FlhF, the third signal recognition particle-GTPase of Bacillus subtilis, is dispensable for protein secretion

Abstract

Bacillus subtilis contains three proteins of the signal recognition particle-GTPase family known as Ffh, FtsY, and FlhF. Here we show that FlhF is dispensable for protein secretion, unlike Ffh and FtsY. Although flhF is located in the fla/che operon, B. subtilis 168 flhF mutant cells assemble flagella and are motile.

FIG. 1.

Conserved domains in proteins of the SRP-GTPase family. The SRP-GTPase family members of yeast (SRP54, SRα), E. coli (P48, FtsY_Ec), and B. subtilis (Ffh, FlhF, FtsY_Bs) are represented schematically. Different domains that can be distinguished are the acidic A domain; the basic B domain, the conserved N domain, the M domain involved in RNA and preprotein binding, and the GTP-binding G domain. The five conserved boxes, G1 to G5, in the G domain, as defined by Eichler and Moll (8), are shown.

FIG. 2.

Extracellular proteome of B. subtilis flhF::cat. The extracellular proteins of the flhF::cat mutant strains and the respective parental strains 168 and DB430 were separated by two-dimensional gel electrophoresis, after which dual-channel fluorescence imaging was used to visualize possible changes in extracellular protein composition (3). Protein spots identified by mass spectrometry and/or N-terminal sequencing are indicated. Green protein spots are predominantly present in the image of the extracellular proteins of the parental strain, red protein spots are predominantly present in the image of the extracellular proteins of the flhF mutant strain, and yellow protein spots are present in similar amounts in both images. (A) Extracellular proteomes of B. subtilis DB430 and DB430 flhF::cat. (B) Variable extracellular levels of prophage-encoded proteins YolA, XkdM, XkdG, and XkdK (top to bottom).

FIG. 3.

Absence of FlhF has no impact on secretion of AmyQ and cellular levels of Ffh and FtsY. The secretion of overproduced AmyQ (A) and the intracellular levels of Ffh and FtsY (B) were analyzed by Western blotting with cellular (c) and/or growth medium (m) fractions of B. subtilis 168 flhF::cat and parental strain 168. d, degradation products of AmyQ.

FIG. 4.

Motility assays. (A) Comparison of the motilities of B. subtilis strains 168, OI2735, 168 flhF::cat, BFA2616 (grown in the absence or presence of 1 mM IPTG), and BFA2616 flhF::cat (grown in the absence or presence of 1 mM IPTG) after 12 h of incubation on 0.27% agar plates at 37°C. (B) Schematic representation of ylxH gene disruption by pMutin2 via Campbell-type integration. lacI, E. coli lacI gene; ori pBR322, origin of replication of plasmid pBR322; ApR, ampicillin resistance marker; EmR, erythromycin resistance marker; t1t2, transcriptional terminators on pMutin2; Pspac, IPTG-dependent promoter; Pfla/che, promoter of the fla/che operon; ylxH′, 3′-truncated ylxH gene; ′ylxH, 5′-truncated ylxH gene; flhF, flhF gene; cheB, cheB gene.