ABSTRACT
BACKGROUND: The WHO's Health Promoting Schools (HPS) framework is based on an understanding of the reciprocal relationship between health and education, and the need to take a holistic approach to health promotion in schools. We aim to clarify the degree to which the HPS framework is reflected in the national policies of eight target countries and the issues surrounding its successful implementation. METHODS: Date were collected through two expert workshops with participants from eight Asian countries: Cambodia, China, Japan, Korea, Lao PDR, Nepal, the Philippines, and Thailand. In the first workshop, data collected on national policy were mapped against the HPS framework. From this, key issues were identified, and follow-up data collection was conducted in each country for a second workshop. RESULTS: We identified a policy shift toward the HPS framework in six out of the eight countries. Neither Japan nor Korea had changed their national policy frameworks to reflect an HPS approach; however, in the latter, model programs had been introduced at a local level. We identified various barriers to successful implementation, especially in relation to mental health and wellbeing. CONCLUSION: Given the recent shift toward the HPS approach in six out of the eight countries in this study, there is a need to conduct research to assess the impact of this framework on the health and wellbeing of students and school staff. At the same time, we call for more dialog in the context of Japan to explore the possible benefits of introducing the HPS framework into schools.
Subject(s)
Health Promotion , School Health Services , Humans , Policy , Schools , ThailandABSTRACT
The bacterial flagellum contains a specialized secretion apparatus in its base that pumps certain protein subunits through the growing structure to their sites of installation beyond the membrane. A related apparatus functions in the injectisomes of gram-negative pathogens to export virulence factors into host cells. This mode of protein export is termed type-III secretion (T3S). Details of the T3S mechanism are unclear. It is energized by the proton gradient; here, a mutational approach was used to identify proton-binding groups that might function in transport. Conserved proton-binding residues in all the membrane components were tested. The results identify residues R147, R154 and D158 of FlhA as most critical. These lie in a small, well-conserved cytoplasmic domain of FlhA, located between transmembrane segments 4 and 5. Two-hybrid experiments demonstrate self-interaction of the domain, and targeted cross-linking indicates that it forms a multimeric array. A mutation that mimics protonation of the key acidic residue (D158N) was shown to trigger a global conformational change that affects the other, larger cytoplasmic domain that interacts with the export cargo. The results are discussed in the framework of a transport model based on proton-actuated movements in the cytoplasmic domains of FlhA.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Type III Secretion Systems/metabolism , Amino Acid Sequence , Flagella/metabolism , Mutation , Protein Conformation , Protein Structure, Tertiary , Protein Transport/physiology , Structure-Activity Relationship , Type III Secretion Systems/physiologyABSTRACT
UNLABELLED: Flagellar biogenesis is a complex process that involves multiple checkpoints to coordinate transcription of flagellar genes with the assembly of the flagellum. In Helicobacter pylori, transcription of the genes needed in the middle stage of flagellar biogenesis is governed by RpoN and the two-component system consisting of the histidine kinase FlgS and response regulator FlgR. In response to an unknown signal, FlgS autophosphorylates and transfers the phosphate to FlgR, initiating transcription from RpoN-dependent promoters. In the present study, export apparatus protein FlhA was examined as a potential signal protein. Deletion of its N-terminal cytoplasmic sequence dramatically decreased expression of two RpoN-dependent genes, flaB and flgE. Optical biosensing demonstrated a high-affinity interaction between FlgS and a peptide consisting of residues 1 to 25 of FlhA (FlhANT). The KD (equilibrium dissociation constant) was 21 nM and was characterized by fast-on (kon = 2.9 × 10(4) M(-1)s(-1)) and slow-off (koff = 6.2 × 10(-4) s(-1)) kinetics. FlgS did not bind peptides consisting of smaller fragments of the FlhANT sequence. Analysis of binding to purified fragments of FlgS demonstrated that the C-terminal portion of the protein containing the kinase domain binds FlhANT. FlhANT binding did not stimulate FlgS autophosphorylation in vitro, suggesting that FlhA facilitates interactions between FlgS and other structures required to stimulate autophosphorylation. IMPORTANCE: The high-affinity binding of FlgS to FlhA characterized in this study points to an additional role for FlhA in flagellar assembly. Beyond its necessity for type III secretion, the N-terminal cytoplasmic sequence of FlhA is required for RpoN-dependent gene expression via interaction with the C-terminal kinase domain of FlgS.
Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Helicobacter pylori/enzymology , Membrane Proteins/metabolism , Protein Kinases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Flagella/chemistry , Flagella/genetics , Flagella/metabolism , Flagellin/genetics , Flagellin/metabolism , Genes, Regulator , Helicobacter pylori/genetics , Helicobacter pylori/metabolism , Histidine Kinase , Kinetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Protein Binding , Protein Kinases/chemistry , Protein Kinases/geneticsABSTRACT
UNLABELLED: Bacterial cell surface glycans, such as capsular polysaccharides and lipopolysaccharides (LPS), influence host recognition and are considered key virulence determinants. The periodontal pathogen Porphyromonas gingivalis is known to display at least three different types of surface glycans: O-LPS, A-LPS, and K-antigen capsule. We have shown that PG0121 (in strain W83) encodes a DNABII histone-like protein and that this gene is transcriptionally linked to the K-antigen capsule synthesis genes, generating a large â¼19.4-kb transcript (PG0104-PG0121). Furthermore, production of capsule is deficient in a PG0121 mutant strain. In this study, we report on the identification of an antisense RNA (asRNA) molecule located within a 77-bp inverted repeat (77bpIR) element located near the 5' end of the locus. We show that overexpression of this asRNA decreases the amount of capsule produced, indicating that this asRNA can impact capsule synthesis in trans. We also demonstrate that deletion of the 77bpIR element and thereby synthesis of the large 19.4-kb transcript also diminishes, but does not eliminate, capsule synthesis. Surprisingly, LPS structures were also altered by deletion of the 77bpIR element, and reactivity to monoclonal antibodies specific to both O-LPS and A-LPS was eliminated. Additionally, reduced reactivity to these antibodies was also observed in a PG0106 mutant, indicating that this putative glycosyltransferase, which is required for capsule synthesis, is also involved in LPS synthesis in strain W83. We discuss our finding in the context of how DNABII proteins, an antisense RNA molecule, and the 77bpIR element may modulate expression of surface polysaccharides in P. gingivalis. IMPORTANCE: The periodontal pathogen Porphyromonas gingivalis displays at least three different types of cell surface glycans: O-LPS, A-LPS, and K-antigen capsule. We have shown using Northern analysis that the K-antigen capsule locus encodes a large transcript (â¼19.4 kb), encompassing a 77-bp inverted repeat (77bpIR) element near the 5' end. Here, we report on the identification of an antisense RNA (asRNA) encoded within the 77bpIR. We show that overexpression of this asRNA or deletion of the element decreases the amount of capsule. LPS structures were also altered by deletion of the 77bpIR, and reactivity to monoclonal antibodies to both O-LPS and A-LPS was eliminated. Our data indicate that the 77bpIR element is involved in modulating both LPS and capsule synthesis in P. gingivalis.
