Bacterial Protein Export Research Articles

2PS028 低温電子線トモグラフィーによって明らかとなった細菌べん毛蛋白質輸送装置の構造と機能(日本生物物理学会第50回年会(2012年度))

2PS028 低温電子線トモグラフィーによって明らかとなった細菌べん毛蛋白質輸送装置の構造と機能(日本生物物理学会第50回年会(2012年度))

2I1436 べん毛輸送装置近傍の局所pHに対するFlil ATPaseの効果(細胞生物的課題II:Prokaryotes,口頭発表,日本生物物理学会第50回年会(2012年度))

2I1436 べん毛輸送装置近傍の局所pHに対するFlil ATPaseの効果(細胞生物的課題II:Prokaryotes,口頭発表,日本生物物理学会第50回年会(2012年度))

Interaction between FliI ATPase and a flagellar chaperone FliT during bacterial flagellar protein export

FliT is a flagellar type III export chaperone specific for the filament-capping protein FliD. The FliT/FliD complex binds to the FliI ATPase of the flagellar export apparatus. The C-terminal α4 helix of FliT controls its interaction with FliI but it remains unknown how it does so. Here, we analysed the FliI-FliT interaction by pull-down assays using GST affinity chromatography. FliT94, missing the C-terminal α4 helix, bound to the extreme N-terminal region of FliI (FliI(EN)) with high affinity and to the C-terminal ATPase domain (FliI(CAT)) with low affinity. The C-terminal α4 helix of FliT suppressed the interaction with FliI(EN). FliH and FliT94 bound to a common binding site on FliI(EN) and hence FliH induced the release of FliI from FliT94 in an ATP-independent manner. FliD increased the binding affinity of FliI(CAT) for FliT. These results raise a possible hypothesis that the FliH/FliI complex binds to the FliT/FliD complex through FliI(CAT) to escort it from the cytoplasm to the export gate made up of six integral membrane proteins and that, upon dissociation of FliD from FliT, FliT94 may bind to FliI(EN) and then FliI may transfer from FliT94 to FliH by the direct competition of FliT94 and FliH for FliI(EN).

1K1324 細菌べん毛タンパク質輸送装置近傍の局所pHの測定(細胞生物的課題1,第49回日本生物物理学会年会)

1K1324 細菌べん毛タンパク質輸送装置近傍の局所pHの測定(細胞生物的課題1,第49回日本生物物理学会年会)

1P204 細菌べん毛蛋白質輸送におけるFliI ATPaseとFliTシャペロンとの相互作用(細胞生物的課題(接着,運動,骨格,伝達,膜),第48回日本生物物理学会年会)

1P204 細菌べん毛蛋白質輸送におけるFliI ATPaseとFliTシャペロンとの相互作用(細胞生物的課題(接着,運動,骨格,伝達,膜),第48回日本生物物理学会年会)

1P207 細菌べん毛特異的蛋白質輸送に関わるFliH-FliI複合体のX線結晶構造解析(細胞生物的課題(接着,運動,骨格,伝達,膜),第48回日本生物物理学会年会)

1P207 細菌べん毛特異的蛋白質輸送に関わるFliH-FliI複合体のX線結晶構造解析(細胞生物的課題(接着,運動,骨格,伝達,膜),第48回日本生物物理学会年会)

Interaction of FliK with the bacterial flagellar hook is required for efficient export specificity switching

FliK-FlhB interaction switches export specificity of the bacterial flagellar protein export apparatus to stop hook protein export at an appropriate timing for hook length control. The hook structure is required for the productive FliK-FlhB interaction to flip the switch but it remains unknown how it works. Here, we characterize the role of FliK in the switching probability in the absence of the hook. When RflH/Flk was missing in the hook mutants, the switching occurred at a low probability. Overproduction of FliK significantly increased the switching probability although not at the wild-type level. An in-frame deletion of residues 129 through 159 of FliK weakened the interaction with the hook protein but not with the hook-capping protein, producing polyhooks with filaments attached. We suggest that temporary association of FliK with the inner surface of the hook during FliK secretion results in a pause in the secretion process to allow the C-terminal switch domain of FliK to be positioned and appropriately oriented near FlhB for catalysing the switch and that RflH/Flk interferes with premature switch by preventing access of cytoplasmic FliK to FlhB and even that of FliK during its secretion until hook length reaches 55 nm; only then FliK(C) passes the RflH/Flk block.

