In a previous article, I discussed the topic of recurring design logic, with a particular emphasis on two-component regulatory systems in bacteria. As I argued there, recurring design logic is a strong prediction on the hypothesis of design but is otherwise a surprising observation. Here, I will discuss another example of recurring design logic: transcriptional regulatory hierarchies in bacteria. 

A transcriptional hierarchy is a regulatory system in which genes are expressed in a specific ordered sequence, ensuring that the appropriate genes are activated at the right time and in the proper order. Transcriptional hierarchies are quite common in prokaryotes, as well as eukaryotes. In this article and subsequent ones, I will focus on examples from bacteria, where genes are organized into operons (defined as a collection of genes that are under the control of a common promoter and are transcribed together). In addition to exhibiting recurring design logic, these transcriptional hierarchies present a significant challenge to evolution, since the specific order of genes along the bacterial chromosome (and their organization into different operons) is crucial to their function.

In this first installment, I will discuss the transcriptional hierarchy that is crucial to the assembly of bacterial flagella.

Flagellar Assembly

The flagellar system in Salmonella has three classes of promoters (promoters are akin to a kind of molecular toggle switch which can initiate gene expression when recognized by RNA polymerase and an associated specialized protein called a “sigma factor”). These three classes of promoters are dubbed “Class I,” “Class II,” and “Class III.”¹ This sequential transcription is coupled to the process of flagellar assembly. Class I contains only two genes in one operon (called FlhD and FlhC). Class II consists of 35 genes across eight operons (including genes involved in the assembly of the hook-basal-body and other components of the flagellum, as well as the export apparatus and two regulatory genes called “FliA” and “FlgM”). Those genes which are involved in the synthesis of the filament are controlled by the Class III promoters.

The Class I promoter drives the expression of a master regulator (particular to the Enterobacteriaceae of which Salmonella is a member) called “FldH4C2.” This enteric master regulator then turns on the Class II promoters in association with a sigma factor, σ⁷⁰ (recall that a sigma factor is a type of protein that enables specific binding of RNA polymerase to gene promoters). The Class II promoters are then responsible for the gene expression of the hook-basal-body subunits and its regulators, including another sigma factor called σ²⁸(which is encoded by a gene called FliA) and its anti-sigma factor, FlgM (anti-sigma factors, as their name suggests, bind to sigma factors to inhibit their transcriptional activity). The sigma factor σ²⁸ is required to activate the Class III promoters, and, indeed, knocking out fliA (which codes for this sigma factor) in Rhodobacter sphaeroides, which employs σ²⁸ and FlgM in a similar manner to Salmonella, disrupts the expression of class III flagellar genes, impairing flagellar assembly and resulting in non-motile cells.^2,3

A Potential Problem

At this juncture, we potentially run into a problem. It makes absolutely no sense to start expressing the flagellin monomers before completion of the hook-basal-body construction. Thus, in order to inhibit the σ²⁸, the anti-sigma factor (FlgM) alluded to above inhibits its activity and prohibits it from interacting with the RNA polymerase holoenzyme complex. When construction of the hook-basal-body is completed, the anti-sigma factor FlgM is secreted through the flagellar structures which are produced by the expression of the Class II hook-basal-body genes. The Class III promoters (which are responsible for the expression of flagellin monomers, the chemotaxis system and the motorforce generators) are then finally activated by σ²⁸ and the flagellum can be completed. A mutant defective in flgM results in “initiation of class 2 and class 3 genes at the same time, eliminating the delay that was observed for class 3 gene in wild type,” leading to defects in flagellar structure.⁴

What triggers the removal of FlgM from the cell? The flagellar export system has two substrate-specificity states: rod-hook-type substrates and filament-type substrates.^5,6 During the process of flagellar assembly, this substrate-specificity switch has to flick from the former of those states to the latter. Proteins that form part of the hook and rod need to be exported before those which form the filament. But how does this switch in substrate-specificity take place?

