# A novel extracytoplasmic function sigma factor (RpoE6) regulates biogenesis of a minor flagellin and other functions in Azospirillum brasilense Sp7

## TL;DR

This study identifies a new sigma factor, RpoE6, that regulates a minor flagellin and other functions in Azospirillum brasilense, a plant growth-promoting bacterium.

## Contribution

The first report of an alternative sigma factor regulating a flagellin in Alphaproteobacteria.

## Key findings

- RpoE6 regulates the expression of the minor flagellin FliC2 in Azospirillum brasilense.
- RpoE6 and its anti-sigma factor Asf6 oppositely regulate FliC2 abundance.
- RpoE6 also controls functions related to glycerol and fructose metabolism.

## Abstract

In Gammaproteobacteria, the biogenesis of the flagellar filaments is regulated by a flagellum-specific sigma factor (FliA). In Alphaproteobacteria, however, a flagellum-specific sigma factor is not known. In our search for a FliA-like sigma factor in Azospirillum brasilense Sp7, we found an extracytoplasmic function sigma factor (RpoE6) located in a gene cluster consisting of FlgK, FlgL, FliD, FliS, and FliC2. A strain lacking RpoE6 (ΔrpoE6) was compromised in expressing a small flagellin, FliC2, which contributes to the polar flagellum structure and function in swimming motility, though there was no detectable defect in swimming motility in swim plates. A non-polar inactivation of this sigma factor also led to a growth defect that depended on aeration. FliC2, like the major flagellin of the polar flagellum, is also likely glycosylated. A hypothetical protein, possibly behaving as an anti-sigma factor (Asf6), encoded downstream of rpoE6, interacts with RpoE6. A mutant lacking Asf6 (Δasf6) overproduces FliC2 and possesses thinner flagellar filaments compared to the wild type, suggesting that the abundance of FliC2 is oppositely regulated by RpoE6 and Asf6. Overexpression of rpoE6 in A. brasilense upregulates many proteins, including those involved in flagellar filament (FliD, FliS, and FliC2), required for growth on fructose and glycerol, as well as in the upregulation of another sigma factor, RpoE8. Our data support a role for RpoE6 in coupling FliC2 expression with genes involved in glycerol and fructose metabolism. Conservation of the genomic organization of rpoE6 in related Alphaproteobacteria suggests a similar function in other species.

Azospirillum brasilense is a common plant growth-promoting rhizobacterium that colonizes crop rhizospheres using chemotaxis via its polar flagellum. However, our understanding of the structure, composition, and biogenesis of the polar flagellum in A. brasilense is limited. Unlike Gammaproteobacteria, where FliA and FlgM regulate flagellum biogenesis, no sigma or anti-sigma factors are known to regulate chemotactic motility in Alphaproteobacteria, including A. brasilense. This study identifies an extracytoplasmic function sigma factor (RpoE6) and its anti-sigma factor (Asf6) which modulate the expression of the minor flagellin FliC2, thereby altering the polar flagellum structure. Unlike FliA and FlgM, this regulatory pair also controls other functions such as glycerol and fructose utilization. To our knowledge, this is the first report of an alternative sigma factor regulating a flagellin in Alphaproteobacteria.