Subject(s)
Gene Expression Regulation, Bacterial/physiology , Polysaccharides, Bacterial/metabolism , Porphyromonas gingivalis/metabolism , Bacterial Capsules/chemistry , Bacterial Capsules/metabolism , Lipopolysaccharides/biosynthesis , Mutation , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/genetics , Reproducibility of Results , TranscriptomeABSTRACT
Interspecies communication between Porphyromonas gingivalis and Streptococcus gordonii underlies the development of synergistic dual species communities. Contact with S. gordonii initiates signal transduction within P. gingivalis that is based on protein tyrosine (de)phosphorylation. In this study, we characterize a bacterial tyrosine (BY) kinase (designated Ptk1) of P. gingivalis and demonstrate its involvement in interspecies signaling. Ptk1 can utilize ATP for autophosphorylation and is dephosphorylated by the P. gingivalis tyrosine phosphatase, Ltp1. Community development with S. gordonii is severely abrogated in a ptk1 mutant of P. gingivalis, indicating that tyrosine kinase activity is required for maximal polymicrobial synergy. Ptk1 controls the levels of the transcriptional regulator CdhR and the fimbrial adhesin Mfa1 which mediates binding to S. gordonii. The ptk1 gene is in an operon with two genes involved in exopolysaccharide synthesis, and similar to other BY kinases, Ptk1 is necessary for exopolysaccharide production in P. gingivalis. Ptk1 can phosphorylate the capsule related proteins PGN_0224, a UDP-acetyl-mannosamine dehydrogenase, and PGN_0613, a UDP-glucose dehydrogenase, in P. gingivalis. Knockout of ptk1 in an encapsulated strain of P. gingivalis resulted in loss of capsule production. Collectively these results demonstrate that the P. gingivalis Ptk1 BY kinase regulates interspecies communication and controls heterotypic community development with S. gordonii through adjusting the levels of the Mfa1 adhesin and exopolysaccharide.
Subject(s)
Microbial Interactions , Porphyromonas gingivalis/enzymology , Porphyromonas gingivalis/growth & development , Protein-Tyrosine Kinases/metabolism , Streptococcus gordonii/growth & development , Adenosine Triphosphate/metabolism , Bacterial Adhesion , Bacterial Capsules/metabolism , Gene Deletion , Phosphorylation , Porphyromonas gingivalis/genetics , Protein Processing, Post-Translational , Protein-Tyrosine Kinases/genetics , Signal TransductionABSTRACT
Porphyromonas gingivalis is a major pathogen in severe and chronic manifestations of periodontal disease, which is one of the most common infections of humans. A central feature of P. gingivalis pathogenicity is dysregulation of innate immunity at the gingival epithelial interface, including suppression of IL-8 production by epithelial cells. NF-κB is a transcriptional regulator that controls important aspects of innate immune responses, and NF-κB RelA/p65 homodimers regulate transcription of IL8. Phosphorylation of the NF-κB p65 subunit protein on the serine 536 residue affects nuclear translocation and transcription of target genes. Here we show that SerB, a haloacid dehalogenase (HAD) family serine phosphatase secreted by P. gingivalis, is produced intracellularly and can specifically dephosphorylate S536 of p65 in gingival epithelial cells. A P. gingivalis mutant lacking SerB was impaired in dephosphorylation of p65 S536, and ectopically expressed SerB bound to p65 and co-localized with p65 in the cytoplasm. Ectopic expression of SerB also resulted in dephosphorylation of p65 with reduced nuclear translocation in TNF-α-stimulated epithelial cells. In contrast, the p105/50 subunit of NF-κB was unaffected by SerB. Co-expression of a constitutively active p65 mutant (S536D) relieved inhibition of nuclear translocation. Both the activity of the IL8 promoter and production of IL-8 were diminished by SerB. Deletion and truncation mutants of SerB lacking the HAD-family enzyme motifs of SerB were unable to dephosphorylate p65, inhibit nuclear translocation or abrogate IL8 transcription. Specific dephosphorylation of NF-κB p65 S536 by SerB, and consequent inhibition of nuclear translocation, provides the molecular basis for a bacterial strategy to manipulate host inflammatory pathways and repress innate immunity at mucosal surfaces.
