The role of planktonic Flavobacteria in processing algal organic matter in coastal East Antarctica revealed using metagenomics and metaproteomics.

Heterotrophic marine bacteria play key roles in remineralizing organic matter generated from primary production. However, far more is known about which groups are dominant than about the cellular processes they perform in order to become dominant. In the Southern Ocean, eukaryotic phytoplankton are the dominant primary producers. In this study we used metagenomics and metaproteomics to determine how the dominant bacterial and archaeal plankton processed bloom material. We examined the microbial community composition in 14 metagenomes and found that the relative abundance of Flavobacteria (dominated by Polaribacter) was positively correlated with chlorophyll a fluorescence, and the relative abundance of SAR11 was inversely correlated with both fluorescence and Flavobacteria abundance. By performing metaproteomics on the sample with the highest relative abundance of Flavobacteria (Newcomb Bay, East Antarctica) we defined how Flavobacteria attach to and degrade diverse complex organic material, how they make labile compounds available to Alphaproteobacteria (especially SAR11) and Gammaproteobacteria, and how these heterotrophic Proteobacteria target and utilize these nutrients. The presence of methylotrophic proteins for archaea and bacteria also indicated the importance of metabolic specialists. Overall, the study provides functional data for the microbial mechanisms of nutrient cycling at the surface of the coastal Southern Ocean.

Ecology of type II secretion in marine gammaproteobacteria.

Compelling findings on the direct association of the type II secretion (T2S) system with different ecological functions in marine bacteria have challenged the traditional view of the T2S pathway, the function of which has been mostly studied in pathogenic bacteria. The availability of a number of whole-genome sequence data sets enabled the analysis of the genetic composition of the T2S system across a number of Vibrios and Alteromonads. The widespread Gammaproteobacteria, in particular the Alteromonadales and the Vibrionales group, are recognized to play significant roles in the cycling of nutrients in coastal and pelagic marine ecosystems and are also found associated with marine eukaryotes. The combined analysis of the role and the genetic composition of the T2S system in Gammaproteobacteria provides important evidence for the significance of the T2S pathway in the ecology of environmental bacteria.

Impact of oil contamination and biostimulation on the diversity of indigenous bacterial communities in soil microcosms.

The aim of this study was to analyse the effect of oil contamination and biostimulation (soil pH raise, and nitrogen, phosphate and sulphur addition) on the diversity of a bacterial community of an acidic Cambisol under Atlantic Forest. The experiment was based on the enumeration of bacterial populations and hydrocarbon degraders in microcosms through the use of conventional plating techniques and molecular fingerprinting of samples directly from the environment. PCR followed by denaturing gradient gel electrophoresis (DGGE) was used to generate microbial community fingerprints employing 16S rRNA gene as molecular marker. Biostimulation led to increases of soil pH (to 7.0) and of the levels of phosphorus and K, Ca, and Mg. Oil contamination caused an increase in soil organic carbon (170-190% higher than control soil). Total bacterial counts were stable throughout the experiment, while MPN counts of hydrocarbon degraders showed an increase in the biostimulated and oil-contaminated soil samples. Molecular fingerprinting performed with 16S rRNA gene PCR and DGGE analysis revealed stable patterns along the 360 days of experiment, showing little change in oil-contaminated microcosms after 90 days. The DGGE patterns of the biostimulated samples showed severe changes due to decreases in the number of bands as compared to the control samples as from 15 days after addition of nutrients to the soil. Results obtained in the present study indicate that the addition of inorganic compounds to soil in conjunction with oil contamination has a greater impact on the bacterial community than oil contamination only.

A metaproteomic assessment of winter and summer bacterioplankton from Antarctic Peninsula coastal surface waters.

A metaproteomic survey of surface coastal waters near Palmer Station on the Antarctic Peninsula, West Antarctica, was performed, revealing marked differences in the functional capacity of summer and winter communities of bacterioplankton. Proteins from Flavobacteria were more abundant in the summer metaproteome, whereas winter was characterized by proteins from ammonia-oxidizing Marine Group I Crenarchaeota. Proteins prevalent in both seasons were from SAR11 and Rhodobacterales clades of Alphaproteobacteria, as well as many lineages of Gammaproteobacteria. The metaproteome data were used to elucidate the main metabolic and energy generation pathways and transport processes occurring at the microbial level in each season. In summer, autotrophic carbon assimilation appears to be driven by oxygenic photoautotrophy, consistent with high light availability and intensity. In contrast, during the dark polar winter, the metaproteome supported the occurrence of chemolithoautotrophy via the 3-hydroxypropionate/4-hydroxybutyrate cycle and the reverse tricarboxylic acid cycle of ammonia-oxidizing archaea and nitrite-oxidizing bacteria, respectively. Proteins involved in nitrification were also detected in the metaproteome. Taurine appears to be an important source of carbon and nitrogen for heterotrophs (especially SAR11), with transporters and enzymes for taurine uptake and degradation abundant in the metaproteome. Divergent heterotrophic strategies for Alphaproteobacteria and Flavobacteria were indicated by the metaproteome data, with Alphaproteobacteria capturing (by high-affinity transport) and processing labile solutes, and Flavobacteria expressing outer membrane receptors for particle adhesion to facilitate the exploitation of non-labile substrates. TonB-dependent receptors from Gammaproteobacteria and Flavobacteria (particularly in summer) were abundant, indicating that scavenging of substrates was likely an important strategy for these clades of Southern Ocean bacteria. This study provides the first insight into differences in functional processes occurring between summer and winter microbial communities in coastal Antarctic waters, and particularly highlights the important role that 'dark' carbon fixation has in winter.

