Angélique Chanal

A novel Sec‐independent periplasmic protein translocation pathway in Escherichia coli

The trimethylamine N‐oxide (TMAO) reductase of Escherichia coli is a soluble periplasmic molybdoenzyme. The precursor of this enzyme possesses a cleavable N‐terminal signal sequence which contains a twin‐arginine motif. By using various moa, mob and mod mutants defective in different steps of molybdocofactor biosynthesis, we demonstrate that acquisition of the molybdocofactor in the cytoplasm is a prerequisite for the translocation of the TMAO reductase. The activation and translocation of the TMAO reductase precursor are post‐translational processes, and activation is dissociable from translocation. The export of the TMAO reductase is driven mainly by the proton motive force, whereas sodium azide exhibits a limited effect on the export. The most intriguing observation is that translocation of the TMAO reductase across the cytoplasmic membrane is independent of the SecY, SecE, SecA and SecB proteins. Depletion of Ffh, a core component of the signal recognition particle of E.coli, appears to have a slight effect on the export of the TMAO reductase. These results strongly suggest that the translocation of the molybdoenzyme TMAO reductase into the periplasm uses a mechanism fundamentally different from general protein translocation.

Exploration of New Geometries in Cellulosome-Like Chimeras

ABSTRACT In this study, novel cellulosome chimeras exhibiting atypical geometries and binding modes, wherein the targeting and proximity functions were directly incorporated as integral parts of the enzyme components, were designed. Two pivotal cellulosomal enzymes (family 48 and 9 cellulases) were thus appended with an efficient cellulose-binding module (CBM) and an optional cohesin and/or dockerin. Compared to the parental enzymes, the chimeric cellulases exhibited improved activity on crystalline cellulose as opposed to their reduced activity on amorphous cellulose. Nevertheless, the various complexes assembled using these engineered enzymes were somewhat less active on crystalline cellulose than the conventional designer cellulosomes containing the parental enzymes. The diminished activity appeared to reflect the number of protein-protein interactions within a given complex, which presumably impeded the mobility of their catalytic modules. The presence of numerous CBMs in a given complex, however, also reduced their performance. Furthermore, a “covalent cellulosome” that combines in a single polypeptide chain a CBM, together with family 48 and family 9 catalytic modules, also exhibited reduced activity. This study also revealed that the cohesin-dockerin interaction may be reversible under specific conditions. Taken together, the data demonstrate that cellulosome components can be used to generate higher-order functional composites and suggest that enzyme mobility is a critical parameter for cellulosome efficiency.

Incorporation of Fungal Cellulases in Bacterial Minicellulosomes Yields Viable, Synergistically Acting Cellulolytic Complexes

ABSTRACT Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.

Potential receptor function of three homologous components, TatA, TatB and TatE, of the twin‐arginine signal sequence‐dependent metalloenzyme translocation pathway in Escherichia coli

The recent determination of the complete Helicobacter pylori genome sequence (Tomb et al., 1997, Nature 388: 539–547) and functional studies on the CAG pathogenicity island (Censini et al., 1997, Proc Natl Acad Sci USA 93: 14648–14653; Covacci et al., 1997, Trends Microbiol 5: 205–208) are major contributions to our understanding of this important pathogen. Current investigations focus on the CAG genes coding for a cytotoxin-associated antigen (CagA, ORF 547) and potential virulence factors such as homologues of Agrobacterium VirB4 (ORF 544), VirB7 (lipoprotein CagT, ORF 532), VirB9 (ORF 528), VirB10 (ORF 527), VirB11 (ORF 525) and VirD4 (ORF 524) proteins. Three additional virb4 gene copies (ORFs 017, 441 and 459) as well as a gene encoding a vacuolating toxin (VacA, ORF 887) are located outside the CAG region. CagA, VacA and a few other proteins were found to be secreted and to enhance the inflammatory response in the gastric mucosa. The presence of vir genes and several GC-rich islands further suggests the existence of an adapted DNA-transfer apparatus for delivering virulence genes across bacterial boundaries. Evidence for conjugation-like DNA-transfer mechanisms between Helicobacter pylori strains has already been demonstrated in vitro (Kuipers et al., 1998, J Bacteriol 180: 2901–2905), but genetic determinants remain unknown. An intriguing aspect is the sequence homology of the CAG-encoded proteins to the membrane pore-forming VirB proteins of the Agrobacterium tumour-inducing (Ti) plasmid for interkingdom export of transfer (T)-DNA from bacteria to plant cells. On the basis of this homology, the CAG island was recently suggested to code for an ancient secretion apparatus that is capable of exporting a variety of proteinaceous material, and possibly also nucleoprotein particles, from Helicobacter pylori (Christie, 1997, Trends Microbiol 5: 264–265). Each of the six proposed proteins in this system is also related to components forming the export machinery for the Bordetella pertussis toxin and broad-host-range DNA plasmids (Pansegrau and Lanka, 1996, Prog Nucleic Acid Res Mol Biol 54: 197–251). Interestingly, members of the VirD4 protein family that have been suggested to link the T-DNA complex directly to the exporting membrane channel have to date been detected only in agrobacterial Ti-plasmid and conjugative plasmid DNA-transfer systems but not in protein transporters. Besides the VirD4 encoded in the CAG island, there is another putative VirD4 homologue (TraG, ORF 1006) present in the Helicobacter pylori sequence. Other VirD proteins representing essential components of a hypothetical DNA-transfer apparatus of Helicobacter pylori have not been identified so far. Likewise, a DNA-processing Molecular Microbiology (1998) 30(3), 673–678