Subject(s)
Interleukin-8/biosynthesis , NF-kappa B/metabolism , Phosphoprotein Phosphatases/metabolism , Porphyromonas gingivalis/metabolism , Transcription Factor RelA/metabolism , Active Transport, Cell Nucleus , Cell Line , Epithelial Cells/metabolism , Humans , Interleukin-8/genetics , Mutation , NF-kappa B p50 Subunit/metabolism , Phosphoprotein Phosphatases/genetics , Phosphorylation , Porphyromonas gingivalis/enzymology , Promoter Regions, Genetic , Transcription Factor RelA/genetics , Transcription, GeneticABSTRACT
Porphyromonas gingivalis is a major etiological agent in chronic and aggressive forms of periodontal disease. The organism is an asaccharolytic anaerobe and is a constituent of mixed species biofilms in a variety of microenvironments in the oral cavity. P. gingivalis expresses a range of virulence factors over which it exerts tight control. High-throughput sequencing technologies provide the opportunity to relate functional genomics to basic biology. In this study we report qualitative and quantitative RNA-Seq analysis of the transcriptome of P. gingivalis. We have also applied RNA-Seq to the transcriptome of a ΔluxS mutant of P. gingivalis deficient in AI-2-mediated bacterial communication. The transcriptome analysis confirmed the expression of all predicted ORFs for strain ATCC 33277, including 854 hypothetical proteins, and allowed the identification of hitherto unknown transcriptional units. Twelve non-coding RNAs were identified, including 11 small RNAs and one cobalamin riboswitch. Fifty-seven genes were differentially regulated in the LuxS mutant. Addition of exogenous synthetic 4,5-dihydroxy-2,3-pentanedione (DPD, AI-2 precursor) to the ΔluxS mutant culture complemented expression of a subset of genes, indicating that LuxS is involved in both AI-2 signaling and non-signaling dependent systems in P. gingivalis. This work provides an important dataset for future study of P. gingivalis pathophysiology and further defines the LuxS regulon in this oral pathogen.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbon-Sulfur Lyases/genetics , Carbon-Sulfur Lyases/metabolism , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Porphyromonas gingivalis/genetics , Gene Deletion , High-Throughput Nucleotide Sequencing , Homoserine/analogs & derivatives , Homoserine/metabolism , Lactones/metabolism , Sequence Analysis, RNA , Signal Transduction/geneticsABSTRACT
Porphyromonas gingivalis, a host-adapted opportunistic pathogen, produces a serine phosphatase, SerB, known to affect virulence, invasion and persistence within the host cell. SerB induces actin filament rearrangement in epithelial cells, but the mechanistic basis of this is not fully understood. Here we investigated the effects of SerB on the actin depolymerizing host protein cofilin. P. gingivalis infection resulted in the dephosphorylation of cofilin in gingival epithelial cells. In contrast, a SerB-deficient mutant of P. gingivalis was unable to cause cofilin dephosphorylation. The involvement of cofilin in P. gingivalis invasion was determined by quantitative image analysis of epithelial cells in which cofilin had been knocked down or knocked in with various cofilin constructs. siRNA-silencing of cofilin led to a significant decrease in numbers of intracellular P. gingivalis marked by an absence of actin colocalization. Transfection with wild-type cofilin or constitutively active cofilin both increased numbers of intracellular bacteria, while constitutively inactive cofilin abrogated invasion. Expression of LIM kinase resulted in reduced P. gingivalis invasion, an effect that was reversed by expression of constitutively active cofilin. These results show that P. gingivalis SerB activity induces dephosphorylation of cofilin, and that active cofilin is required for optimal invasion into gingival epithelial cells.
Subject(s)
Actin Depolymerizing Factors/metabolism , Host-Pathogen Interactions , Phosphoric Monoester Hydrolases/metabolism , Porphyromonas gingivalis/pathogenicity , Actin Cytoskeleton/metabolism , Actin Depolymerizing Factors/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacteroidaceae Infections/metabolism , Bacteroidaceae Infections/microbiology , Cells, Cultured , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Humans , Keratinocytes/metabolism , Keratinocytes/microbiology , Lim Kinases/genetics , Lim Kinases/metabolism , Phosphoric Monoester Hydrolases/genetics , Phosphorylation , Plasmids/genetics , Plasmids/metabolism , Porphyromonas gingivalis/enzymology , Porphyromonas gingivalis/genetics , Porphyromonas gingivalis/metabolism , RNA Interference , TransfectionABSTRACT
Staphylococcus aureus spreads on soft agar surfaces and forms giant colonies. Here, we examined the inhibitory role of extracellular DNA on the colony spreading activity. The double-deletion mutation of nuc1 and nuc2, which encode secretory nucleases, increased extracellular DNA and showed a decreased ability to form giant colonies. The addition of DNase I or micrococcal nuclease to the soft agar restored the ability of the nuc1-nuc2 double mutant to form giant colonies. In addition, the promoter activities of nuc1 and nuc2 in the wild-type strain were elevated in the peripheral region of the giant colony. These findings suggest that the digestion of extracellular DNA by secretory nucleases is required for the colony spreading activity of S. aureus.
