Biofilm formation as a microbial strategy to assimilate particulate substrates.

In most ecosystems, a large part of the organic carbon is not solubilized in the water phase. Rather, it occurs as particles made of aggregated hydrophobic and/or polymeric natural or man-made organic compounds. These particulate substrates are degraded by extracellular digestion/solubilization implemented by heterotrophic bacteria that form biofilms on them. Organic particle-degrading biofilms are widespread and have been observed in aquatic and terrestrial natural ecosystems, in polluted and man-driven environments and in the digestive tracts of animals. They have central ecological functions as they are major players in carbon recycling and pollution removal. The aim of this review is to highlight bacterial adhesion and biofilm formation as central mechanisms to exploit the nutritive potential of organic particles. It focuses on the mechanisms that allow access and assimilation of non-dissolved organic carbon, and considers the advantage provided by biofilms for gaining a net benefit from feeding on particulate substrates. Cooperative and competitive interactions taking place in biofilms feeding on particulate substrates are also discussed.

AupA and AupB Are Outer and Inner Membrane Proteins Involved in Alkane Uptake in Marinobacter hydrocarbonoclasticus SP17.

This study describes the functional characterization of two proteins, AupA and AupB, which are required for growth on alkanes in the marine hydrocarbonoclastic bacterium Marinobacter hydrocarbonoclasticus The aupA and aupB genes form an operon whose expression was increased upon adhesion to and biofilm formation on n-hexadecane. AupA and AupB are outer and inner membrane proteins, respectively, which are able to interact physically. Mutations in aupA or/and aupB reduced growth on solid paraffin and liquid n-hexadecane, while growth on nonalkane substrates was not affected. In contrast, growth of aup mutants on n-hexadecane solubilized in Brij 58 micelles was completely abolished. Mutant cells had also lost the ability to bind to n-hexadecane solubilized in Brij 58 micelles. These results support the involvement of AupA and AupB in the uptake of micelle-solubilized alkanes and provide the first evidence for a cellular process involved in the micellar uptake pathway. The phylogenetic distribution of the aupAB operon revealed that it is widespread in marine hydrocarbonoclastic bacteria of the orders Oceanospirillales and Alteromonadales and that it is present in high copy number (up to six) in some Alcanivorax strains. These features suggest that Aup proteins probably confer a selective advantage in alkane-contaminated seawater.IMPORTANCE Bacteria are the main actors of the biological removal of hydrocarbons in seawater, and so, it is important to understand how they degrade hydrocarbons and thereby mitigate marine environmental damage. Despite a considerable amount of literature about the dynamic of microbial communities subjected to hydrocarbon exposure and the isolation of strains that degrade hydrocarbons, most of the genetic determinants and molecular mechanisms of bacterial hydrocarbon uptake remain unknown. This study identifies two genes, aupA and aupB, in the hydrocarbonoclastic bacterium Marinobacter hydrocarbonoclasticus that are present frequently in multiple copies in most of the marine hydrocarbon-degrading bacteria for which the genomic sequence is available. AupA and AupB are two novel membrane proteins interacting together that are involved in the uptake of alkanes dissolved in surfactant micelles. The function and the phylogenetic distribution of aupA and aupB suggest that they might be one attribute of the remarkable adaptation of marine hydrocarbonoclastic bacteria that allow them to take advantage of hydrocarbons.

The extracellular matrix of the oleolytic biofilms of Marinobacter hydrocarbonoclasticus comprises cytoplasmic proteins and T2SS effectors that promote growth on hydrocarbons and lipids.

The assimilation of the nearly water insoluble substrates hydrocarbons and lipids by bacteria entails specific adaptations such as the formation of oleolytic biofilms. The present article reports that the extracellular matrix of an oleolytic biofilm formed by Marinobacter hydrocarbonoclasticus at n-hexadecane-water interfaces is largely composed of proteins typically cytoplasmic such as translation factors and chaperones, and a lesser amount of proteins of unknown function that are predicted extra-cytoplasmic. Matrix proteins appear to form a structured film on hydrophobic interfaces and were found mandatory for the development of biofilms on lipids, alkanes and polystyrene. Exo-proteins secreted through the type-2 secretion system (T2SS) were shown to be essential for the formation of oleolytic biofilms on both alkanes and triglycerides. The T2SS effector involved in biofilm formation on triglycerides was identified as a lipase. In the case of biofilm formation on n-hexadecane, the T2SS effector is likely involved in the mass transfer, capture or transport of alkanes. We propose that M. hydrocarbonoclasticus uses cytoplasmic proteins released by cell lysis to form a proteinaceous matrix and dedicated proteins secreted through the T2SS to act specifically in the assimilation pathways of hydrophobic substrates.

