Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli

Abstract

How do organisms assess the degree of completion of a large structure, especially an extracellular structure such as a flagellum? Bacteria can do this. Mutants that lack key components needed early in assembly fail to express proteins that would normally be added at later assembly stages. In some cases, the regulatory circuitry is able to sense completion of structures beyond the cell surface, such as completion of the external hook structure. In Salmonella and Escherichia coli, regulation occurs at both transcriptional and posttranscriptional levels. One transcriptional regulatory mechanism involves a regulatory protein, FlgM, that escapes from the cell (and thus can no longer act) through a complete flagellum and is held inside when the structure has not reached a later stage of completion. FlgM prevents late flagellar gene transcription by binding the flagellum-specific transcription factor sigma(28). FlgM is itself regulated in response to the assembly of an incomplete flagellum known as the hook-basal body intermediate structure. Upon completion of the hook-basal body structure, FlgM is exported through this structure out of the cell. Inhibition of sigma(28)-dependent transcription is relieved, and genes required for the later assembly stages are expressed, allowing completion of the flagellar organelle. Distinct posttranscriptional regulatory mechanisms occur in response to assembly of the flagellar type III secretion apparatus and of ring structures in the peptidoglycan and lipopolysaccharide layers. The entire flagellar regulatory pathway is regulated in response to environmental cues. Cell cycle control and flagellar development are codependent. We discuss how all these levels of regulation ensure efficient assembly of the flagellum in response to environmental stimuli.

FIG. 1

Chromosomal locations of the operons that make up the flagellar regulon of S. enterica serovar Typhimurium. The operons are labeled E, M, or L depending on whether they are expressed early, middle, or late in the temporal induction pathway (see text).

FIG. 2

Flagellar transcriptional hierarchy coupled to flagellar assembly. There are more than 50 genes in the flagellar and chemotaxis regulon. These genes are transcribed in operons of three temporal classes, early, middle, and late (57, 71). The early genes are included in the master flagellar operon, flhDC. The FlhC and FlhD proteins form a heteromultimeric complex that directs ς⁷⁰-dependent transcription of class 2 promoters of the middle and some late genes (78). The middle operons encode structural and assembly proteins required for the biosynthesis of the flagellar motor intermediate structure, also known as the hook-basal body (85). In addition to hook-basal body proteins, two competing regulatory proteins, FlgM and FliA (ς²⁸), are also transcribed from class 2 promoters. The fliA gene encodes an alternative transcription factor, ς²⁸, which is specifically required for class 3 promoter transcription. Genes whose products are required late in flagellar assembly, including the external filament (flagellar propeller), are primarily transcribed from class 3 promoters. The FlgM protein binds to ς²⁸ directly to prevent class 3 promoter transcription until after hook-basal body completion. Once the hook-basal body is complete, FlgM is secreted from the cell to free ς²⁸, and class 3 transcription occurs. In this way, the external filament (propeller) is not made until there is a motor (hook-basal body) for it to polymerize onto. A number of late flagellar genes, including flgK, flgL, flgM, flgN, fliD, fliS, and fliT, are expressed in both middle and late operons.

FIG. 3

Structure of flagellar class 2 and class 3 promoters. Class 2 promoters are dependent on ς⁷⁰ and the tetrameric FlhD₂C₂ transcriptional activation complex for transcription. Class 3 promoters are dependent on the alternative transcription factor ς²⁸ for transcription. The data for class 2 promoters are primarily taken from Ikebe et al. (44). Transcriptional start site mapping was determined as follows: flgA_S, flgB_S, flhB_S, fliE_S, and fliL_S are from reference ; fliA_S is from references and ; fliD_S is from reference ; flhB_E and fliL_E are from reference ; and fliA_E is from reference . The primary transcriptional start site for the characterized class 2 promoters are underlined and labeled +1. The subscripts E and S after each gene designate whether the promoter corresponds to a flagellar promoter from E. coli or S. enterica serovar Typhimurium, respectively. Sequences that closely match consensus GCAATAA and TTATTCC sequences within known and presumed FlhDC binding regions are labeled by arrows. DNA regions protected by the binding of the FlhDC complex are boxed.

FIG. 4

Organization of the ς⁷⁰ and ς²⁸ transcription factors. The ς⁷⁰ family of transcription factors contain four conserved regions that are subdivided within a given region (79). Region 1 is absent in ς²⁸, but regions 2, 3, and 4 are conserved between the two ς factors. The degree of homology between ς⁷⁰ and ς²⁸ is indicated within the ς²⁸ structure as follows: hatching, >50% homology; stippling, between 25 and 50% homology; blank, <25% homology. Homologous residue groups are S and T; R and K; N and Q; F, Y, and W; and I, L, V, and M.

FIG. 5

DNA sequence analysis of FlgM-insensitive mutations in ς²⁸ (; Chadsey and Hughes, Submitted) (A) Distribution of single amino acid changes in ς²⁸ that allow expression of a fliC-lac transcriptional fusion in a hook-basal body mutant strain (FlgM-inhibitory conditions). The 239-amino-acid ς²⁸ protein is represented by a horizontal bar. Conserved regions of the ς⁷⁰ family are boxed. Possible structural motifs and putative domain functions based on results with other ς factors are indicated below the protein (79, 86, 112). (B) Conserved helix-turn-helix motif of region 4.2 of ς²⁸. FlgM bypass mutants are indicated by black circles. Note that selection was for FlgM bypass ς²⁸ mutants that still transcribed the fliC promoter. Thus, amino acids within the DNA recognition helix thought to contact the −35 region of the class 3 promoter DNA are not affected.

FIG. 6

Model for the coupling of flgM translation from the class 3 flgMN transcript to secretion by translational regulators Flk and FlgN. This model combines both mRNA and amino acid secretion signals for the FlgM substrate. The hook-basal body is the secretion channel. Some ς²⁸-dependent transcription of class 3 flgMN occurs prior to hook-basal body completion. Secretion of FlgM expressed from the class 3 promoter prior to hook-basal body completion is coupled to mRNA translation by a ribosome/mRNA receptor and a flagellar type III secretion chaperone (right). After hook-basal body completion, secretion can also occur without cotranslation (left). In the case of cotranslation secretion (left), prior to hook-basal body completion, the flgM class 3 transcript (left) is targeted to the secretion channel by the ribosomal protein S1 homologue Flk (or another component of the flagellar type III secretion apparatus), where translation occurs. Both Flk and FlgN facilitate class 3 flgM translation prior to hook-basal body completion. The secretion chaperone FlgN may cooperate with Flk to bind the class 3 flgM transcript. The translated FlgM protein may be held at the secretion channel by a flagellum-specific type III secretion chaperone to keep the nascent secretion substrate from diffusing into the cytoplasm before secretion occurs, using the N-terminal amino acid secretion signal. The FlgN protein is one possible secretion chaperone for this purpose. In the case of translation uncoupled to secretion (right), only the amino acid secretion signal(s) within FlgM targets it to the secretion channel. The class 2 flgM transcript (right) is translated in the cytoplasm using the S1 ribosomal subunit protein. Cytoplasmic FlgM inhibits ς²⁸-dependent transcription. Cytoplasmic FlgM can be targeted to the flagellar secretion channel through amino acid secretion signals. The amino acid signal may include interaction with just the secretion channel or both the chaperone and the secretion channel to facilitate secretion. OM, outer membrane; PG, peptidoglycan; IM, inner membrane.