Claire-Lise Santini

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.

Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli

The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl2, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but over‐expression of MobB increases the level of factor X. MobB, therefore, can participate in nitrate reductase activation. The narJ protein is not a component of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A. Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity. Although the mob gene products are necessary for the biosynthesis of all E. coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A. The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein. NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis is complete.

NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli

The formation of active membrane‐bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ‐6His polypeptide to the cell supernatant of a narJ strain. Once the apo‐enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro. NarJ protein specifically recognizes the catalytic NarG subunit. Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent‐open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the apo‐nitrate reductase, NarJ is then dissociated from the activated enzyme.

Biogenesis of actin-like bacterial cytoskeletal filaments destined for positioning prokaryotic magnetic organelles

Magnetosomes comprise a magnetic nanocrystal surrounded by a lipid bilayer membrane. These unique prokaryotic organelles align inside magnetotactic bacterial cells and serve as an intracellular compass allowing the bacteria to navigate along the geomagnetic field in aquatic environments. Cryoelectron tomography of Magnetospirillum strains has revealed that the magnetosome chain is surrounded by a network of filaments that may be composed of MamK given that the filaments are absent in the mamK mutant cells. The process of the MamK filament assembly is unknown. Here we prove the authenticity of the MamK filaments and show that MamK exhibits linear distribution inside Magnetospirillum sp. cells even in the area without magnetosomes. The mamK gene alone is sufficient to direct the synthesis of straight filaments in Escherichia coli, and one extremity of the MamK filaments is located at the cellular pole. By using dual fluorescent labeling of MamK, we found that MamK nucleates at multiple sites and assembles into mosaic filaments. Time-lapse experiments reveal that the assembly of the MamK filaments is a highly dynamic and kinetically asymmetrical process. MamK bundles might initiate the formation of a new filament or associate to one preexistent filament. Our results demonstrate the mechanism of biogenesis of prokaryotic cytoskeletal filaments that are structurally and functionally distinct from the known MreB and ParM filaments. In addition to positioning magnetosomes, other hypothetical functions of the MamK filaments in magnetotaxis might include anchoring magnetosomes and being involved in magnetic reception.

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

Enzymatic and physiological properties of the tungsten‐substituted molybdenum TMAO reductase from Escherichia coli

The trimethylamine N‐oxide (TMAO) reductase of Escherichia coli is a molybdoenzyme that catalyses the reduction of the TMAO to trimethylamine (TMA) with a redox potential of + 130 mV. We have successfully substituted the molybdenum with tungsten and obtained an active tungsto‐TMAO reductase. Kinetic studies revealed that the catalytic efficiency of the tungsto‐substituted TMAO reductase (W‐TorA) was increased significantly (twofold), although a decrease of about 50% in its kcat was found compared with the molybdo‐TMAO reductase (Mo‐TorA). W‐TorA is more sensitive to high pH, is less sensitive to high NaCl concentration and is more heat resistant than Mo‐TorA. Most importantly, the W‐TorA becomes capable of reducing sulphoxides and supports the anaerobic growth of a bacterial host on these substrates. The evolutionary implication and mechanistic significance of the tungsten substitution are discussed.

Topology determination and functional analysis of the Escherichia coli TatC protein.

The TatC protein is an essential component of the bacterial Tat system. By using alkaline phosphatase and β‐glucuronidase fusions we found that TatC contains four transmembrane helices. Three insertions of Ala‐Ser dipeptide at the cytoplasmic N‐ and C‐termini and in the cytoplasmic loop had no or only partial effect on the TatC function. In contrast, five of seven insertions in the two periplasmic loops abolished the Tat function. Four insertions analyzed had no effect on the stability of the altered TatC proteins or on membrane assembly of the TatA and TatB proteins. These data provide a novel base for more detailed studies of the mechanism of the Tat system.

Export of Thermus thermophilus alkaline phosphatase via the twin-arginine translocation pathway in Escherichia coli

The bacterial twin‐arginine translocation (Tat) pathway is distinct from the Sec system by its remarkable capacity to export folded enzymes. To address the question whether the two systems are capable of translocating homologous enzymes catalyzing the same reaction, we cloned the tap gene encoding Thermus thermophilus alkaline phosphatase (Tap) and expressed it in Escherichia coli. Unlike the alkaline phosphatase of E. coli, which is translocated through the Sec system and then activated in the periplasm, Tap was exported exclusively via the Tat pathway and active Tap precursor was observed in the cytoplasm. These results demonstrate that two sequence and functional related enzymes are exported by distinct protein transport systems, which may play an integral role in the bacterial adaptation to their environment during the evolution.

Requirement for phospholipids of the translocation of the trimethylamine N-oxide reductase through the Tat pathway in Escherichia coli.

Trimethylamine N‐oxide reductase (TorA) is an anaerobically synthesized molybdoenzyme. It is translocated across the cytoplasmic membrane in a folded conformation via the Tat pathway of Escherichia coli. The requirement for phospholipids for the export of this enzyme was analyzed in the pgsA and pss mutants lacking anionic phospholipids and phosphatidylethanolamine, respectively. Anaerobic growth did not influence phospholipid composition of the pgsA and pss mutants. Interestingly, both pgsA and pss mutations severely retarded the translocation of TorA into the periplasm. Therefore, translocation of proteins through the Tat pathway is dependent on the anionic phospholipids and on lipid polymorphism.

Mutants of Escherichia coli specifically deficient in respiratory formate dehydrogenase activity.

Escherichia coli K12 mutants lacking phenazine-methosulphate-linked formate dehydrogenase (FDH-PMS) activity, but still capable of producing normal levels of benzyl-viologen-linked formate dehydrogenase (FDH-BV) and nitrate reductase activities, have been isolated following P1 localized mutagenesis. The relevant mutations mapped with the same cotransduction frequency close to the rhaD gene, at 88 min on the E. coli chromosome. They were further subdivided into two classes. Class I consisted of six fdhD mutants which synthesized an inactive FDH-PMS protein with the same subunit composition as the wild-type enzyme. In contrast, class II contained four fdhE mutants totally devoid of this antigen. Construction of merodiploid strains harbouring various combinations of the mutated alleles, fdhE on the episome and fdhD on the chromosome, led to the restoration of FDH-PMS activity by complementation of the products encoded by the respective wild-type alleles. Difference spectroscopy suggested that both fdhD and fdhE mutants contained normal amounts of the cytochrome b559 associated with FDH-PMS although the cytochrome had lost its capacity for formate-dependent reduction.