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 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.

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.

Behavior of Marinobacter hydrocarbonoclasticus SP17 cells during initiation of biofilm formation at the alkane-water interface.

Hexadecane assimilation by Marinobacter hydrocarbonoclasticus SP17 occurs through the formation of a biofilm at the alkane-water interface. In this study we focused on the interactions of cells with the alkane-water interface occurring during initiation of biofilm development. The behavior of cells at the interface was apprehended by investigating alterations of the mechanical properties of the interface during cell adsorption, using dynamic drop tensiometry measurements. It was found that after having reached the hexadecane-water interface, by a purely thermal diffusion process, cells released surface-active compounds (SACs) resulting in the formation of an interfacial visco-elastic film. Release of SACs was an active process requiring protein synthesis. This initial interaction occurred on metabolizable as well as non-metabolizable alkanes, indicating that at this stage cells are not affected by the nature of the alkane forming the interface. In contrast, at a later stage, the nature of the interface turned out to exert control over the behavior of the cells. The availability of a metabolizable alkane at the interface influenced cell activity, as revealed by cell cluster formation and differences in the interfacial elasticity.

Proteomic analysis of Marinobacter hydrocarbonoclasticus SP17 biofilm formation at the alkane-water interface reveals novel proteins and cellular processes involved in hexadecane assimilation.

Many hydrocarbon-degrading bacteria form biofilms at the hydrocarbon-water interface to overcome the weak accessibility of these poorly water-soluble substrates. In order to gain insight into the cellular functions involved, we undertook a proteomic analysis of Marinobacter hydrocarbonoclasticus SP17 biofilm developing at the hexadecane-water interface. Biofilm formation on hexadecane led to a global change in cell physiology involving modulation of the expression of 576 out of 1144 detected proteins when compared with planktonic cells growing on acetate. Biofilm cells overproduced a protein encoded by MARHY0478 that contains a conserved domain belonging to the family of the outer membrane transporters of hydrophobic compounds. Homologs of MARHY0478 were exclusively found in marine bacteria degrading alkanes or possessing alkane degradation genes, and hence presumably constitute a family of alkane transporters specific to marine bacteria. Interestingly, we also found that sessile cells growing on hexadecane overexpressed type VI secretion system components. This secretion system has been identified as a key factor in virulence and in symbiotic interaction with host organisms. This observation is the first experimental evidence of the contribution of a type VI secretion system to environmental adaptation, and raises the intriguing question about the role of this secretion machine in alkane assimilation.

Cytoplasmic wax ester accumulation during biofilm-driven substrate assimilation at the alkane--water interface by Marinobacter hydrocarbonoclasticus SP17.

During growth on n-alkanes, the marine bacterium Marinobacter hydrocarbonoclasticus SP17 formed a biofilm at the alkane-water interface. We showed that hexadecane degradation was correlated with biofilm development and that alkane uptake is localized in the biofilm but not in the bulk medium. Biofilms were observed in cultures on metabolizable n-alkanes (C8-C28) and n-alcohols (C12 and C16), but were formed neither on non-metabolizable alkanes (pristane, heptamethylnonane and n-C32) nor on inert substrata (glass, polystyrene and Permanox). This substratum specificity indicates that biofilm formation is determined by the presence of an interface between an insoluble substrate and the aqueous phase. Simultaneously with biofilm growth, planktonic cells were released from the biofilm. Detached cells were in a non-growing state, implying that the growing population was exclusively located within the biofilm. Planktonic and sessile cells exhibited differences in their ultrastructure and lipid content. Biofilm cells contained a large amount of wax esters (0.47mg/mg protein) in rounded or irregularly shaped cytoplasmic inclusions, whereas detached cells displayed rod-shaped inclusions and contained 5 times fewer wax esters (0.10mg/mg protein) than their sessile counterparts. This study points out the inter-relationship between biofilm formation, insoluble substrate uptake and lipid storage.

Rhodococcus pyridinovorans MW3, a bacterium producing a nitrile hydratase.

Rhodococcus pyridinovorans MW3 was isolated from an arable land of manioc from the Congo for its ability to transform acrylonitrile to acrylamide. This strain contains a cobalt nitrile hydratase (NHase) showing high sequence homology with NHases so far described. The specific NHase activity was 97 U mg(-1) dry wt. NHase production by R. pyridinovorans MW3 was urea and Co-dependent. The NHase was active for acrylamide up to 60% (w/v) indicating its potential for acrylamide production.

