FliZ Is a posttranslational activator of FlhD4C2-dependent flagellar gene expression

Flagellar assembly proceeds in a sequential manner, beginning at the base and concluding with the filament. A critical aspect of assembly is that gene expression is coupled to assembly. When cells transition from a nonflagellated to a flagellated state, gene expression is sequential, reflecting the manner in which the flagellum is made. A key mechanism for establishing this temporal hierarchy is the sigma(28)-FlgM checkpoint, which couples the expression of late flagellar (P(class3)) genes to the completion of the hook-basal body. In this work, we investigated the role of FliZ in coupling middle flagellar (P(class2)) gene expression to assembly in Salmonella enterica serovar Typhimurium. We demonstrate that FliZ is an FlhD(4)C(2)-dependent activator of P(class2)/middle gene expression. Our results suggest that FliZ regulates the concentration of FlhD(4)C(2) posttranslationally. We also demonstrate that FliZ functions independently of the flagellum-specific sigma factor sigma(28) and the filament-cap chaperone/FlhD(4)C(2) inhibitor FliT. Furthermore, we show that the previously described ability of sigma(28) to activate P(class2)/middle gene expression is, in fact, due to FliZ, as both are expressed from the same overlapping P(class2) and P(class3) promoters at the fliAZY locus. We conclude by discussing the role of FliZ regulation with respect to flagellar biosynthesis based on our characterization of gene expression and FliZ's role in swimming and swarming motility.     

The functional cycle and regulation of the Thermus thermophilus DnaK chaperone system

The Escherichia coli DnaK (DnaKEco) chaperone cycle is tightly regulated by the cochaperones DnaJ, which stimulates ATP hydrolysis, and GrpE, which acts as a nucleotide exchange factor. The Thermus thermophilus DnaK (DnaKTth) system additionally comprises the DnaK-DnaJ assembly factor (DafATth) that is mediating formation of a 300 kDa DnaKTth. DnaJTth.DafATth complex.A model peptide derived from the tumor suppressor protein p53 was used to dissect the regulation of the individual kinetic key steps of the DnaKTth nucleotide/chaperone cycle. As with DnaKEco the DnaKTth.ATP complex binds substrates with reduced affinity and large exchange rates compared to the DnaKTth.ADP.Pi state. In contrast to DnaKEco, ADP-Pi release is slow compared to the rate of hydrolysis, reversing the balance of the two functional nucleotide states. Whereas GrpETth stimulates nucleotide release from DnaKTth, DnaJTth does not accelerate ATP hydrolysis under various experimental conditions. However, it exerts influence on the interaction of DnaKTth with substrates: in the presence of DafATth, DnaJTth inhibits substrate binding, and substrate already bound to DnaKTth is displaced by DnaJTth and DafATth, indicating competitive binding of DnaJTth/DafATth and substrate. It thus appears that the DnaKTth. DnaJTth.DafATth complex as isolated from T. thermophilus does not represent the active species in the DnaKTth chaperone cycle. Isothermal titration calorimetry showed that the ternary complex of DnaKTth, DnaJTth and DafATth is assembling with high affinity, whereas binary complexes of DnaKTth and DnaJTth or DafATth were not detectable, indicating highly synergistic formation of the 300 kDa DnaKTth. DnaJTth.DafATth complex.Based on these results, a model describing the DnaKTth chaperone cycle and its regulation by cochaperones is proposed where DnaKTth. DnaJTth.DafATth constitutes the resting state, and a DnaKTth. substrate.DnaJTth complex is the active chaperone species. The novel factor DafATth that mediates interaction of DnaKTth with DnaJTth would thus serve as a "template" to stabilise the ternary DnaKTth.DafATth.DnaJTth complex until it is replaced by substrate proteins under heat shock conditions.     

Interaction of the DnaK and DnaJ chaperone system with a native substrate,P1 RepA

DnaK, the Hsp70 chaperone of Escherichia coli interacts with protein substrates in an ATP-dependent manner, in conjunction with DnaJ and GrpE co-chaperones, to carry out protein folding, protein remodeling, and assembly and disassembly of multisubunit protein complexes. To understand how DnaJ targets specific proteins for recognition by the DnaK chaperone system, we investigated the interaction of DnaJ and DnaK with a known natural substrate, bacteriophage P1 RepA protein. By characterizing RepA deletion derivatives, we found that DnaJ interacts with a region of RepA located between amino acids 180 and 200 of the 286-amino acid protein. A peptide corresponding to amino acids 180-195 inhibited the interaction of RepA and DnaJ. Two site-directed RepA mutants with alanine substitutions in this region were about 4-fold less efficiently activated for oriP1 DNA binding by DnaJ and DnaK than wild type RepA. We also identified by deletion analysis a site in RepA, in the region of amino acids 35-49, which interacts with DnaK. An alanine substitution mutant in amino acids 36-39 was constructed and found defective in activation by DnaJ and DnaK. Taken together the results suggest that DnaJ and DnaK interact with separate sites on RepA.     