Subject(s)
DNA, Bacterial/metabolism , Staphylococcus aureus/growth & development , Staphylococcus aureus/metabolism , Agar , Culture Media/chemistry , Deoxyribonucleases/genetics , Deoxyribonucleases/metabolism , Gene Deletion , Humans , HydrolysisABSTRACT
Interspecies signalling between Porphyromonas gingivalis and Streptococcus gordonii serves to constrain development of dual species communities. Contact with S. gordonii propagates a tyrosine phosphorylation-dependent signal within P. gingivalis that culminates in reduced transcription of adhesin and signalling genes. Here we demonstrate the involvement of the P. gingivalis orphan LuxR family transcription factor PGN_1373, which we designate CdhR, in this control pathway. Expression of cdhR is elevated following contact with S. gordonii; however, regulation of cdhR did not occur in a mutant lacking the tyrosine phosphatase Ltp1, indicating that CdhR and Ltp1 are components of the same regulon. Contact between S. gordonii and a CdhR mutant resulted in increased transcription of mfa, encoding the subunit of the short fimbriae, along with higher levels of Mfa protein. Expression of luxS, encoding AI-2 synthase, was also increased in the cdhR mutant after contact with S. gordonii. The Mfa adhesive function and AI-2-dependent signalling participate in the formation and development of dual species communities, and consistent with this the cdhR mutant displayed elevated accumulation on a substratum of S. gordonii. Recombinant CdhR protein bound to upstream regulatory regions of both mfa and luxS, indicating that CdhR has a direct effect on gene expression. LuxS was also found to participate in a positive feedback loop that suppresses CdhR expression. Interaction of Mfa fimbriae with S. gordonii is necessary to initiate signalling through CdhR. These results reveal CdhR to be an effector molecule in a negative regulatory network that controls P. gingivalis-S. gordonii heterotypic communities.
Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Porphyromonas gingivalis/metabolism , Signal Transduction , Streptococcus gordonii/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Base Sequence , Molecular Sequence Data , Porphyromonas gingivalis/genetics , Protein Binding , Streptococcus gordonii/genetics , Transcription Factors/geneticsABSTRACT
A molecular ruler, FliK, controls the length of the flagellar hook. FliK measures hook length and catalyses the secretion-substrate specificity switch from rod-hook substrate specificity to late substrate secretion, which includes the filament subunits. Here, we show normal hook-length control and filament assembly in the complete absence of the C-ring thus refuting the previous 'cup' model for hook-length control. Mutants of C-ring components, which are reported to produce short hooks, show a reduced rate of hook-basal body assembly thereby allowing for a premature secretion-substrate specificity switch. Unlike fliK null mutants, hook-length control in an autocleavage-defective mutant of flhB, the protein responsible for the switch to late substrate secretion, is completely abolished. FliK deletion variants that retain the ability to measure hook length are secreted thus demonstrating that FliK directly measures rod-hook length during the secretion process. Finally, we present a unifying model accounting for all published data on hook-length control in which FliK acts as a molecular ruler that takes measurements of rod-hook length while being intermittently secreted during the assembly process of the hook-basal body complex.
Subject(s)
Bacterial Proteins/metabolism , Flagella/metabolism , Salmonella enterica/metabolism , Bacterial Proteins/genetics , Gene Deletion , Models, Biological , Salmonella enterica/geneticsABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Flagella/metabolism , Protein Transport , Salmonella/metabolism , Bacterial Proteins/genetics , Flagella/genetics , Models, Molecular , Mutation , Salmonella/genetics , Sequence Deletion , Substrate SpecificityABSTRACT
Salmonella flgG point mutations produce filamentous rod structures whose lengths are determined by FliK. FliK length variants produce rods with lengths proportional to the corresponding FliK molecular size, suggesting that FliK controls the length of not only the hook but also the rod by the same molecular mechanism.
