Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization

Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F1/V1-ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, cross-linking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa (monomer); II, approximately 300 kDa (putative hexamer); III, 575 kDa (dodecamer); and IV, approximately 3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (>700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane.     

Cloning, purification and characterization of a functional anthracycline glycosyltransferase

We have cloned the gene that encodes a novel glucosyl transferase (AraGT) involved in rhamnosylation of the polyketide antibiotic Aranciamycin in Streptomyces echinatus. AraGT comprises two domains characteristic of bacterial glycosyltranferases. AraGT was synthesized in E. coli as a decahistidinyl-tagged polypeptide. Purified AraGT is dimeric, displays a T(mapp) of 30 degrees C and can glycosylate the aglycone of an Aranciamycin derivative as shown by liquid chromatography and mass spectrometry. The availability of functional AraGT will allow the generation Aranciamycin-based combinatorial libraries.     

The Preprotein Binding Domain of SecA Displays Intrinsic Rotational Dynamics

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Long-Lived Folding Intermediates Predominate the Targeting-Competent Secretome

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Disorder-order folding transitions underlie catalysis in the helicase motor of SecA

SecA is a helicase-like motor that couples ATP hydrolysis with the translocation of extracytoplasmic protein substrates. As in most helicases, this process is thought to occur through nucleotide-regulated rigid-body movement of the motor domains. NMR, thermodynamic and biochemical data show that SecA uses a novel mechanism wherein conserved regions lining the nucleotide cleft undergo cycles of disorder-order transitions while switching among functional catalytic states. The transitions are regulated by interdomain interactions mediated by crucial 'arginine finger' residues located on helicase motifs. Furthermore, we show that the nucleotide cleft allosterically communicates with the preprotein substrate-binding domain and the regulatory, membrane-inserting C domain, thereby allowing for the coupling of the ATPase cycle to the translocation activity. The intrinsic plasticity and functional disorder-order folding transitions coupled to ligand binding seem to provide a precise control of the catalytic activation process and simple regulation of allosteric mechanisms.     

Evaluation of the fully automated Bactec MGIT 960 system for the susceptibility testing of Mycobacterium tuberculosis to first-line drugs: a multicenter study

The accuracy of the Bactec MGIT 960 system for susceptibility testing of 177 clinical isolates of Mycobacterium tuberculosis to first line drugs (isoniazid, rifampicin, ethambutol and streptomycin) was compared with the agar reference method. The sensitivity, the ability to detect resistance, of the MGIT system was 100%, while the specificity, the ability to detect susceptibility, ranged from 98.6% to 100% for all drugs tested.     

Escherichia coli SecA truncated at its termini is functional and dimeric

Terminal residues in SecA, the dimeric ATPase motor of bacterial preprotein translocase, were proposed to be required for function and dimerization. To test this, we generated truncation mutants of the 901aa long SecA of Escherichia coli. We now show that deletions of carboxy-terminal domain (CTD), the extreme CTD of 70 residues, or of the N-terminal nonapeptide or of both, do not compromise protein translocation or viability. Deletion of additional C-terminal residues upstream of CTD compromised function. Functional truncation mutants like SecA9-861 are dimeric, conformationally similar to SecA, fully competent for nucleotide and SecYEG binding and for ATP catalysis. Our data demonstrate that extreme terminal SecA residues are not essential for SecA catalysis and dimerization.     

Purification of a functional mature region from a SecA-dependent preprotein

Most of the bacterial proteins that are active in extracytoplasmic locations are translocated through the inner membrane by the Sec translocase. Translocase comprises a membrane "pore" and the peripheral ATPase SecA. Where preproteins bind to SecA and how they activate translocation ATPase remains elusive. To address this central question we have purified to homogeneity the mature and preprotein parts of an exported protein (pCH5EE). pCH5EE satisfies a minimal size required for protein translocation and its membrane insertion is SecA-dependent. Purified pCH5EE and CH5EE can form physical complexes with SecA and can functionally suppress the elevated ATPase of a constitutively activated mutant. These properties render pCH5EE and CH5EE unique tools for the biochemical mapping of the preprotein binding site on SecA.     

Phylogenetic analysis of a gene cluster encoding an additional, rhizobial-like type III secretion system that is narrowly distributed among Pseudomonas syringae strains

ABSTRACT: BACKGROUND: The central role of Type III secretion systems (T3SS) in bacteria-plant interactions is well established, yet unexpected findings are being uncovered through bacterial genome sequencing. Some Pseudomonas syringae strains possess an uncharacterized cluster of genes encoding putative components of a second T3SS (T3SS-2) in addition to the well characterized Hrc1 T3SS which is associated with disease lesions in host plants and with the triggering of hypersensitive response in non-host plants. The aim of this study is to perform an in silico analysis of T3SS-2, and to compare it with other known T3SSs. RESULTS: Based on phylogenetic analysis and gene organization comparisons, the T3SS-2 cluster of the P. syringae pv. phaseolicola strain is grouped with a second T3SS found in the pNGR234b plasmid of Rhizobium sp. These additional T3SS gene clusters define a subgroup within the Rhizobium T3SS family. Although, T3SS-2 is not distributed as widely as the Hrc1 T3SS in P. syringae strains, it was found to be constitutively expressed in P. syringae pv phaseolicola through RT-PCR experiments. CONCLUSIONS: The relatedness of the P. syringae T3SS-2 to a second T3SS from the pNGR234b plasmid of Rhizobium sp., member of subgroup II of the rhizobial T3SS family, indicates common ancestry and/or possible horizontal transfer events between these species. Functional analysis and genome sequencing of more rhizobia and P. syringae pathovars may shed light into why these bacteria maintain a second T3SS gene cluster in their genome.