Abstract
In addition to its role in DNA damage repair and recombination, the RecA protein, through its interaction with CheW, is involved in swarming motility, a form of flagella-dependent movement across surfaces. In order to better understand how SOS response modulates swarming, in this work the location of RecA and CheW proteins within the swarming cells has been studied by using super-resolution microscopy. Further, and after in silico docking studies, the specific RecA and CheW regions associated with the RecA-CheW interaction have also been confirmed by site-directed mutagenesis and immunoprecipitation techniques. Our results point out that the CheW distribution changes, from the cell poles to foci distributed in a helical pattern along the cell axis when SOS response is activated or RecA protein is overexpressed. In this situation, the CheW presents the same subcellular location as that of RecA, pointing out that the previously described RecA storage structures may be modulators of swarming motility. Data reported herein not only confirmed that the RecA-CheW pair is essential for swarming motility but it is directly involved in the CheW distribution change associated to SOS response activation. A model explaining not only the mechanism by which DNA damage modulates swarming but also how both the lack and the excess of RecA protein impair this motility is proposed.
Highlights
RecA is a multifunctional protein present in almost all members of the Bacteria domain (Eisen, 1995)
The location of RecA and CheW proteins within SOS response activated-S. enterica swarming cells was analyzed by using 3Dstimulated emission depletion microscopy (3D-Stimulated Emission Depletion (STED)), a superresolution fluorescence imaging technique that increases axial resolution by up to 20–40 nm in biological samples (Han and Ha, 2015)
In agreement with E. coli cells grown in liquid medium under non-DNA-damaging conditions (Greenfield et al, 2009), in the non-mitomycin C-treated S. enterica swarming cells, the CheW protein was majorly located at the cell poles (Figure 1B)
Summary
RecA is a multifunctional protein present in almost all members of the Bacteria domain (Eisen, 1995). Swarming motility is the rapid, organized multicellular translocation of bacteria across a moist surface. It is powered by rotating flagella (Henrichsen, 1972) and is widely distributed through the Bacteria Domain (Harshey, 1994). CheW couples the transmembrane methyl-accepting chemoreceptor protein trimers of dimers (MCPs) to CheA, a histidine kinase that transfers the signal to the CheY response regulator, which acts on the flagellar motor by switching flagellar rotation according to the stimuli detected by the MCPs (Boukhvalova et al, 2002; Sourjik and Wingreen, 2012). To avoid saturation of the sensory system, the chemoreceptor signal is reset by the activity of a methyltransferase (CheR) and a methylesterase (CheB), both of which are located in the vicinity of the chemoreceptors and which restore pre-stimulus activity through reversible covalent modification of the MCPs (Sourjik and Wingreen, 2012)
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