Acylation of the Type 3 Secretion System Translocon Using a Dedicated Acyl Carrier Protein.

Bacterial pathogens often deliver effectors into host cells using type 3 secretion systems (T3SS), the extremity of which forms a translocon that perforates the host plasma membrane. The T3SS encoded by Salmonella pathogenicity island 1 (SPI-1) is genetically associated with an acyl carrier protein, IacP, whose role has remained enigmatic. In this study, using tandem affinity purification, we identify a direct protein-protein interaction between IacP and the translocon protein SipB. We show, by mass spectrometry and radiolabelling, that SipB is acylated, which provides evidence for a modification of the translocon that has not been described before. A unique and conserved cysteine residue of SipB is identified as crucial for this modification. Although acylation of SipB was not essential to virulence, we show that this posttranslational modification promoted SipB insertion into host-cell membranes and pore-forming activity linked to the SPI-1 T3SS. Cooccurrence of acyl carrier and translocon proteins in several γ- and ß-proteobacteria suggests that acylation of the translocon is conserved in these other pathogenic bacteria. These results also indicate that acyl carrier proteins, known for their involvement in metabolic pathways, have also evolved as cofactors of new bacterial protein lipidation pathways.

The aerobic respiratory chain of the acidophilic archaeon Ferroplasma acidiphilum: A membrane-bound complex oxidizing ferrous iron.

The extremely acidophilic archaeon Ferroplasma acidiphilum is found in iron-rich biomining environments and is an important micro-organism in naturally occurring microbial communities in acid mine drainage. F. acidiphilum is an iron oxidizer that belongs to the order Thermoplasmatales (Euryarchaeota), which harbors the most extremely acidophilic micro-organisms known so far. At present, little is known about the nature or the structural and functional organization of the proteins in F. acidiphilum that impact the iron biogeochemical cycle. We combine here biochemical and biophysical techniques such as enzyme purification, activity measurements, proteomics and spectroscopy to characterize the iron oxidation pathway(s) in F. acidiphilum. We isolated two respiratory membrane protein complexes: a 850 kDa complex containing an aa3-type cytochrome oxidase and a blue copper protein, which directly oxidizes ferrous iron and reduces molecular oxygen, and a 150 kDa cytochrome ba complex likely composed of a di-heme cytochrome and a Rieske protein. We tentatively propose that both of these complexes are involved in iron oxidation respiratory chains, functioning in the so-called uphill and downhill electron flow pathways, consistent with autotrophic life. The cytochrome ba complex could possibly play a role in regenerating reducing equivalents by a reverse ('uphill') electron flow. This study constitutes the first detailed biochemical investigation of the metalloproteins that are potentially directly involved in iron-mediated energy conservation in a member of the acidophilic archaea of the genus Ferroplasma.

An ArsR/SmtB family member is involved in the regulation by arsenic of the arsenite oxidase operon in Thiomonas arsenitoxydans.

The genetic organization of the aioBA operon, encoding the arsenite oxidase of the moderately acidophilic and facultative chemoautotrophic bacterium Thiomonas arsenitoxydans, is different from that of the aioBA operon in the other arsenite oxidizers, in that it encodes AioF, a metalloprotein belonging to the ArsR/SmtB family. AioF is stabilized by arsenite, arsenate, or antimonite but not molybdate. Arsenic is tightly attached to AioF, likely by cysteine residues. When loaded with arsenite or arsenate, AioF is able to bind specifically to the regulatory region of the aio operon at two distinct positions. In Thiomonas arsenitoxydans, the promoters of aioX and aioB are convergent, suggesting that transcriptional interference occurs. These results indicate that the regulation of the aioBA operon is more complex in Thiomonas arsenitoxydans than in the other aioBA containing arsenite oxidizers and that the arsenic binding protein AioF is involved in this regulation. On the basis of these data, a model to explain the tight control of aioBA expression by arsenic in Thiomonas arsenitoxydans is proposed.

DnaJ (Hsp40 protein) binding to folded substrate impacts KplE1 prophage excision efficiency.

