We have shown that FliZ is an FlhD4C2-dependent activator of Pclass2 activity that acts, at least in part, at the level of FlhD4C2 protein. Deleting fliZ causes approximately a 50% decrease in Pclass2 activity and FlhC protein levels. Likewise, overexpressing FliZ roughly doubles Pclass2 activity. Furthermore, we have shown that FliZ functions independently of σ28 and FliT. In the case of σ28, we have shown that it does not directly activate Pclass2 promoters. Rather, the ability of σ28 to influence Pclass2 activity is indirect through FliZ, as the two are under the control of both Pclass2 and Pclass3 promoters. This indicates that fliZ expression is partly responsive to σ28 activity and, as a consequence, late protein secretion. Finally, an interesting finding was that FliZ had a strong effect on swarming motility for one specific medium recipe. In particular, we found that a ΔfliZ mutant was unable to swarm on TB medium supplemented with Tween 80. However, only a moderate reduction in swarming was observed when a more traditional recipe was used. These results potentially imply that the specific environment places differential demands on noncanonical flagellar regulators such FliZ.
We still do not know how FliZ increases FlhD4
protein levels. Our data suggest that FliZ does not directly increase protein translation rates but rather enhances the stability of FlhD4
. Note that as we measured only FlhC protein levels and translation rates, it is not yet clear whether FliZ is also affecting FlhD. As for a possible mechanism, sequence analysis indicates that FliZ shares no similarity to any other known regulator. However, FliZ does possess a SAM-like phage integrase domain (PFAM PF02988) (14
). The presence of this domain suggests that FliZ is a DNA-binding protein. In the related insect pathogen Xenorhabdus nematophila
, FliZ was recently shown to bind to the flhDC
promoter and increase the rate of transcription (38
). As the FliZ proteins from S. enterica
serovar Typhimurium and X. nematophila
share 56% sequence identify, these results suggest that FliZ is also a transcription factor in S. enterica
serovar Typhimurium. However, transcriptional regulation of the flagellar genes in S. enterica
serovar Typhimurium and X. nematophila
is different despite the fact that the organisms share common regulators. For example, transcription of the fliAZ
operon in X. nematophila
dependent, whereas the equivalent fliAZY
operon is under independent control of both Pclass2
promoters in S. enterica
serovar Typhimurium (26
). Likewise, our data show that FliZ is not a transcriptional regulator of the flhDC
operon in S. enterica
serovar Typhimurium, whereas it is in X. nematophila
. Furthermore, our data suggest that FliZ regulates the FlhD4
complex posttranslationally. Based on the results from X. nematophila
regarding FliZ being a DNA-binding protein, the most likely mechanism in S. enterica
serovar Typhimurium is that FliZ increases the expression of some protein that stabilizes FlhD4
or, alternatively, inhibits the expression of a protease such as ClpXP that degrades it. Furthermore, such promiscuity in FliZ DNA binding between the organisms is not altogether unreasonable, as the greatest difference between the two FliZ orthologs is at the C terminus, the region predicted to bind DNA based on the homology to the SAM-like phage integrase domain (38
In the overall context of assembly, our data suggest the following model for FliZ-dependent regulation. Prior to completion of the HBB, FlhD4C2 can only partially activate Pclass2 promoters due to low protein levels arising from weak FliZ expression. However, when the first few HBBs are completed and FlgM is secreted at a sufficient rate such that σ28 is active, FliZ expression then increases. Note that FliZ is under the control of both Pclass2 and Pclass3 promoters, where the latter is the dominant promoter. Increased FliZ expression then leads to increased stability of FlhD4C2 and, as a consequence, further activation of the Pclass2 promoters. This mechanism suggests that Pclass2 activity is coupled to assembly. Unlike the case for Pclass3 promoters, where the coupling results in a strict binary checkpoint, Pclass2 activity is “tuned” in response to assembly. In other words, the cell increases Pclass2 activity once the first few HBBs are complete. In support of this model, we have recently shown that in HBB mutants Pclass2 expression rates are reduced, consistent with reduced FliZ regulation prior to HBB completion (Brown et al., submitted).
The current model for flagellar gene regulation focuses predominantly on initiation, when cells transition from a nonflagellated to a flagellated state (1
). Key to regulating initiation is the σ28
-FlgM checkpoint. Once cells already possess flagella, however, this checkpoint is no longer relevant, as FlgM is being continuously secreted from the cell. The fact that cells already possess flagella does not mean that the assembly process no longer needs to be regulated. Rather, different regulatory tasks and their associated mechanisms become necessary. In particular, cells need to ensure that they have sufficient flagella, not too many or too few. Furthermore, every time the cell divides, the progeny need to reinitiate the assembly process to ensure that they have sufficient flagella for efficient motility. This regulation means that cells need to continually monitor assembly and adjust gene expression accordingly. We speculate that FliZ is one element of this regulation, providing cells with a mechanism for increasing gene expression in response to assembly.
Note that FliT has the reciprocal effect of FliZ. Like fliZ expression, fliT expression is under the control of both Pclass2 and Pclass3 promoters, where again the latter is dominant. This means that fliT is maximally expressed when its Pclass3 promoter is active, concomitant with the completion of the first few HBBs. Unlike FliZ, however, FliT is a negative regulator of FlhD4C2 activity. Furthermore, FliD, the filament cap protein, regulates FliT activity. Prior to secretion, FliD binds FliT and is thought to prevent FliT from inhibiting FlhD4C2. Note that FliZ is indirectly regulated by late protein secretion, as its expression is controlled by σ28. As both FliZ and FliT are activated by the same signal, namely, late protein secretion, an open question is how these two antagonizing regulators operate. In fact, for FliT, both its expression (promoter activity) and function (loss of inhibition by FliD secretion) are responsive to protein secretion. One possibility is that these two regulators are dominant at different secretion rates, where by secretion rate we mean the total amount of protein secreted from a cell per unit time. In particular, we imagine that FliZ is dominant at low secretion rates and that FliT is dominant at high secretion rates, for example, in cases when the cell possesses too few or too many active HBBs, respectively. Such a mechanism would enable cells to regulate flagellum abundance if we assume that the secretion rate is proportional to number of flagella. In other words, this mechanism would enable cells to increase gene expression when there are too few flagella, due to the action of FliZ at low secretion rates, and decrease gene expression when there are too many, due to the action of FliT at high secretion rates.