The Issue of Secretion in Heterologous Expression of Clostridium cellulolyticum Cellulase-Encoding Genes in Clostridium acetobutylicum ATCC 824

ABSTRACT The genes encoding the cellulases Cel5A, Cel8C, Cel9E, Cel48F, Cel9G, and Cel9M from Clostridium cellulolyticum were cloned in the C. acetobutylicum expression vector pSOS952 under the control of a Gram-positive constitutive promoter. The DNA encoding the native leader peptide of the heterologous cellulases was maintained. The transformation of the solventogenic bacterium with the corresponding vectors generated clones in the cases of Cel5A, Cel8C, and Cel9M. Analyses of the recombinant strains indicated that the three cellulases are secreted in an active form to the medium. A large fraction of the secreted cellulases, however, lost the C-terminal dockerin module. In contrast, with the plasmids pSOS952-cel9E, pSOS952-cel48F, and pSOS952-cel9G no colonies were obtained, suggesting that the expression of these genes has an inhibitory effect on growth. The deletion of the DNA encoding the leader peptide of Cel48F in pSOS952-cel48F, however, generated strains of C. acetobutylicum in which mature Cel48F accumulates in the cytoplasm. Thus, the growth inhibition observed when the wild-type cel48F gene is expressed seems related to the secretion of the cellulase. The weakening of the promoter, the coexpression of miniscaffoldin-encoding genes, or the replacement of the native signal sequence of Cel48F by that of secreted heterologous or endogenous proteins failed to generate strains secreting Cel48F. Taken together, our data suggest that a specific chaperone(s) involved in the secretion of the key family 48 cellulase, and probably Cel9G and Cel9E, is missing or insufficiently synthesized in C. acetobutylicum.

Specific Inhibition of the Translocation of a Subset of Escherichia coli TAT Substrates by the TorA Signal Peptide

The SufI protein and the trimethylamine N-oxide reductase (TorA) are the two best-characterized prototype proteins exported by the Escherichia coli TAT system. Whereas SufI does not contain cofactors, TorA is a molybdo-enzyme and the acquisition of the molybdo-cofactor is a prerequisite for its translocation. The overproduction of each protein leads to the saturation of its translocation, but it was unknown if the overproduction of one substrate could saturate the TAT apparatus and block thus the translocation of other TAT substrates. Here, we showed that the overproduction of SufI saturated only its own translocation, but had no effect of the translocation of TorA and other TAT substrate analyzed. To dissect the saturation mechanism of TorA translocation, we shortened by about one-third of the TorA protein and removed nine consensus molybdo-cofactor-binding ligands. Like SufI, the truncated TorA (TorA502) did not contain cofactor and would not compete with the full length TorA for molybdo-cofactor acquisition. The overproduction of TorA502 completely inhibited the export of the full length TorA and dimethyl sulfoxide (DMSO) reductase, but had no effect on the translocation of SufI, nitrate-induced formate dehydrogenase and hydrogenase-2. Importantly, deletion of the twin-arginine signal peptide of TorA502 abolished the inhibitory effect. Moreover, the overproduction of the TorA signal peptide fused to the green fluorescence protein (GFP) was sufficient to block the TorA translocation. These results demonstrated that the twin-arginine signal peptide of the TorA protein specifically inhibits the translocation of a subset of TAT substrates, probably at the step of their targeting to the TAT apparatus.