Temperate phages mediate gene transfer and can modify the properties of their host organisms through the acquisition of novel genes, a process called lysogeny. The KplE1 prophage is one of the 10 prophage regions in Escherichia coli K12 MG1655. KplE1 is defective for lysis but fully competent for site-specific recombination. The TorI recombination directionality factor is strictly required for prophage excision from the host genome. We have previously shown that DnaJ promotes KplE1 excision by increasing the affinity of TorI for its site-specific recombination DNA target. Here, we provide evidence of a direct association between TorI and DnaJ using in vitro cross-linking assays and limited proteolysis experiments that show that this interaction allows both proteins to be transiently protected from trypsin digestion. Interestingly, NMR titration experiments showed that binding of DnaJ involves specific regions of the TorI structure. These regions, mainly composed of α-helices, are located on a surface opposite the DNA-binding site. Taken together, we propose that DnaJ, without the aid of DnaK/GrpE, is capable of increasing the efficiency of KplE1 excision by causing a conformational stabilization that allows TorI to adopt a more favorable conformation for binding to its specific DNA target.

Arsenite oxidase from Ralstonia sp. 22: characterization of the enzyme and its interaction with soluble cytochromes.

We characterized the aro arsenite oxidation system in the novel strain Ralstonia sp. 22, a beta-proteobacterium isolated from soil samples of the Salsigne mine in southern France. The inducible aro system consists of a heterodimeric membrane-associated enzyme reacting with a dedicated soluble cytochrome c(554). Our biochemical results suggest that the weak association of the enzyme to the membrane probably arises from a still unknown interaction partner. Analysis of the phylogeny of the aro gene cluster revealed that it results from a lateral gene transfer from a species closely related to Achromobacter sp. SY8. This constitutes the first clear cut case of such a transfer in the Aro phylogeny. The biochemical study of the enzyme demonstrates that it can accommodate in vitro various cytochromes, two of which, c(552) and c(554,) are from the parent species. Cytochrome c(552) belongs to the sox and not the aro system. Kinetic studies furthermore established that sulfite and sulfide, substrates of the sox system, are both inhibitors of Aro activity. These results reinforce the idea that sulfur and arsenic metabolism are linked.

Modulation of cellulosome composition in Clostridium cellulolyticum: adaptation to the polysaccharide environment revealed by proteomic and carbohydrate-active enzyme analyses.

Clostridium cellulolyticum is a model mesophilic anaerobic bacterium that efficiently degrades plant cell walls. The recent genome release offers the opportunity to analyse its complete degradation system. A total of 148 putative carbohydrate-active enzymes were identified, and their modular structures and activities were predicted. Among them, 62 dockerin-containing proteins bear catalytic modules from numerous carbohydrate-active enzymes' families and whose diversity reflects the chemical and structural complexity of the plant carbohydrate. The composition of the cellulosomes produced by C. cellulolyticum upon growth on different substrates (cellulose, xylan, and wheat straw) was investigated by LC MS/MS. The majority of the proteins encoded by the cip-cel operon, essential for cellulose degradation, were detected in all cellulosome preparations. In the presence of wheat straw, the natural and most complex of the substrates studied, additional proteins predicted to be involved in hemicellulose degradation were produced. A 32-kb gene cluster encodes the majority of these proteins, all harbouring carbohydrate-binding module 6 or carbohydrate-binding module 22 xylan-binding modules along dockerins. This newly identified xyl-doc gene cluster, specialised in hemicellulose degradation, comes in addition of the cip-cel operon for plant cell wall degradation. Hydrolysis efficiencies determined on the different substrates corroborates the finding that cellulosome composition is adapted to the growth substrate.

Carbonylated proteins are detectable only in a degradation-resistant aggregate state in Escherichia coli.