ATP-induced FliI hexamerization facilitates bacterial flagellar protein export

FliI ATPase forms a homo-hexamer to fully exert its ATPase activity, facilitating bacterial flagellar protein export. However, it remains unknown how FliI hexamerization is linked to protein export. Here, we analyzed the capability of ring formation by FliI and its catalytic mutant variants. Compared to ATP a non-hydrolysable ATP analog increased the probability of FliI hexamerization. In contrast, FliI(E221Q), which retained the affinity for ATP but has lost ATPase activity, efficiently formed the hexamer even in the presence of ATP. The mutations, which reduced the binding affinity for ATP, significantly abolished the ring formation. These results indicate that ATP-binding induces FliI hexamerization and that the release of ADP and Pi destabilizes the ring structure. FliI(E221Q) facilitated flagellar protein export in the absence of the FliH regulator of the export apparatus although not at the wild-type FliI level while the other did not. We propose that FliI couples ATP binding and hydrolysis to its assembly–disassembly cycle to efficiently initiate the flagellar protein export cycle.

Genetic Suppressors and Recovery of Repressed Biochemical Memory

Many of the Reflections articles in this journal have focused on particular biological problems that the authors have devoted their careers to or on particular techniques that have facilitated their biological studies. Although I began my career as a biochemist, one of the most common threads to my research has been the exploitation of a genetic approach, the analysis of suppressor mutations. Particularly in the last 20 years, the success of this approach has led me into biological problems that required a remembering or relearning of my early training in biochemistry. This article will describe the voyage from chemistry and biochemistry to genetics and then to a recovery of remnants of my biochemical memory. As a graduate student working under Lowell Hager at Harvard in the late 1950s, my thesis research was chemical and biochemical, as I performed the chemical synthesis and studied the biosynthesis of the chlorinated mold product caldariomycin (2,2-dichloro-1,3-cyclopentanediol) (1–3). With his postdoctoral fellow Paul Shaw, Lowell had discovered an enzyme, chloroperoxidase, that was capable of utilizing chloride ion to chlorinate organic compounds (4). Part of my work was to determine how this enzyme might be involved in the biosynthesis of caldariomycin. Apparently, the work became somewhat well known. This I learned when, toward the end of my Ph.D. work, I attended a FASEB meeting in Atlantic City to give a short presentation. As I walked into the conference center on the first day, a scientist passing by me stopped to look at my badge, hesitated a moment, and then commented “Beckwith... hmmm, oh yes, I remember... chlorination!” Despite this really quite limited chemical/biochemical notoriety, I became more and more fascinated by bacterial genetics in graduate school. I proceeded to do postdoctoral work in several bacterial genetics laboratories.

The Accessory SecA2 System of Mycobacteria Requires ATP Binding and the Canonical SecA1

In bacteria, the majority of exported proteins are transported by the general Sec pathway from their site of synthesis in the cytoplasm across the cytoplasmic membrane. The essential SecA ATPase powers this Sec-mediated export. Mycobacteria possess two nonredundant SecA homologs: SecA1 and SecA2. In pathogenic Mycobacterium tuberculosis and the nonpathogenic model mycobacterium Mycobacterium smegmatis, SecA1 is essential for protein export and is the "housekeeping" SecA, whereas SecA2 is an accessory SecA that exports a specific subset of proteins. In M. tuberculosis the accessory SecA2 pathway plays a role in virulence. In this study, we uncovered basic properties of the mycobacterial SecA2 protein and its pathway for exporting select proteins. By constructing secA2 mutant alleles that encode proteins defective in ATP binding, we showed that ATP binding is required for SecA2 function. SecA2 mutant proteins unable to bind ATP were nonfunctional and dominant negative. By evaluating the subcellular distribution of each SecA, SecA1 was shown to be equally divided between cytosolic and cell envelope fractions, whereas SecA2 was predominantly localized to the cytosol. Finally, we showed that the canonical SecA1 has a role in the process of SecA2-dependent export. The accessory SecA2 export system is important to the physiology and virulence of mycobacteria. These studies help establish the mechanism of this new type of specialized protein export pathway.