Subject(s)
Bacterial Proteins/metabolism , Flagella/ultrastructure , Salmonella enterica/cytology , Salmonella enterica/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial/physiology , Recombinant Proteins/metabolism , Salmonella enterica/geneticsABSTRACT
Upon completion of the flagellar hook-basal body (HBB) structure, the flagellar type III secretion system switches from secreting rod/hook-type to filament-type substrates. The secretion specificity switch has been reported to occur prematurely (prior to HBB completion) in flk-null mutants (P. Aldridge, J. E. Karlinsey, E. Becker, F. F. Chevance, and K. T. Hughes, Mol. Microbiol. 60:630-643, 2006) and in distal rod gene gain-of-function mutants (flgG* mutants) that produce filamentous rod structures (F. F. Chevance, N. Takahashi, J. E. Karlinsey, J. Gnerer, T. Hirano, R. Samudrala, S. Aizawa, and K. T. Hughes, Genes Dev. 21:2326-2335, 2007). A fusion of beta-lactamase (Bla) to the C terminus of the filament-type secretion substrate FlgM was used to select for mutants that would secrete FlgM-Bla into the periplasmic space and show ampicillin resistance (Ap(r)). Ap(r) resulted from null mutations in the flhE gene, C-terminal truncation mutations in the flhA gene, null and dominant mutations in the flk gene, and flgG* mutations. All mutant classes required the hook length control protein (FliK) and the rod cap protein (FlgJ) for the secretion specificity switch to occur. However, neither the hook (FlgE) nor the hook cap (FlgD) protein was required for premature FlgM-Bla secretion in the flgG* and flk mutant strains, but it was in the flhE mutants. Unexpectedly, when deletions of either flgE or flgD were introduced into flgG* mutant strains, filaments were able to grow directly on the filamentous rod structures.
Subject(s)
Bacterial Proteins/genetics , Flagella/metabolism , Flagellin/metabolism , Membrane Proteins/genetics , Mutation , Salmonella typhimurium/genetics , Salmonella typhimurium/metabolism , Bacterial Proteins/metabolism , Flagella/genetics , Flagellin/genetics , Membrane Proteins/metabolism , Protein TransportABSTRACT
Bacterial flagella contain a specialized secretion apparatus that functions to deliver the protein subunits that form the filament and other structures to outside the membrane. This apparatus is related to the injectisome used by many gram-negative pathogens and symbionts to transfer effector proteins into host cells; in both systems this export mechanism is termed 'type III' secretion. The flagellar secretion apparatus comprises a membrane-embedded complex of about five proteins, and soluble factors, which include export-dedicated chaperones and an ATPase, FliI, that was thought to provide the energy for export. Here we show that flagellar secretion in Salmonella enterica requires the proton motive force (PMF) and does not require ATP hydrolysis by FliI. The export of several flagellar export substrates was prevented by treatment with the protonophore CCCP, with no accompanying decrease in cellular ATP levels. Weak swarming motility and rare flagella were observed in a mutant deleted for FliI and for the non-flagellar type-III secretion ATPases InvJ and SsaN. These findings show that the flagellar secretion apparatus functions as a proton-driven protein exporter and that ATP hydrolysis is not essential for type III secretion.
