bacterial strains representing taxonomic groups consistently detected in corals for their ability to influence larval settlement in the coral Porites astreoides. We identified one α-proteobacterial strain, Roseivivax sp. 46E8, which significantly increased larval settlement in P. astreoides. Logarithmic growth phase (log phase) cell cultures of Roseivivax sp. 46E8 and filtrates (0.22µm) from log phase Roseivivax sp. 46E8 cultures significantly increased settlement, suggesting that an extracellular settlement factor is produced during active growth phase. Filtrates from log phase cultures of two other bacterial isolates, Marinobacter sp. 46E3, and Cytophaga sp. 46B6, also significantly increased settlement, but the cell cultures themselves did not. Monospecific biofilms of the three strains did not result in significant increases in larval settlement. Organic and aqueous/methanol extracts of Roseivivax sp. 46E8 cultures did not affect larval settlement. Examination of filtrates from cell cultures showed that Roseivivax sp. 46E8 spontaneously generated virus-like particles in log and stationary phase growth. Though the mechanism of settlement enhancement by Roseivivax sp. 46E8 is not yet elucidated, our findings point to a new aspect of coral-Roseobacter interactions that should be further investigated, especially in naturally occurring, complex microbial biofilms on reef surfaces.

Microbial community transcriptional networks are conserved in three domains at ocean basin scales.

Planktonic microbial communities in the ocean are typically dominated by several cosmopolitan clades of Bacteria, Archaea, and Eukarya characterized by their ribosomal RNA gene phylogenies and genomic features. Although the environments these communities inhabit range from coastal to open ocean waters, how the biological dynamics vary between such disparate habitats is not well known. To gain insight into the differential activities of microbial populations inhabiting different oceanic provinces we compared the daily metatranscriptome profiles of related microbial populations inhabiting surface waters of both a coastal California upwelling region (CC) as well as the oligotrophic North Pacific Subtropical Gyre (NPSG). Transcriptional networks revealed that the dominant photoautotrophic microbes in each environment (Ostreococcus in CC, Prochlorococcus in NPSG) were central determinants of overall community transcriptome dynamics. Furthermore, heterotrophic bacterial clades common to both ecosystems (SAR11, SAR116, SAR86, SAR406, and Roseobacter) displayed conserved, genome-wide inter- and intrataxon transcriptional patterns and diel cycles. Populations of SAR11 and SAR86 clades in particular exhibited tightly coordinated transcriptional patterns in both coastal and pelagic ecosystems, suggesting that specific biological interactions between these groups are widespread in nature. Our results identify common diurnally oscillating behaviors among diverse planktonic microbial species regardless of habitat, suggesting that highly conserved temporally phased biotic interactions are ubiquitous among planktonic microbial communities worldwide.

Acyl-homoserine lactone quorum sensing in the Roseobacter clade.

Members of the Roseobacter clade are ecologically important and numerically abundant in coastal environments and can associate with marine invertebrates and nutrient-rich marine snow or organic particles, on which quorum sensing (QS) may play an important role. In this review, we summarize current research progress on roseobacterial acyl-homoserine lactone-based QS, particularly focusing on three relatively well-studied representatives, Phaeobacter inhibens DSM17395, the marine sponge symbiont Ruegeria sp. KLH11 and the dinoflagellate symbiont Dinoroseobacter shibae. Bioinformatic survey of luxI homologues revealed that over 80% of available roseobacterial genomes encode at least one luxI homologue, reflecting the significance of QS controlled regulatory pathways in adapting to the relevant marine environments. We also discuss several areas that warrant further investigation, including studies on the ecological role of these diverse QS pathways in natural environments.

Adaptation of Phaeobacter inhibens DSM 17395 to growth with complex nutrients.

Phaeobacter inhibens DSM 17395, a member of the Roseobacter clade, was studied for its adaptive strategies to complex and excess nutrient supply, here mimicked by cultivation with Marine Broth (MB). During growth in process-controlled fermenters, P. inhibens DSM 17395 grew faster (3.6-fold higher µmax ) and reached higher optical densities (2.2-fold) with MB medium, as compared to the reference condition of glucose-containing mineral medium. Apparently, in the presence of MB medium, metabolism was tuned to maximize growth rate at the expense of efficiency. Comprehensive proteomic analysis of cells harvested at ½ ODmax identified 1783 (2D DIGE, membrane and extracellular protein-enriched fractions, shotgun) different proteins (50.5% coverage), 315 (based on 2D DIGE) of which displayed differential abundance profiles. Moreover, 145 different metabolites (intra- and extracellular combined) were identified, almost all of which (140) showed abundance changes. During growth with MB medium, P. inhibens DSM 17395 specifically formed the various proteins required for utilization of phospholipids and several amino acids, as well as for gluconeogenesis. Metabolic tuning on amino acid utilization is also reflected by massive discharge of urea to dispose the cell of excess ammonia. Apparently, P. inhibens DSM 17395 modulated its metabolism to simultaneously utilize diverse substrates from the complex nutrient supply.