The marine bacterium Marinobacter hydrocarbonoclasticus SP17 degrades a wide range of lipids and hydrocarbons through the formation of oleolytic biofilms with distinct gene expression profiles.

Hydrophobic organic compounds (mainly lipids and hydrocarbons) represent a significant part of the organic matter in marine waters, and their degradation has an important impact in the carbon fluxes within oceans. However, because they are nearly insoluble in the water phase, their degradation by microorganisms occurs at the interface with water and thus requires specific adaptations such as biofilm formation. We show that Marinobacter hydrocarbonoclasticus SP17 develops biofilms, referred to as oleolytic biofilms, on a large variety of hydrophobic substrates, including hydrocarbons, fatty alcohols, fatty acids, triglycerides, and wax esters. Microarray analysis revealed that biofilm growth on n-hexadecane or triolein involved distinct genetic responses, together with a core of common genes that might concern general mechanisms of biofilm formation. Biofilm growth on triolein modulated the expression of hundreds of genes in comparison with n-hexadecane. The processes related to primary metabolism and genetic information processing were downregulated. Most of the genes that were overexpressed on triolein had unknown functions. Surprisingly, their genome localization was restricted to a few regions identified as putative genomic islands or mobile elements. These results are discussed with regard to the adaptive responses triggered by M. hydrocarbonoclasticus SP17 to occupy a specific niche in marine ecosystems.

Identification of novel transcription factors regulating secondary cell wall formation in Arabidopsis.

The presence of lignin in secondary cell walls (SCW) is a major factor preventing hydrolytic enzymes from gaining access to cellulose, thereby limiting the saccharification potential of plant biomass. To understand how lignification is regulated is a prerequisite for selecting plant biomass better adapted to bioethanol production. Because transcriptional regulation is a major mechanism controlling the expression of genes involved in lignin biosynthesis, our aim was to identify novel transcription factors (TFs) dictating lignin profiles in the model plant Arabidopsis. To this end, we have developed a post-genomic approach by combining four independent in-house SCW-related transcriptome datasets obtained from (1) the fiber cell wall-deficient wat1 Arabidopsis mutant, (2) Arabidopsis lines over-expressing either the master regulatory activator EgMYB2 or (3) the repressor EgMYB1 and finally (4) Arabidopsis orthologs of Eucalyptus xylem-expressed genes. This allowed us to identify 502 up- or down-regulated TFs. We preferentially selected those present in more than one dataset and further analyzed their in silico expression patterns as an additional selection criteria. This selection process led to 80 candidates. Notably, 16 of them were already proven to regulate SCW formation, thereby validating the overall strategy. Then, we phenotyped 43 corresponding mutant lines focusing on histological observations of xylem and interfascicular fibers. This phenotypic screen revealed six mutant lines exhibiting altered lignification patterns. Two of them [Bel-like HomeoBox6 (blh6) and a zinc finger TF] presented hypolignified SCW. Three others (myb52, myb-like TF, hb5) showed hyperlignified SCW whereas the last one (hb15) showed ectopic lignification. In addition, our meta-analyses highlighted a reservoir of new potential regulators adding to the gene network regulating SCW but also opening new avenues to ultimately improve SCW composition for biofuel production.

Cells dispersed from Marinobacter hydrocarbonoclasticus SP17 biofilm exhibit a specific protein profile associated with a higher ability to reinitiate biofilm development at the hexadecane-water interface.

Biofilm formation by marine hydrocarbonoclastic bacteria is commonly observed and has been recognized as an important mechanism for the biodegradation of hydrocarbons. In order to colonize new oil-water interfaces, surface-attached communities of hydrocarbonoclastic bacteria must release cells into the environment. Here we explored the physiology of cells freshly dispersed from a biofilm of Marinobacter hydrocarbonoclasticus developing at the hexadecane-water interface, by combining proteomic and physiological approaches. The comparison of the dispersed cells' proteome with those of biofilm, logarithmic- and stationary-phase planktonic cells indicated that dispersed cells had lost most of the biofilm phenotype and expressed a specific proteome. Two proteins involved in cell envelope maturation, DsbA and CtpA, were exclusively detected in dispersed cells, suggesting a reshaping of the cell envelopes during biofilm dispersal. Furthermore, dispersed cells exhibited a higher affinity for hexadecane and initiated more rapidly biofilm formation on hexadecane than the reference planktonic cells. Interestingly, storage wax esters were rapidly degraded in dispersed cells, suggesting that their observed physiological properties may rely on reserve mobilization. Thus, by promoting oil surface colonization, cells emigrating from the biofilm could contribute to the success of marine hydrocarbonoclastic bacteria in polluted environments.