Carbonylation is currently used as a marker for irreversible protein oxidative damage. Several studies indicate that carbonylated proteins are more prone to degradation than their nonoxidized counterparts. In this study, we observed that in Escherichia coli, more than 95% of the total carbonyl content consisted of insoluble protein and most were cytosolic proteins. We thereby demonstrate that, in vivo, carbonylated proteins are detectable mainly in an aggregate state. Finally, we show that detectable carbonylated proteins are not degraded in vivo. Here we propose that some carbonylated proteins escape degradation in vivo by forming carbonylated protein aggregates and thus becoming nondegradable. In light of these findings, we provide evidence that the accumulation of nondegradable carbonylated protein presented in an aggregate state contributes to the increases in carbonyl content observed during senescence.

Responses of the marine diatom Thalassiosira pseudonana to changes in CO2 concentration: a proteomic approach.

The concentration of CO2 in many aquatic systems is variable, often lower than the KM of the primary carboxylating enzyme Rubisco, and in order to photosynthesize efficiently, many algae operate a facultative CO2 concentrating mechanism (CCM). Here we measured the responses of a marine diatom, Thalassiosira pseudonana, to high and low concentrations of CO2 at the level of transcripts, proteins and enzyme activity. Low CO2 caused many metabolic pathways to be remodeled. Carbon acquisition enzymes, primarily carbonic anhydrase, stress, degradation and signaling proteins were more abundant while proteins associated with nitrogen metabolism, energy production and chaperones were less abundant. A protein with similarities to the Ca2+/ calmodulin dependent protein kinase II_association domain, having a chloroplast targeting sequence, was only present at low CO2. This protein might be a specific response to CO2 limitation since a previous study showed that other stresses caused its reduction. The protein sequence was found in other marine diatoms and may play an important role in their response to low CO2 concentration.

The intriguing CP12-like tail of adenylate kinase 3 from Chlamydomonas reinhardtii.

UNLABELLED: Adenylate kinases (ADK) are key enzymes that maintain the energetic balance in cellular compartments by catalyzing the reaction: AMP + ATP↔2 ADP. Here, we analyzed the chloroplast ADK 3 from the green alga, Chlamydomonas reinhardtii for the first time. This enzyme bears a C-terminal extension that is highly similar to the C-terminal end of the intrinsically disordered protein CP12 that plays a major role in the redox regulation of key enzymes of the Calvin-Benson cycle like glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase. The only other known example of a CP12-like extension is found in the GapB isoform of GAPDH, where it is responsible for the autonomous redox regulation of the higher plant A2 B2 GAPDH. In this study, we show that the CP12-like tail is not involved in the redox regulation of ADK 3, but contributes greatly to its stability, and is essential for the post-translational modification of the Cys221 residue by glutathione. This report highlights the fact that the C-terminal part of the CP12 protein can act as a moonlighting, intrinsically disordered module conferring additional capabilities to the proteins to which it is added. ENZYMES: Adenylate kinase (ADK, EC 2.7.4.3) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13).

Expression of terminal oxidases under nutrient-starved conditions in Shewanella oneidensis: detection of the A-type cytochrome c oxidase.

Shewanella species are facultative anaerobic bacteria that colonize redox-stratified habitats where O2 and nutrient concentrations fluctuate. The model species Shewanella oneidensis MR-1 possesses genes coding for three terminal oxidases that can perform O2 respiration: a bd-type quinol oxidase and cytochrome c oxidases of the cbb3-type and the A-type. Whereas the bd- and cbb3-type oxidases are routinely detected, evidence for the expression of the A-type enzyme has so far been lacking. Here, we investigated the effect of nutrient starvation on the expression of these terminal oxidases under different O2 tensions. Our results reveal that the bd-type oxidase plays a significant role under nutrient starvation in aerobic conditions. The expression of the cbb3-type oxidase is also modulated by the nutrient composition of the medium and increases especially under iron-deficiency in exponentially growing cells. Most importantly, under conditions of carbon depletion, high O2 and stationary-growth, we report for the first time the expression of the A-type oxidase in S. oneidensis, indicating that this terminal oxidase is not functionally lost. The physiological role of the A-type oxidase in energy conservation and in the adaptation of S. oneidensis to redox-stratified environments is discussed.