Subject(s)
Energy Metabolism , Flagella/metabolism , Proton-Motive Force/physiology , Salmonella enterica/metabolism , Adenosine Triphosphate/metabolism , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Energy Metabolism/drug effects , Flagella/chemistry , Hydrogen-Ion Concentration , Mutation/genetics , Protein Transport/drug effects , Protein Transport/genetics , Proton-Motive Force/drug effects , Proton-Translocating ATPases/metabolism , Salmonella enterica/enzymology , Salmonella enterica/geneticsABSTRACT
A bacterial cell tethered through a flagellum on a glass slide rotates its cell body in either counter-clockwise or clockwise at around 10 Hz. To analyze the detailed manner of rotation, we have constructed and expressed yellow fluorescent protein (YFP)-FliN fusion protein in a fliN deletion mutant, resulting in the recovery of motility of the Fla(-) mutant cells. The tethered cells that incorporated the fusion protein in the flagellar motor rotate around one of the fluorescent spots. Tracing the center spot of a rotating cell, we have found that the rotating circles of the tethered cells were often distorted, and that the cell has seldom rotated smoothly but gyrated around the center point. The radii of the gyrating circles were 100-200 nm for the wild-type cells, and 50 nm for the cells carrying short hooks, suggesting that the flexibility of the hook is responsible for asymmetrical rotation. These observations indicate that tethered cells almost always interact with the glass surface in one cycle of rotation, where the length and flexibility of the hook have an important role.
Subject(s)
Escherichia coli/physiology , Salmonella typhimurium/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/physiology , Cell Movement , Escherichia coli/genetics , Flagella/physiology , Flagella/ultrastructure , Gene Deletion , Glass/chemistry , Isopropyl Thiogalactoside/pharmacology , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Microspheres , Models, Biological , Particle Size , Recombinant Fusion Proteins/metabolism , Rotation , Salmonella typhimurium/genetics , Time FactorsABSTRACT
The rod component of the bacterial flagellum polymerizes from the inner membrane across the periplasmic space and stops at a length of 25 nm at the outer membrane. Bushing structures, the P- and L-rings, polymerize around the distal rod and form a pore in the outer membrane. The flagellar hook structure is then added to the distal rod growing outside the cell. Hook polymerization stops after the rod-hook structure reaches approximately 80 nm in length. This study describes mutants in the distal rod protein FlgG that fail to terminate rod growth. The mutant FlgG subunits continue to polymerize close to the length of the normal rod-hook structure of 80 nm. These filamentous rod structures have multiple P-rings and fail to form the L-ring pore at the outer membrane. The flagella grow within the periplasm similar to spirochete flagella. This provides a simple method to evolve intracellular flagella as in spirochetes. The mechanism that couples rod growth termination to the ring assembly and outer membrane penetration exemplifies the importance of stopping points in the construction of a complex macromolecular machine that facilitate efficient coupling to the next step in the assembly pathway.
Subject(s)
Biological Evolution , Cell Membrane/metabolism , Flagella/metabolism , Spirochaetales/physiology , Amino Acid Sequence , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/physiology , Flagella/chemistry , Models, Biological , Models, Molecular , Molecular Sequence Data , Periplasm/metabolism , Sequence Homology, Amino AcidABSTRACT
The 5'-untranslated region (5'UTR) of the fliC flagellin gene of Salmonella contains sequences critical for efficient fliC mRNA translation coupled to assembly. In a previous study we used targeted mutagenesis of the 5' end of the fliC gene to isolate single base changes defective in fliC gene translation. This identified a predicted stem-loop structure, SL2, as an effector of normal fliC mRNA translation. A single base change (-38C:U) in the fliC 5'UTR resulted in a mutant that is defective in fliC mRNA translation and was chosen for this study. Motile (Mot+) revertants of the -38C:T mutant were isolated and characterized, yielding several unexpected results. Second-site suppressors that restored fliC translation and motility included mutations that disrupt a RNA duplex stem formed between RNA sequences in the fliC 5'UTR SL2 region (including a precise deletion of SL2) and bases early within the fliC-coding region. A stop codon mutation at position 80 of flgM also suppressed the -38C:T motility defect, while flgM mutants defective in anti-sigma28 activity had no effect on fliC translation. One remarkable mutation in the fliC 5'UTR (-15G:A) results in a translation defect by itself but, in combination with the -38C:U mutation, restores normal translation. These results suggests signals intrinsic to the fliC mRNA that have both positive and negative effects on fliC translation involving both RNA structure and interacting proteins.