EgMYB1, an R2R3 MYB transcription factor from eucalyptus negatively regulates secondary cell wall formation in Arabidopsis and poplar.

• The eucalyptus R2R3 transcription factor, EgMYB1 contains an active repressor motif in the regulatory domain of the predicted protein. It is preferentially expressed in differentiating xylem and is capable of repressing the transcription of two key lignin genes in vivo. • In order to investigate in planta the role of this putative transcriptional repressor of the lignin biosynthetic pathway, we overexpressed the EgMYB1 gene in Arabidopsis and poplar. • Expression of EgMYB1 produced similar phenotypes in both species, with stronger effects in transgenic Arabidopsis plants than in poplar. Vascular development was altered in overexpressors showing fewer lignified fibres (in phloem and interfascicular zones in poplar and Arabidopsis, respectively) and reduced secondary wall thickening. Klason lignin content was moderately but significantly reduced in both species. Decreased transcript accumulation was observed for genes involved in the biosynthesis of lignins, cellulose and xylan, the three main polymers of secondary cell walls. Transcriptomic profiles of transgenic poplars were reminiscent of those reported when lignin biosynthetic genes are disrupted. • Together, these results strongly suggest that EgMYB1 is a repressor of secondary wall formation and provide new opportunities to dissect the transcriptional regulation of secondary wall biosynthesis.

Anaerobic biodegradation of BTEX by original bacterial communities from an underground gas storage aquifer.

BTEX biodegradation by an indigenous deep subsurface microbial community was evaluated in a water sample collected in the area of an underground gas storage. Five different sulfate-reducing microbial communities able to use at least either benzene, toluene, ethylbenzene, or xylene (BTEX) compounds were studied. A total of 21 different bacterial phylotypes were identified, each community containing three to nine bacterial phylotypes. Archaeal phylotypes were retrieved from only three communities. The analysis of 16S rRNA gene sequences showed that i) these consortia were mainly composed of novel species, some of which belonging to bacterial groups not previously suspected to be involved in BTEX anaerobic degradation, ii) three consortia were dominated by an uncultured Pelobacter sp. previously detected in biodegraded oil reservoirs, iii) a deeply branching species distantly affiliated to Thermotogales was abundant in two consortia, and that iv) Firmicutes related to the Desulfotomaculum and Carboxydocella genera represented the only three detectable phylotypes in a toluene-degrading consortium. This work shows that subdominant microbial populations present in a deep subsurface aquifer used for seasonal underground gas storage could be involved in the natural attenuation of the traces of BTEX coinjected with methane in the deep subsurface.

Overexpression of EgROP1, a Eucalyptus vascular-expressed Rac-like small GTPase, affects secondary xylem formation in Arabidopsis thaliana.

To better understand the genetic control of secondary xylem formation in trees we analysed genes expressed during Eucalyptus xylem development. Using eucalyptus xylem cDNA libraries, we identified EgROP1, a member of the plant ROP family of Rho-like GTPases. These signalling proteins are central regulators of many important processes in plants, but information on their role in xylogenesis is scarce. Quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) confirmed that EgROP1 was preferentially expressed in the cambial zone and differentiating xylem in eucalyptus. Genetic mapping performed in a eucalyptus breeding population established a link between EgROP1 sequence polymorphisms and quantitative trait loci (QTLs) related to lignin profiles and fibre morphology. Overexpression of various forms of EgROP1 in Arabidopsis thaliana altered anisotropic cell growth in transgenic leaves, but most importantly affected vessel element and fibre growth in secondary xylem. Patches of fibre-like cells in the secondary xylem of transgenic plants showed changes in secondary cell wall thickness, lignin and xylan composition. These results suggest a role for EgROP1 in fibre cell morphology and secondary cell wall formation making it a good candidate gene for marker-based selection of eucalyptus trees.

A new genomic resource dedicated to wood formation in Eucalyptus.