Describing and modelling ozone-dependent variation in flavonoid content of bean (Phaseolus vulgaris cv. Bergamo) leaves: a particular dose-response relationship analysis.

We investigated the ozone-dependent variation in the amount of a flavonoid accumulated by bean leaves (Phaseolus vulgaris L. cv. Bergamo). The phenolic response was modelled with special regard to different ozone exposure indexes. Using open-top chamber technology, six atmospheres of increasing ozone concentration were tested. Four successive harvests were carried out during a 33-d experiment. Primary and first trifoliate leaves were collected. Visible foliar injuries were recorded and the quantification of an ozone-responsive flavonoid was achieved by HPLC. Ozone significantly increased the amount of kaempferol glucuronide, which normally decreased with leaf ageing. Depending on the leaf type, this increase occurred either before or after the appearance of visible foliar damage. A linear regression could account for the ozone dose-phenolic response relationship. However, with respect to leaf type, the agreement between the model and observed values was influenced by the way in which ozone dose was calculated. Among the ozone exposure indexes tested, only the index with the highest threshold (AOT60) was appropriate to make the phenolic response linear in the case of primary leaves while in the case of first trifoliate leaves, this index always displayed the poorest adjustment compared with SUM00, SUM60, and AOT40 indexes. The study of the relationship suggests that sensitivity to ozone could be dependent on leaf type.

Ozone-induced oxidation of Rubisco: from an ELISA quantification of carbonyls to putative pathways leading to oxidizing mechanisms.

In an attempt to detect a possible relationship between protein oxidation and ozone (O3) atmospheric concentration, we used a sensitive enzyme-linked immunosorbent assay (ELISA) method for measuring carbonyl formation in amino acid residues that constitute Rubisco (EC 4.1.1.39) small subunit (Rubisco-SSU). Using open-top chamber technology, bean plants (Phaseolus vulgaris L. cv. Bergamo) were exposed for 21 d (from emergence) to four different atmospheres characterized by average daylight O3 concentrations of 12, 70, 89 and 109nL L-1. Rubisco-SSU extracted from primary leaves was fixed specifically on wells coated with anti-SSU antibodies. Aldehydes and ketones, previously derivatized with 2,4-dinitrophenylhydrazine (DNP), were quantified with anti-DNP antibodies conjugated with alkaline phosphatase. A significant positive O3 effect on carbonyl formation was detected, and the number of carbonyls was found to be linearly increased (r2=0.82) when plotted against increasing external O3 dose expressed as accumulated exposure over a threshold of 40 nL L-1 h (AOT40). Furthermore, these O3-induced oxidative modifications were connected with a significant reduction in the amount of native SSU that linearly decreased (r2=0.95) as AOT40 increased from 0 to about 8295 nL L-1 h. A possible pathway leading to oxidation of Rubisco is proposed, with special reference to O3 reactivity.

The response of vacuolar phenolic content of common bean (Phaseolus vulgaris cv. Bergamo) to a chronic ozone exposure: questions and hypotheses.

Using open-top chamber technology, we investigated the foliar phenolic response of common bean (Phaseolus vulgaris L. cv. Bergamo) to a chronic, moderate ozone stress. Three atmospheric concentrations of ozone were tested: non-filtered air (NF) prevailing at the experimental site, and non-filtered air supplied with 40 (NF+40) and 60 nL L-1 ozone (NF+60), respectively. Both constitutive and ozone-induced non-polymerized phenolics were considered with regards to pollutant concentration, exposure time, leaf type (primary or trifoliate), and leaf growth. The biomass of primary leaves was unaffected by the tested ozone concentrations, whereas dry mass of first and second trifoliate leaves significantly decreased as atmospheric ozone increased. Characteristic symptoms were observed on the upper surface of leaves from the two ozone-supplied treatments. Their severity reflected both leaf exposure time and ozone concentration. As a whole, the total content of foliar soluble constitutive phenolics remained unchanged as the ozone increased, even for leaves almost totally covered with dark-brown discolourations. Nonetheless, among the three main detected phenolics, the accumulation of the kaempferol derivative could be significantly stimulated by ozone. Also, six ozone-induced phenolics could be synthesized by leaves exposed to the two pollutant-enriched atmospheres, and their elicitation and amount were closely connected with both exposure time and ozone concentration.