Régis Grimaud

Methionine sulfoxide reductases protect Ffh from oxidative damages in Escherichia coli

In proteins, methionine residues are primary targets for oxidation. Methionine oxidation is reversed by methionine sulfoxide reductases A and B, a class of highly conserved enzymes. Ffh protein, a component of the ubiquitous signal recognition particle, contains a methionine‐rich domain, interacting with a small 4.5S RNA. In vitro analyses reported here show that: (i) oxidized Ffh is unable to bind 4.5S RNA, (ii) oxidized Ffh contains methionine sulfoxide residues, (iii) oxidized Ffh is a substrate for MsrA and MsrB enzymes; and (iv) MsrA/B repairing activities allow oxidized Ffh to recover 4.5S RNA‐binding abilities. In vivo analyses reveal that: (i) Ffh synthesized in the msrA msrB mutant contains methionine sulfoxide residues and is unstable, (ii) msrA msrB mutant requires high levels of Ffh synthesis for growth and (iii) msrA msrB mutation leads to defects in Ffh‐dependent targeting of MalF. We conclude that MsrA and MsrB are required to repair Ffh oxidized by reactive oxygen species produced by aerobic metabolism, establishing an as‐yet undescribed link between protein targeting and oxidation.

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.

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.

Genome Sequence of the Marine Bacterium Marinobacter hydrocarbonoclasticus SP17, Which Forms Biofilms on Hydrophobic Organic Compounds

Marinobacter hydrocarbonoclasticus SP17 forms biofilms specifically at the interface between water and hydrophobic organic compounds (HOCs) that are used as carbon and energy sources. Biofilm formation at the HOC-water interface has been recognized as a strategy to overcome the low availability of these nearly water-insoluble substrates. Here, we present the genome sequence of SP17, which could provide further insights into the mechanisms of enhancement of HOCs assimilation through biofilm formation.

Biosynthesis, purification and analysis of selenomethionyl calmodulin by gel electrophoresis-laser ablation-ICP-MS and capillary HPLC-ICP-MS peptide mapping following in-gel tryptic digestion

A biosynthesis method was developed to produce a standard of a selenium-containing protein. It consisted of the expression of calmodulin in Escherichia coli culture in the presence of selenomethionine, which allowed the replacement of all methionine residues by selenomethionine. The resulting 17 kDa protein containing 8 selenomethionine residues was purified by two-step hydrophobic interaction chromatography. The selenomethionyl calmodulin was subsequently used to develop a method for the characterization of selenium-containing proteins (detected in the polyacrylamide gel by laser ablation-ICP-MS) by means of peptide mapping using capillary HPLC-ICP-MS. The monitoring of the 80Se isotope using an ICP mass spectrometer equipped with a collision cell allowed as little as 0.3 pg as Se (1.3 ng ml−1 in the analysed solution) to be detected in the gel. The band containing the protein of interest was excised, the protein was digested with trypsin and the Se-containing peptides were analyzed by capillary HPLC-ICP-MS. The sensitivity of the method was at least a factor of 5 higher than that of capillary LC-electrospray MS/MS in similar conditions. Some of the selenopeptides detected by capillary LC-ICP MS could nevertheless be identified by retention time matching using a set of peptides generated by trypsin digestion from the concentrated selenomethionyl calmodulin standard.

Physiological adaptation of Desulfitobacterium hafniense strain TCE1 to tetrachloroethene respiration.

ABSTRACT Desulfitobacterium spp. are ubiquitous organisms with a broad metabolic versatility, and some isolates have the ability to use tetrachloroethene (PCE) as terminal electron acceptor. In order to identify proteins involved in this organohalide respiration process, a comparative proteomic analysis was performed. Soluble and membrane-associated proteins obtained from cells of Desulfitobacterium hafniense strain TCE1 that were growing on different combinations of the electron donors lactate and hydrogen and the electron acceptors PCE and fumarate were analyzed. Among proteins increasingly expressed in the presence of PCE compared to fumarate as electron acceptor, a total of 57 proteins were identified by mass spectrometry analysis, revealing proteins involved in stress response and associated regulation pathways, such as PspA, GroEL, and CodY, and also proteins potentially participating in carbon and energy metabolism, such as proteins of the Wood-Ljungdahl pathway and electron transfer flavoproteins. These proteomic results suggest that D. hafniense strain TCE1 adapts its physiology to face the relative unfavorable growth conditions during an apparent opportunistic organohalide respiration.

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.

Imaging Bacterial Cells and Biofilms Adhering to Hydrophobic Organic Compound–Water Interfaces

Assimilation of Hydrophobic Organic Compound (HOC) entails frequently the formation of biofilm at the HOC-water interface. Knowledge on the behavior of cells at the oil-water interface and within the structured biofilm is therefore important to understand the degradation of the HOC in ecosystems. The adhesion and biofilm formation on oil-water interface is best documented by microscopic observations. In this chapter we thus describe two methods for observation of bacterial cells and biofilms growing at the HOC-water interface. The first method uses CLSM (confocal laser scanning microscopy) to obtain in situ images of biofilm developing on thin paraffin strip which offers a flat transparent surface allowing imaging directly through the bottom of the culture dish without sampling. Alternatively, the biofilm can be grown on a paraffin strip deposited on a glass microscope slide and then imaged from the top when high resolution is needed. The second method addresses the problematic of the ultrastructure of biofilm developing on HOC. It enables to obtain by TEM (transmission electron microscopy) images of cross sections of biofilms with identification of the side in contact with the HOC.

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

ABSTRACT 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. 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.

Impact of temperature on Marinobacter hydrocarbonoclasticus SP17 morphology and biofilm structure during growth on alkanes

Alkanes are widespread pollutants found in soil, freshwater and marine environments. Marinobacter hydrocarbonoclasticus (Mh) strain SP17 is a marine bacterium able to use many hydrophobic organic compounds, including alkanes, through the production of biofilms that allow their poor solubility to be overcome. This study pointed out that temperature is an environmental factor that strongly affects the biofilm formation and morphology of Mh on the model alkanes, hexadecane and paraffin. We showed that Mh biofilm formation and accumulation of intracytoplasmic inclusions are higher on solid alkanes (hexadecane at 10 °C and paraffin at 10 °C and 30 °C) than on liquid alkane (hexadecane at 30 °C) or soluble substrate (lactate at both temperatures). We also found that Mh produces more extracellular polymeric substances at 30 °C than at 10 °C on alkanes and none on lactate. We observed that bacterial length is significantly higher at 10 °C than at 30 °C on lactate and hexadecane. On paraffin, at 30 °C, the cell morphology is markedly altered by large rounded or irregularly shaped cytoplasmic inclusions. Altogether, the results showed that Mh is able to adapt and use alkanes as a carbon source, even at low temperature.