BACKGROUND: Renowned for their fast growth, valuable wood properties and wide adaptability, Eucalyptus species are amongst the most planted hardwoods in the world, yet they are still at the early stages of domestication because conventional breeding is slow and costly. Thus, there is huge potential for marker-assisted breeding programs to improve traits such as wood properties. To this end, the sequencing, analysis and annotation of a large collection of expressed sequences tags (ESTs) from genes involved in wood formation in Eucalyptus would provide a valuable resource. RESULTS: We report here the normalization and sequencing of a cDNA library from developing Eucalyptus secondary xylem, as well as the construction and sequencing of two subtractive libraries (juvenile versus mature wood and vice versa). A total of 9,222 high quality sequences were collected from about 10,000 cDNA clones. The EST assembly generated a set of 3,857 wood-related unigenes including 2,461 contigs (Cg) and 1,396 singletons (Sg) that we named 'EUCAWOOD'. About 65% of the EUCAWOOD sequences produced matches with poplar, grapevine, Arabidopsis and rice protein sequence databases. BlastX searches of the Uniref100 protein database allowed us to allocate gene ontology (GO) and protein family terms to the EUCAWOOD unigenes. This annotation of the EUCAWOOD set revealed key functional categories involved in xylogenesis. For instance, 422 sequences matched various gene families involved in biosynthesis and assembly of primary and secondary cell walls. Interestingly, 141 sequences were annotated as transcription factors, some of them being orthologs of regulators known to be involved in xylogenesis. The EUCAWOOD dataset was also mined for genomic simple sequence repeat markers, yielding a total of 639 putative microsatellites. Finally, a publicly accessible database was created, supporting multiple queries on the EUCAWOOD dataset. CONCLUSION: In this work, we have identified a large set of wood-related Eucalyptus unigenes called EUCAWOOD, thus creating a valuable resource for functional genomics studies of wood formation and molecular breeding in this economically important genus. This set of publicly available annotated sequences will be instrumental for candidate gene approaches, custom array development and marker-assisted selection programs aimed at improving and modulating wood properties.

Transcript profiling of a xylem vs phloem cDNA subtractive library identifies new genes expressed during xylogenesis in Eucalyptus.

Eucalyptus is one of the world's main sources of biomass. The genus includes species representing the principle hardwood trees used for pulp and paper. Here, we aimed to identify genes specifically expressed in differentiating secondary xylem compared with phloem. We constructed a xylem vs phloem subtractive library (Xp) that generated 263 unique sequences. By transcript profiling of xylem, phloem, vascular cambium and leaves using macroarrays, we classified the 263 unigenes into distinct tissue-specific groups. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed the differential expression of representative expressed sequence tags (ESTs). A total of 87 unigenes were preferentially expressed in xylem. They were involved in functional categories known to play roles in xylogenesis, such as hormone signaling and metabolism, secondary cell wall thickening and proteolysis. Some of these genes, including unknown genes, may be considered xylem-specific and they are likely to control important functions in xylogenesis. These data shed light on the cellular functions of xylem cells and, importantly, provide us with a portfolio of Eucalyptus xylem genes that may be major players in the control of wood formation and quality.

Transcript profiling of Eucalyptus xylem genes during tension wood formation.

Tension wood formed in response to gravitational force is a striking example of the plasticity of angiosperm wood. In this study our goal was to characterize the early changes in gene expression during tension wood formation in Eucalyptus. Using cDNA array technology, transcript profiling of 231 genes preferentially expressed in differentiating Eucalyptus xylem was followed from 6 h to 1 wk of a tension time course of artificially bent Eucalyptus trees. 196 genes were differentially regulated between control and bent trees, some exhibiting distinctive expression patterns related to changes in secondary cell wall structure and composition. For instance, expression of a cellulose synthase gene was well correlated with the appearance of the G-layers. Cluster correlation analysis revealed differential regulation of lignin biosynthetic genes and may also be used to help infer the function of unknown gene products. Eucalyptus wood transcriptome analysis during tension wood formation not only provided new clues into the transcriptional regulatory network of genes preferentially expressed in xylem, but also highlighted candidate genes responsible for the genetic and environmentally induced variation of wood quality traits.

Identification of genes preferentially expressed during wood formation in Eucalyptus.

Wood is the most abundant biological resource on earth and it is also an important raw material for a major global industry with rapidly increasing demand. The genus Eucalyptus includes the most widely used tree species for industrial plantation, mainly for making pulp and paper. With the aim of identifying major genes involved in wood formation in Eucalyptus , we have developed a targeted approach of functional genomics based on the isolation of xylem preferentially expressed genes by subtractive PCR. Transcript profiling using cDNA arrays and analysis of variance (ANOVA) were used to identify differentially expressed ESTs between secondary xylem and leaves. Real-time RT-PCR was performed to confirm the differential expression of representative EST. Of 224 independent EST sequences obtained, 81% were preferentially expressed in xylem. One-third of the ESTs exhibiting homologies with proteins of known function fell into two main classes highlighting the importance of the auxin signalling through ubiquitin-dependent proteolysis on one hand, and of the enzymes involved in cell wall biosynthesis and remodelling, on the other. The functions of the genes represented by the remaining 61% of ESTs should be of great interest for future research. This systematic analysis of genes involved in wood formation in Eucalyptus provides valuable insights into the molecular mechanisms involved in secondary xylem differentiation as well as new candidate-genes for wood quality improvement.