Analysis of the essential cell division gene ftsL of Bacillus subtilis by mutagenesis and heterologous complementation - PubMed (original) (raw)

Analysis of the essential cell division gene ftsL of Bacillus subtilis by mutagenesis and heterologous complementation

J Sievers et al. J Bacteriol. 2000 Oct.

Abstract

The ftsL gene is required for the initiation of cell division in a broad range of bacteria. Bacillus subtilis ftsL encodes a 13-kDa protein with a membrane-spanning domain near its N terminus. The external C-terminal domain has features of an alpha-helical leucine zipper, which is likely to be involved in the heterodimerization with another division protein, DivIC. To determine what residues are important for FtsL function, we used both random and site-directed mutagenesis. Unexpectedly, all chemically induced mutations fell into two clear classes, those either weakening the ribosome-binding site or producing a stop codon. It appears that the random mutagenesis was efficient, so many missense mutations must have been generated but with no phenotypic effect. Substitutions affecting hydrophobic residues in the putative coiled-coil domain, introduced by site-directed mutagenesis, also gave no observable phenotype except for insertion of a helix-breaking proline residue, which destroyed FtsL function. ftsL homologues cloned from three diverse Bacillus species, Bacillus licheniformis, Bacillus badius, and Bacillus circulans, could complement an ftsL null mutation in B. subtilis, even though up to 66% of the amino acid residues of the predicted proteins were different from B. subtilis FtsL. However, the ftsL gene from Staphylococcus aureus (whose product has 73% of its amino acids different from those of the B. subtilis ftsL product) was not functional. We conclude that FtsL is a highly malleable protein that can accommodate a large number of sequence changes without loss of function.

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Figures

FIG. 1

FIG. 1

Strain 2007 constructed for mutagenesis of ftsL. (A) Schematic representation of the genetic organization of strain 2007. The chromosomal pbpB operon contains four genes, yllB, yllC, ftsL, and pbpB. The chromosomal copy of ftsL is disrupted by a neo cassette, whose promoter also provides transcription of the downstream pbpB gene. An IPTG-inducible wild-type copy of ftsL is placed in the amyE locus. The φ105J125 prophage, integrated elsewhere in the chromosome, allows transformation with φ105J506 phage DNA and the insertion by a double-crossover event (dashed lines) of a second copy of ftsL (ftsL*) under the control of the xylose-inducible Pxyl promoter. (B and C) Phase-contrast images of 2007 grown in the presence of the inducer IPTG (B) and after ca. three generations in its absence (C). Bar, 5 μm.

FIG. 2

FIG. 2

Comparison of the FtsL proteins of B. subtilis (FtsLBs), B. licheniformis (FtsLBl), B. badius (FtsLBb), B. circulans (FtsLBc), and S. aureus (FtsLSa). The alignment was performed using the CLUSTAL W method, with residues identical to the B. subtilis sequence shown in gray boxes. The membrane-spanning domain and the a and d positions of the heptad repeat of the putative coiled-coil domain are shown above the alignment.

FIG. 3

FIG. 3

Complementation of a B. subtilis ftsL null mutation by ftsL alleles of S. aureus, B. licheniformis, B. circulans, and B. badius. Shown are phase-contrast images of strains 2044, 2045, 2046, and 2047 grown at 37°C in PAB supplemented with either IPTG (left panel) or xylose (central panel) or not supplemented with any inducer (right panel). IPTG induces the expression of the wild-type gene, whereas the foreign ftsL alleles are transcribed in the presence of xylose. As expected, all four strains were filamented in unsupplemented medium (i.e., when FtsL is depleted). Bar, 5 μm.

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