Microbial oxidative sulfur metabolism: biochemical evidence of the membrane-bound heterodisulfide reductase-like complex of the bacterium Aquifex aeolicus.

The Hdr (heterodisulfide reductase)-like enzyme is predicted, from gene transcript profiling experiments previously published, to be essential in oxidative sulfur metabolism in a number of bacteria and archaea. Nevertheless, no biochemical and physicochemical data are available so far about this enzyme. Genes coding for it were identified in Aquifex aeolicus, a Gram-negative, hyperthermophilic, chemolithoautotrophic and microaerophilic bacterium that uses inorganic sulfur compounds as electron donor to grow. We provide biochemical evidence that this Hdr-like enzyme is present in this sulfur-oxidizing prokaryote (cultivated with thiosulfate or elemental sulfur). We demonstrate, by immunolocalization and cell fractionation, that Hdr-like enzyme is associated, presumably monotopically, with the membrane fraction. We show by co-immunoprecipitation assay or partial purification, that the Hdr proteins form a stable complex composed of at least five subunits, HdrA, HdrB1, HdrB2, HdrC1 and HdrC2, present in two forms of high molecular mass on native gel (∼240 and 450 kDa). These studies allow us to propose a revised model for dissimilatory sulfur oxidation pathways in A. aeolicus, with Hdr predicted to generate sulfite.

Phosphoribulokinase from Chlamydomonas reinhardtii: a Benson-Calvin cycle enzyme enslaved to its cysteine residues.

Phosphoribulokinase (PRK) in the green alga Chlamydomonas reinhardtii is a finely regulated and well-studied enzyme of the Benson-Calvin cycle. PRK can form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small chloroplast protein CP12. This study aimed to determine the molecular determinants on PRK involved in the complex and the mechanism of action of a recently described novel regulation of PRK that involves glutathionylation. A combination of mass spectrometry, mutagenesis and activity analyses showed that Cys16, besides its role as the binding site of ATP, was also the site for S-glutathionylation. Previous kinetic analysis of the C55S mutant showed that in the oxidized inactive form of PRK, this residue formed a disulfide bridge with the Cys16 residue. This is the only bridge reported for PRK in the literature. Our data show for the first time that a disulfide bridge between Cys243 and Cys249 on PRK is required to form the PRK-GAPDH-CP12 complex. These results uncover a new mechanism for the PRK-GAPDH-CP12 formation involving a thiol disulfide exchange reaction with CP12 and identify Cys16 of PRK as a target of glutathionylation acting against oxidative stress. Although Cys16 is the key residue involved in binding ATP and acting as a defense against oxidative damage, the formation of the algal ternary complex requires the formation of another disulfide bridge on PRK involving Cys243 and Cys249.

The primary pathway for lactate oxidation in Desulfovibrio vulgaris.

The ability to respire sulfate linked to lactate oxidation is a key metabolic signature of the Desulfovibrio genus. Lactate oxidation by these incomplete oxidizers generates reductants through lactate dehydrogenase (LDH) and pyruvate-ferredoxin oxidoreductase (PFOR), with the latter catalyzing pyruvate conversion into acetyl-CoA. Acetyl-CoA is the source of substrate-level phosphorylation through the production of ATP. Here, we show that these crucial steps are performed by enzymes encoded by a nonacistronic transcriptional unit named now as operon luo (for lactate utilization operon). Using a combination of genetic and biochemical techniques, we assigned a physiological role to the operon genes DVU3027-28 and DVU3032-33. The growth of mutant Δ26-28 was highly disrupted on D-lactate, whereas the growth of mutant Δ32-33 was slower on L-lactate, which could be related to a decrease in the activity of D-lactate or L-lactate oxidase in the corresponding mutants. The DVU3027-28 and DVU3032-33 genes thus encode functional D-LDH and L-LDH enzymes, respectively. Scanning of the genome for lactate utilization revealed several lactate permease and dehydrogenase homologs. However, transcriptional compensation was not observed in any of the mutants except for lactate permease. Although there is a high degree of redundancy for lactate oxidase, it is not functionally efficient in LDH mutants. This result could be related to the identification of several operon enzymes, including LDHs, in the PFOR activity bands, suggesting the occurrence of a lactate-oxidizing supermolecular structure that can optimize the performance of lactate utilization in Desulfovibrio species.