Analysis of the Pseudoalteromonas tunicata genome reveals properties of a surface-associated life style in the marine environment.

BACKGROUND: Colonisation of sessile eukaryotic host surfaces (e.g. invertebrates and seaweeds) by bacteria is common in the marine environment and is expected to create significant inter-species competition and other interactions. The bacterium Pseudoalteromonas tunicata is a successful competitor on marine surfaces owing primarily to its ability to produce a number of inhibitory molecules. As such P. tunicata has become a model organism for the studies into processes of surface colonisation and eukaryotic host-bacteria interactions. METHODOLOGY/PRINCIPAL FINDINGS: To gain a broader understanding into the adaptation to a surface-associated life-style, we have sequenced and analysed the genome of P. tunicata and compared it to the genomes of closely related strains. We found that the P. tunicata genome contains several genes and gene clusters that are involved in the production of inhibitory compounds against surface competitors and secondary colonisers. Features of P. tunicata's oxidative stress response, iron scavenging and nutrient acquisition show that the organism is well adapted to high-density communities on surfaces. Variation of the P. tunicata genome is suggested by several landmarks of genetic rearrangements and mobile genetic elements (e.g. transposons, CRISPRs, phage). Surface attachment is likely to be mediated by curli, novel pili, a number of extracellular polymers and potentially other unexpected cell surface proteins. The P. tunicata genome also shows a utilisation pattern of extracellular polymers that would avoid a degradation of its recognised hosts, while potentially causing detrimental effects on other host types. In addition, the prevalence of recognised virulence genes suggests that P. tunicata has the potential for pathogenic interactions. CONCLUSIONS/SIGNIFICANCE: The genome analysis has revealed several physiological features that would provide P. tunciata with competitive advantage against other members of the surface-associated community. We have also identified properties that could mediate interactions with surfaces other than its currently recognised hosts. This together with the detection of known virulence genes leads to the hypothesis that P. tunicata maintains a carefully regulated balance between beneficial and detrimental interactions with a range of host surfaces.

Profiling the secretome of the marine bacterium Pseudoalteromonas tunicata using amine-specific isobaric tagging (iTRAQ).

The eukaryote-associated marine bacterium Pseudoalteromonas tunicata produces a range of target-specific compounds that inhibit different types of marine organisms including invertebrate larvae and algal spores, as well as a broad spectrum of fungi, protozoa, and bacteria. The ability to produce such bioactive compounds is correlated to the expression of a yellow and a purple pigment in P. tunicata. To investigate the regulation and biosynthesis of the pigments and bioactive compounds, the expressed secretome of the pigmented wild-type P. tunicata and a nonpigmented mutant (wmpD-) defective in the type-II secretion pathway were compared. Secreted proteins were digested with trypsin, labeled using amine-specific isobaric tagging reagents (iTRAQ), and identified using two-dimensional SCX and nano C18 RP liquid-chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS). The iTRAQ labeling experiments enabled accurate measurement of the proteins identified in this work. A sequence-base prediction of P. tunicata secretome was also obtained and compared to the expressed proteome to determine the role of the type-II secretion pathway in this bacterium. Our results suggest that this secretion pathway has a role in iron transport and acquisition in P. tunicata.

Biofilm development and cell death in the marine bacterium Pseudoalteromonas tunicata.

The newly described green-pigmented bacterium Pseudoalteromonas tunicata (D2) produces target-specific inhibitory compounds against bacteria, algae, fungi, and invertebrate larvae and is frequently found in association with living surfaces in the marine environment. As part of our studies on the ecology of P. tunicata and its interaction with marine surfaces, we examined the ability of P. tunicata to form biofilms under continuous culture conditions within the laboratory. P. tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water channels. Remarkably, we observed a repeatable pattern of cell death during biofilm development of P. tunicata, similar to that recently reported for biofilms of Pseudomonas aeruginosa (J. S. Webb et al., J. Bacteriol. 185:4585-4595, 2003). Killing and lysis occurred inside microcolonies, apparently resulting in the formation of voids within these structures. A subpopulation of viable cells was always observed within the regions of killing in the biofilm. Moreover, extensive killing in mature biofilms appeared to result in detachment of the biofilm from the substratum. A novel 190-kDa autotoxic protein produced by P. tunicata, designated AlpP, was found to be involved in this biofilm killing and detachment. A Delta alpP mutant derivative of P. tunicata was generated, and this mutant did not show cell death during biofilm development. We propose that AlpP-mediated cell death plays an important role in the multicellular biofilm development of P. tunicata and subsequent dispersal of surviving cells within the marine environment.