Subject(s)
Bacterial Proteins/genetics , Flagellin/genetics , Protein Biosynthesis , Salmonella typhimurium/genetics , 5' Untranslated Regions/genetics , 5' Untranslated Regions/metabolism , Bacterial Proteins/physiology , Base Sequence , Flagella , Flagellin/biosynthesis , Flagellin/metabolism , Gene Expression Regulation , Locomotion , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Open Reading Frames , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , Repressor Proteins , Salmonella typhimurium/physiologyABSTRACT
In an effort to delineate the precise mechanisms underlying the organ-specific expression of photosynthesis genes, Arabidopsis lines homozygous for each transgene construct made with the gene for hygromycin B phosphotransferase or beta-glucuronidase (GUS) placed under control of the promoter of the nuclear gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBCS-3B) were constructed. Furthermore, activation tagging with T-DNA possessing quadruply repeated enhancers derived from the cauliflower mosaic virus 35S promoter was applied to a transgenic line of Arabidopsis. Mutants resistant to hygromycin B during the growth of calli generated from non-green roots on callus-inducing medium resulted from the expression of hygromycin B phosphotransferase driven by the RBCS-3B promoter. Three mutant lines, ces101 to ces103 (callus expression of RBCS), were obtained from approximately 4,000 calli resistant to a selectable marker for transformation. The active transcription driven by the RBCS-3B promoter in all the calli of ces mutants was confirmed by expression of both the GUS reporter gene and endogenous RBCS-3B. Chlorophyll and carotenoids, as well as light-dependent O(2) evolution, have been detected in the calli of all ces mutants. The loci where T-DNA was integrated in the ces101 line were determined by thermal asymmetric interlaced (TAIL)-PCR. The introduction of a DNA fragment harboring the gene for receptor-like kinase placed under the influence of enhancers into the parental line reproduced the phenotype of ces mutants. We have thus concluded that CES101 is a receptor-like kinase. The strategy presented in this investigation may promise to select a greater number of ces mutants.
Subject(s)
Arabidopsis/cytology , Arabidopsis/genetics , Cell Differentiation , Gene Expression Regulation, Plant , Genes, Plant/genetics , Mutation/genetics , Photosynthesis/genetics , Chlorophyll/metabolism , Exons/genetics , Genes, Reporter/genetics , Genetic Vectors , Glucuronidase/metabolism , Introns/genetics , Phenotype , Plant Leaves/genetics , Plant Roots/genetics , Plants, Genetically Modified , Promoter Regions, Genetic/genetics , Ribulose-Bisphosphate Carboxylase/genetics , Tissue Culture Techniques , Transcription, Genetic , Transformation, GeneticABSTRACT
The length of the flagellar hook is regulated; it is 55 +/- 6 nm long in Salmonella. Five genes involved in hook-length regulation are fliK, flhB, fliG, fliM and fliN. The last four genes encode structural components of the protein export apparatus in the flagellar base, whereas FliK is soluble and secreted during flagellar assembly. The role of FliK, however, remains ambiguous. We constructed two kinds of FliK variants: N-terminally truncated FliK protein and FliK N-terminally fused with cyan fluorescent protein (CFP-FliK). Both N-terminally truncated FliK missing the first 99 amino acids (aa) and CFP-FliK fusion variants partially complemented a fliK null (polyhook) mutant to produce cells with filaments, allowing cells to swim; the hooks, however, were not normal but were polyhooks. When the N-terminally defective FliK variants were expressed at high levels, the average polyhook length was shortened coming close to the length of the wild-type hook, independently of the sizes of the FliK variants. These FliK variants were not secreted. CFP-FliK fusion proteins were observed to homogeneously distribute in the cytoplasm. We conclude that FliK does not need to be exported to control hook length and is unlikely to be a ruler; instead, we conclude that FliK controls hook length by the timely switching of secretion modes of the flagellar type III secretion system by the FliK C-terminal domain, and that the N-terminal region is dispensable for hook length control.