A biochemical approach to study the role of the terminal oxidases in aerobic respiration in Shewanella oneidensis MR-1.

The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb 3-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb 3-type oxidase is the major terminal oxidase under aerobic conditions while both cbb 3-type and bd-type oxidases are involved in respiration at low-O2 tensions. On the contrary, the low O2-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa 3-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa 3-type oxidase in S. oneidensis MR-1 are discussed.

The intrinsically disordered C-terminal region of Arabidopsis thaliana TCP8 transcription factor acts both as a transactivation and self-assembly domain.

TCPs are plant specific transcription factors with non-canonical basic helix-loop-helix domains. While Arabidopsis thaliana has 24 TCPs involved in cell proliferation and differentiation, their mode of action has not been fully elucidated. Using bioinformatic tools, we demonstrate that TCP transcription factors belong to the intrinsically disordered proteins (IDP) family and that disorder is higher in class I TCPs than in class II TCPs. In particular, using bioinformatic and biochemical approaches, we have characterized TCP8, a class I TCP. TCP8 exhibits three intrinsically disordered regions (IDR) made of more than 50 consecutive residues, in which phosphorylable Ser residues are mainly clustered. Phosphorylation of Ser-211 that belongs to the central IDR was confirmed by mass spectrometry. Yeast two-hybrid assays also showed that the C-terminal IDR corresponds to a transactivation domain. Moreover, biochemical experiments demonstrated that TCP8 tends to oligomerize in dimers, trimers and higher-order multimers. Bimolecular fluorescence complementation (BiFC) experiments carried out on a truncated form of TCP8 lacking the C-terminal IDR indicated that it is effectively required for the pronounced self-assembly of TCP8. These data were reinforced by the prediction of a coiled coil domain in this IDR. The C-terminal IDR acts thus as an oligomerization domain and also a transactivation domain. Moreover, many Molecular Recognition Features (MoRFs) were predicted, indicating that TCP8 could interact with several partners to fulfill a fine regulation of transcription in response to various stimuli.

Dynamics of the intrinsically disordered protein CP12 in its association with GAPDH in the green alga Chlamydomonas reinhardtii: a fuzzy complex.

CP12 is a widespread regulatory protein of oxygenic photosynthetic organisms that contributes to the regulation of the Calvin cycle by forming a supra-molecular complex with at least two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). CP12 shares some similarities with intrinsically disordered proteins (IDPs) depending on its redox state. In this study, site-directed spin labeling (SDSL) combined with EPR spectroscopy was used to probe the dynamic behavior of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH, the first step towards ternary complex formation. The two N-terminal cysteine residues were labeled using the classical approach while the tyrosine located at the C-terminal end of CP12 was modified following an original procedure. The results show that the label grafted at the C-terminal extremity is in the vicinity of the interaction site whereas the N-terminal region remains fully disordered upon binding to GAPDH. In conclusion, GAPDH-CP12 is a fuzzy complex, in which the N-terminal region of CP12 keeps a conformational freedom in the bound form. This fuzziness could be one of the keys to facilitate binding of PRK to CP12-GAPDH and to form the ternary supra-molecular complex.

Interplay between three RND efflux pumps in doxycycline-selected strains of Burkholderia thailandensis.