Improving olefin tolerance and production in E. coli using native and evolved AcrB

Microorganisms can be engineered for the production of chemicals utilized in the polymer industry. However many such target compounds inhibit microbial growth and might correspondingly limit production levels. Here, we focus on compounds that are precursors to bioplastics, specifically styrene and representative alpha‐olefins; 1‐hexene, 1‐octene, and 1‐nonene. We evaluated the role of the Escherichia coli efflux pump, AcrAB‐TolC, in enhancing tolerance towards these olefin compounds. AcrAB‐TolC is involved in the tolerance towards all four compounds in E. coli. Both styrene and 1‐hexene are highly toxic to E. coli. Styrene is a model plastics precursor with an established route for production in E. coli (McKenna and Nielsen, 2011). Though our data indicates that AcrAB‐TolC is important for its optimal production, we observed a strong negative selection against the production of styrene in E. coli. Thus we used 1‐hexene as a model compound to implement a directed evolution strategy to further improve the tolerance phenotype towards this alpha‐olefin. We focused on optimization of AcrB, the inner membrane domain known to be responsible for substrate binding, and found several mutations (A279T, Q584R, F617L, L822P, F927S, and F1033Y) that resulted in improved tolerance. Several of these mutations could also be combined in a synergistic manner. Our study shows efflux pumps to be an important mechanism in host engineering for olefins, and one that can be further improved using strategies such as directed evolution, to increase tolerance and potentially production. Biotechnol. Bioeng. 2015;112: 879–888.

Effect of O2, H2 and redox potential on the activity and synthesis of hydrogenase 2 in Escherichia coli.

The aim of this work was to study the influence of O2 with special emphasis on low oxygen tension, the effect of H2 under various conditions of oxygen tension and the influence of the redox potential in the growth medium on hydrogenase 2 of Escherichia coli. The hydrogenase activity and the content of the large (HybC) and small (HybO) subunits of hydrogenase 2 were compared during turbidostat cultivation in a wild strain and mutant HDK103 lacking hydrogenases 1 and 3. No hydrogenase 2 activity in the mutant HDK103 was observed under aerobic conditions, but it was maximal under anaerobic conditions and half-maximal at an oxygen tension of approximately 4 mbar as is common for enzymes of anaerobic respiration. The content of hydrogenase 2 in both the strains was maximal under anaerobic conditions. In the wild strain, H2 addition enhanced hydrogenase activity and the HybO content under microaerobic conditions only. Under anaerobic conditions endogenous H2 production hindered this effect. Under aerobic conditions, the 02-related negative effect seemed to dominate over the H2-related positive effect. By contrast, in the mutant HDK103, hydrogen influenced neither hydrogenase 2 activity nor its content. A possible role of hydrogenase I in the response of hydrogenase 2 to hydrogen is discussed. Under conditions of different O2 tension, hydrogenase activity in both strains correlated inversely with the value of the redox potential of the medium. The presence of H2 changed this dependence. Thus, the value of the redox potential itself is not a controlling factor for hydrogenase 2.

Engineering Cellulase Activity into Clostridium acetobutylicum

Clostridium acetobutylicum produces substantial amounts of butanol, and an engineered cellulolytic strain of the bacterium would be an attractive candidate for biofuel production using consolidated bioprocessing. Recent studies have shown that this solventogenic bacterium can be used as a host for heterologous production and secretion of individual cellulosomal components, termed the minicellulosome. Their secretion yields range from 0.3 to 15 mg/L. Nevertheless, it appeared that key cellulosomal enzymes such as family GH48 processive enzymes and members of the large family of GH9 cellulases probably necessitate specific chaperone(s) for translocation and secretion, that is/are absent in the solventogenic bacterium. Heterologous secretion of the latter enzymes, however, can be obtained by grafting specific combinations of scaffoldin modules at the N-terminus of these cellulases, which are then used as cargo domains.