Characterization of the fliL gene in the flagellar regulon of Escherichia coli and Salmonella typhimurium.

filL is a small gene of unknown function that lies within the beginning of a large flagellar operon of Salmonella typhimurium and Escherichia coli. A spontaneous fliL mutant of S. typhimurium, containing a frameshift mutation about 40% from the 3' end of the gene, was moderately motile but swarmed poorly, suggesting that FliL might be a component of the flagellar motor or switch. However, in-frame deletions of the E. coli gene, including an essentially total deletion, had little or no effect on motility or chemotaxis. Thus, FliL does not appear to have a major role in flagellar structure or function and is therefore unlikely to be a component of the motor or switch; the effect on motility caused by truncation of the gene is probably an indirect one.     

Hook-length control of the export-switching machinery involves a double-locked gate in Salmonella typhimurium flagellar morphogenesis.

During flagellar morphogenesis in Salmonella typhimurium, the genes involved in filament assembly are expressed fully only after completion of hook-basal body assembly. This coupling of gene expression to morphogenesis is achieved by exporting the flagellum-specific anti-sigma factor, FlgM, out of the cell through the mature hook-basal body structure. Therefore, the flagellum-specific export apparatus must be able to sense the assembly state of the flagellar structure and to turn on FlgM export at a specific stage of hook assembly. It has been suggested that FlhB may act as the molecular switch which mediates this ordered export. Here, I report genetic evidence that in addition to FlhB, the product of a newly identified gene, rflH, is involved in the negative regulation of FlgM export. FlgM is released through the basal body structure lacking the hook and the filament only when the flhB and rflH genes are both defective. Therefore, the export gate for FlgM should be double locked by FlhB and RflH. The rflH gene is located at around 52 min, where no flagellum-related gene has been found. I propose a revised model of the export-switching machinery which consists of two systems, the hook-length signal transduction pathway and the double-locked gate for FlgM export.     

The importance of having thermosensor control in the DnaK chaperone system

In addition to the sigma(32)-mediated heat shock response, the DnaK/DnaJ/GrpE molecular chaperone system of Escherichia coli directly adapts to elevated temperatures by sequestering a higher fraction of substrate. This immediate heat shock response is due to the differential temperature dependence of the activity of DnaJ, which stimulates the hydrolysis of DnaK-bound ATP, and the activity of GrpE, which facilitates ADP/ATP exchange and converts DnaK from its high-affinity ADP-liganded state into its low-affinity ATP-liganded state. GrpE acts as thermosensor with its ADP/ATP exchange activity decreasing above 40 degrees C. To assess the importance of this reversible thermal adaptation for the chaperone action of the DnaK/DnaJ/GrpE system during heat shock, we used glucose-6-phosphate dehydrogenase and luciferase as substrates. We compared the performance of wild-type GrpE as a component of the chaperone system with that of GrpE R40C. In this mutant, the thermosensing helices are stabilized with an intersubunit disulfide bond and its nucleotide exchange activity thus increases continuously with increasing temperature. Wild-type GrpE with intact thermosensor proved superior to GrpE R40C with desensitized thermosensor. The chaperone system with wild-type GrpE yielded not only a higher fraction of refolding-competent protein at the end of a heat shock but also protected luciferase more efficiently against inactivation during heat shock. Consistent with their differential thermal behavior, the protective effects of wild-type GrpE and GrpE R40C diverged more and more with increasing temperature. Thus, the direct thermal adaptation of the DnaK chaperone system by thermosensing GrpE is essential for efficient chaperone action during heat shock.     

Authentic precursors to ribosomal subunits accumulate in Escherichia coli in the absence of functional DnaK chaperone

Escherichia coli dnaK-ts mutants are defective in the late stages of ribosome biogenesis at high temperature. Here, we show that the 21S, 32S and 45S ribosomal particles that accumulate in the dnaK756-ts mutant at 44 degrees C contain unprocessed forms of their 16S and 23S rRNAs (partially processed in the case of 45S particles). Their 5S rRNA stoichiometry and ribosomal protein composition are typical of the genuine ribosomal precursors found in a wild-type (dnaK+) strain. Despite the lack of a functional DnaK, a very slow maturation of these 21S, 32S and 45S particles to structurally and functionally normal 30S and 50S ribosomal subunits still occurs at high temperature. This conversion is accompanied by the processing of p16S and p23S rRNAs to their mature forms. We conclude that: (i) 21S, 32S and 45S particles are not dead-end particles, but true precursors to active ribosomes (21S particles are converted to 30S subunits, and 32S and 45S to 50S subunits); (ii) DnaK is not absolutely necessary for ribosome biogenesis, but accelerates the late steps of this process considerably at high temperature; and (iii) 23S rRNA processing depends on the stage reached in the stepwise assembly of the 50S subunit, not directly on DnaK.