BACKGROUND: Efflux systems are involved in multidrug resistance in most Gram-negative non-fermentative bacteria. We have chosen Burkholderia thailandensis to dissect the development of multidrug resistance phenotypes under antibiotic pressure. METHODOLOGY/PRINCIPAL FINDINGS: We used doxycycline selection to obtain several resistant B. thailandensis variants. The minimal inhibitory concentrations of a large panel of structurally unrelated antibiotics were determined ± the efflux pump inhibitor phenylalanine-arginine ß-naphthylamide (PAßN). Membrane proteins were identified by proteomic method and the expressions of major efflux pumps in the doxycycline selected variants were compared to those of the parental strains by a quantitative RT-PCR analysis. Doxycycline selected variants showed a multidrug resistance in two major levels corresponding to the overproduction of two efflux pumps depending on its concentration: AmrAB-OprA and BpeEF-OprC. The study of two mutants, each lacking one of these pumps, indicated that a third pump, BpeAB-OprB, could substitute for the defective pump. Surprisingly, we observed antagonistic effects between PAßN and aminoglycosides or some ß-lactams. PAßN induced the overexpression of AmrAB-OprA and BpeAB-OprB pump genes, generating this unexpected effect. CONCLUSIONS/SIGNIFICANCE: These results may account for the weak activity of PAßN in some Gram-negative species. We clearly demonstrated two antagonistic effects of this molecule on bacterial cells: the blocking of antibiotic efflux and an increase in efflux pump gene expression. Thus, doxycycline is a very efficient RND efflux pump inducer and PAßN may promote the production of some efflux pumps. These results should be taken into account when considering antibiotic treatments and in future studies on efflux pump inhibitors.

CP12 from Chlamydomonas reinhardtii, a permanent specific "chaperone-like" protein of glyceraldehyde-3-phosphate dehydrogenase.

A new role is reported for CP12, a highly unfolded and flexible protein, mainly known for its redox function with A(4) glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both reduced and oxidized CP12 can prevent the in vitro thermal inactivation and aggregation of GAPDH from Chlamydomonas reinhardtii. This mechanism is thus not redox-dependent. The protection is specific to CP12, because other proteins, such as bovine serum albumin, thioredoxin, and a general chaperone, Hsp33, do not fully prevent denaturation of GAPDH. Furthermore, CP12 acts as a specific chaperone, since it does not protect other proteins, such as catalase, alcohol dehydrogenase, or lysozyme. The interaction between CP12 and GAPDH is necessary to prevent the aggregation and inactivation, since the mutant C66S that does not form any complex with GAPDH cannot accomplish this protection. Unlike the C66S mutant, the C23S mutant that lacks the N-terminal bridge is partially able to protect and to slow down the inactivation and aggregation. Tryptic digestion coupled to mass spectrometry confirmed that the S-loop of GAPDH is the interaction site with CP12. Thus, CP12 not only has a redox function but also behaves as a specific "chaperone-like protein" for GAPDH, although a stable and not transitory interaction is observed. This new function of CP12 may explain why it is also present in complexes involving A(2)B(2) GAPDHs that possess a regulatory C-terminal extension (GapB subunit) and therefore do not require CP12 to be redox-regulated.

The cellulosomes from Clostridium cellulolyticum: identification of new components and synergies between complexes.

Cellulosomes produced by Clostridium cellulolyticum grown on cellulose were purified and separated using anion-exchange chromatography. SDS/PAGE analysis of six fractions showed variations in their cellulosomal protein composition. Hydrolytic activity on carboxymethyl cellulose, xylan, crystalline cellulose and hatched straw differed from one fraction to another. Fraction F1 showed a high level of activity on xylan, whereas fractions F5 and F6 were most active on crystalline cellulose and carboxymethyl cellulose, respectively. Several cellulosomal components specific to fractions F1, F5 and F6 were investigated using MS analysis. Several hemicellulases were identified, including three xylanases in F1, and several cellulases belonging to glycoside hydrolase families 9 and 5 and, a cystein protease inhibitor were identified in F5 and F6. Synergies were observed when two or three fractions were combined. A mixture containing fractions F1, F3 and F6 showed the most divergent cellulosomal composition, the most synergistic effects and the highest level of activity on straw (the most heterogeneous substrate tested). These findings show that on complex substrates such as straw, synergies occur between differently composed cellulosomes and the degradation efficiency of the cellulosomes is correlated with their enzyme diversity.