Role of the N-terminal domain of FliI ATPase in bacterial flagellar protein export

FliI, the ATPase involved in bacterial flagellar protein export, forms a complex with its regulator FliH in the cytoplasm and hexamerizes upon docking to the export gate composed of integral membrane proteins. The extreme N-terminal region of FliI is involved not only in its interaction with FliH but also in its oligomerization, but the regulatory mechanism of oligomerization remains unclear. Using in-frame 10-residue deletions within the 100 residues of the N-terminal domain, we demonstrate that the first 20 residues are required for FliH binding and that the conformation of the N-terminal domain is sensitive to the export function, even though the oligomerization and FliH-binding ability are retained and the ATPase activity is maintained in most of the deletion variants.

2SA2-05 水素イオンで動く細菌べん毛蛋白質輸送装置の動作機構(2SA2 タンパク質の膜透過輸送の最前線,第47回日本生物物理学会年会)

2SA2-05 水素イオンで動く細菌べん毛蛋白質輸送装置の動作機構(2SA2 タンパク質の膜透過輸送の最前線,第47回日本生物物理学会年会)

2P-142 細菌べん毛蛋白質輸送装置構成蛋白質FlhBのC末細胞質ドメインの機能解析(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

2P-142 細菌べん毛蛋白質輸送装置構成蛋白質FlhBのC末細胞質ドメインの機能解析(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

Bacterial Protein Secretion and Trafficking Down Under

In July 2008, Australian microbiologists braved wintry Melbourne weather to attend the Annual Scientific Meeting of the Australian Society for Microbiology. The meeting attracted more than 1100 delegates from around the Asia–Pacific region and offered over 400 scientific presentations covering the latest research findings from leading local and international research and clinical laboratories. The theme of the meeting, ‘Changing Times’, highlighted the immense challenges ahead for the microbiology community in responding quickly and appropriately to often unpredictable environmental, political, public health and regulatory changes in the face of microbes that are changing just as rapidly. Keynote speakers addressed many of these issues within their presentations including Nobel Laureate, Peter Doherty (Department of Microbiology & Immunology, University of Melbourne, Australia) who delivered the opening address. Mike Levine (Center for Vaccine Development, Baltimore, USA) gave the Commonwealth Serum Laboratory-sponsored Bazeley Oration and used his invaluable clinical insight to discuss the history and future development of typhoid vaccines as well as the use of Salmonella as a vaccine carrier, and the construction and testing of new-generation Salmonella vaccines. Alan Cowman (Walter and Eliza Hall Institute, Australia) gave the prestigious Rubbo Oration and presented a stunning overview of malaria biology as well as recent progress on mechanisms to control this devastating infectious disease. The meeting also hosted a special ‘Infection Control Day’, which comprised a series of workshops and presentations designed to draw attention to the problems of antibiotic resistance and the measures needed to reduce hospital-acquired infections and their transmission. In the area of microbial and molecular pathogenesis, a major topic of the meeting was the secretion of bacterial proteins and their trafficking within infected cells. There are now at least six recognized bacterial systems (Type I–VI) dedicated to the export of bacterial proteins that often play a direct role in the interaction of pathogens with their hosts. The study of these secretion systems and their substrates is therefore an essential part of understanding how a pathogen causes disease. In some cases, the export of a protein leads to the construction of zones within the bacterial membranes that serve as an adhesive organelle to contact host surfaces. In other cases, the secreted factors are translocated directly into a eukaryotic cell, where they interfere with host cell function. In this instance, a bacterial protein may need to cross two bacterial membranes and one or more mammalian cell membranes to reach a target site. The substrate specificity of secretion systems is remarkable and a single secretion system may be dedicated to a single substrate, only a few substrates or, in the case of Legionella, more than 150 proteins. Current work on the function of bacterial secretion systems and their substrates at the molecular and structural level is driven by the expectation of greater insight into how pathogens evolve and cause disease.