A membrane-bound protein called FlhB is the key player in this process. There is also a flagellar hook-length protein which is responsible for ensuring that the hook length is of the right size (around 55nm) called FliK.⁷This same protein is also responsible for initiating the switch export substrate specificity. As it turns out, without FliK, both the ability to switch and export filament and the hook length control are completely lost. ^8,9 FliK has two key domains, i.e., the N-terminal and C-terminal domains. During hook assembly, FliKN functions as a molecular sensor and transmitter of information on hook length. When the hook reaches the right length, the information is transmitted to FliKC and FliKCT, resulting in a conformational change, which in turn results in FliKCT binding to FlhBC. This, in turn, results in a conformational change in FlhBC, bringing about the substrate specificity switch.

The Challenge to Evolution

By an unguided process of tinkering, it seems quite implausible that the genes would be appropriately arranged along the chromosome in a manner that corresponds to the timing of their expression. Not only must the necessary genes be organized into a hierarchical transcriptional cascade to ensure proper timing and coordination of assembly, but the specific order of the genes along their respective operons is also significant. Indeed, the genes are typically arranged in a manner that corresponds to the order in which their protein products are required for flagellar construction. Such a process would require a significant amount of trial-and-error. On the other hand, on the hypothesis that flagellar assembly is a genuinely engineered system, designed by an intelligent mind, this is precisely the sort of apparatus we might expect to see.

Notes

1. Das C, Mokashi C, Mande SS, Saini S. Dynamics and Control of Flagella Assembly in Salmonella typhimurium. Front Cell Infect Microbiol. 2018 Feb 8;8:36. doi: 10.3389/fcimb.2018.00036. PMID: 29473025; PMCID: PMC5809477.
2. Martin AC, Gould M, Byles E, Roberts MA, Armitage JP. Two chemosensory operons of Rhodobacter sphaeroides are regulated independently by sigma 28 and sigma 54. J Bacteriol. 2006 Nov;188(22):7932-40. doi: 10.1128/JB.00964-06. Epub 2006 Sep 8. PMID: 16963577; PMCID: PMC1636300.
3. Wilkinson DA, Chacko SJ, Vénien-Bryan C, Wadhams GH, Armitage JP. Regulation of flagellum number by FliA and FlgM and role in biofilm formation by Rhodobacter sphaeroides. J Bacteriol. 2011 Aug;193(15):4010-4. doi: 10.1128/JB.00349-11. Epub 2011 Jun 3. PMID: 21642454; PMCID: PMC3147513.
4. Das C, Mokashi C, Mande SS, Saini S. Dynamics and Control of Flagella Assembly in Salmonella typhimurium. Front Cell Infect Microbiol. 2018 Feb 8;8:36. doi: 10.3389/fcimb.2018.00036. PMID: 29473025; PMCID: PMC5809477.
5. Minamino T, Doi H, Kutsukake K. Substrate specificity switching of the flagellum-specific export apparatus during flagellar morphogenesis in Salmonella typhimurium. Biosci Biotechnol Biochem. 1999 Jul;63(7):1301-3. doi: 10.1271/bbb.63.1301. PMID: 10478459.
6. Ferris HU, Minamino T. Flipping the switch: bringing order to flagellar assembly. Trends Microbiol. 2006 Dec;14(12):519-26. doi: 10.1016/j.tim.2006.10.006. Epub 2006 Oct 25. PMID: 17067800.
7. Waters RC, O’Toole PW, Ryan KA. The FliK protein and flagellar hook-length control. Protein Sci. 2007 May;16(5):769-80. doi: 10.1110/ps.072785407. PMID: 17456739; PMCID: PMC2206646.
8. Williams AW, Yamaguchi S, Togashi F, Aizawa SI, Kawagishi I, Macnab RM. Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium. J Bacteriol. 1996 May;178(10):2960-70. doi: 10.1128/jb.178.10.2960-2970.1996. PMID: 8631688; PMCID: PMC178035.
9. Muramoto K, Makishima S, Aizawa SI, Macnab RM. Effect of cellular level of FliK on flagellar hook and filament assembly in Salmonella typhimurium. J Mol Biol. 1998 Apr 10;277(4):871-82. doi: 10.1006/jmbi.1998.1659. PMID: 9545378.