1P-202 蛍光イメージング法を用いたサルモネラ菌べん毛特異的ATPaseの基部体への離合集散機構の解析(細胞生物学的課題(1),第46回日本生物物理学会年会)

1P-202 蛍光イメージング法を用いたサルモネラ菌べん毛特異的ATPaseの基部体への離合集散機構の解析(細胞生物学的課題(1),第46回日本生物物理学会年会)

1P-174 細菌べん毛蛋白質輸送におけるプロトン駆動力の2成分の異なる使われ方(細胞生物学的課題(1),第46回日本生物物理学会年会)

1P-174 細菌べん毛蛋白質輸送におけるプロトン駆動力の2成分の異なる使われ方(細胞生物学的課題(1),第46回日本生物物理学会年会)

Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export

Translocation of many soluble proteins across cell membranes occurs in an ATPase-driven manner. For construction of the bacterial flagellum responsible for motility, most of the components are exported by the flagellar protein export apparatus. The FliI ATPase is required for this export, and its ATPase activity is regulated by FliH; however, it is unclear how the chemical energy derived from ATP hydrolysis is used for the export process. Here we report that flagellar proteins of Salmonella enterica serovar Typhimurium are exported even in the absence of FliI. A fliH fliI double null mutant was weakly motile. Certain mutations in FlhA or FlhB, which form the core of the export gate, substantially improved protein export and motility of the double null mutant. Furthermore, proton motive force was essential for the export process. These results suggest that the FliH-FliI complex facilitates only the initial entry of export substrates into the gate, with the energy of ATP hydrolysis being used to disassemble and release the FliH-FliI complex from the protein about to be exported. The rest of the successive unfolding/translocation process of the substrates is driven by proton motive force.

β-Lactamase can function as a reporter of bacterial protein export during Mycobacterium tuberculosis infection of host cells

Mycobacterium tuberculosis is an intracellular pathogen that is able to avoid destruction by host immune defences. Exported proteins of M. tuberculosis, which include proteins localized to the bacterial surface or secreted into the extracellular environment, are ideally situated to interact with host factors. As a result, these proteins are attractive candidates for virulence factors, drug targets and vaccine components. Here we describe a beta-lactamase reporter system capable of identifying exported proteins of M. tuberculosis during growth in host cells. Because beta-lactams target bacterial cell-wall synthesis, beta-lactamases must be exported beyond the cytoplasm to protect against these drugs. When used in protein fusions, beta-lactamase can report on the subcellular location of another protein as measured by protection from beta-lactam antibiotics. Here we demonstrate that a truncated TEM-1 beta-lactamase lacking a signal sequence for export ('BlaTEM-1) can be used in this manner directly in a mutant strain of M. tuberculosis lacking the major beta-lactamase, BlaC. The 'BlaTEM-1 reporter conferred beta-lactam resistance when fused to both Sec and Tat export signal sequences. We further demonstrate that beta-lactamase fusion proteins report on protein export while M. tuberculosis is growing in THP-1 macrophage-like cells. This genetic system should facilitate the study of proteins exclusively exported in the host environment by intracellular M. tuberculosis.

Mycobacterial virulence and specialized secretion: same story, different ending

The genomic region lost during the attenuation of BCG vaccine encodes a newly discovered secretion system conserved among gram-positive bacteria. A series of papers has now dissected the components of this system, revealing a unique Mycobacterium tuberculosis–specific signal for export of bacterial proteins into the host.

Feature‐based reappraisal of the Bacillus subtilis exoproteome

Proteomics-based verification of computer-assisted predictions on bacterial protein export have indicated that problems occur with the distinction between (Sec-type) signal peptides that govern protein secretion, and lipoprotein signal peptides or amino-terminal membrane anchors that cause protein retention in the membrane. Therefore, the main aim of this study was to investigate whether feature-based predictions by the SecretomeP (SecP) algorithm will aid the proteomics-based analysis of protein export in Bacillus subtilis. The SecP algorithm is trained to recognize features such as secondary structure and disordered regions, which are generally present in secreted proteins. The results showed that membrane-retained proteins receive, in general, high SecP scores, similar to the scores of secretory proteins. Importantly, the SecP algorithm aided in the re-evaluation of a class of previously identified proteins that remain attached to the membrane despite the presence of an apparent Sec-type signal peptide. These so-called 'Sec-attached' proteins receive on average a lower SecP score, and several of these proteins could be unmasked as transmembrane proteins by combined SecP and signal peptide analyses. Finally, the present study suggests that feature-based outlier analysis may provide leads towards the discovery of novel special-purpose pathways for bacterial protein export.