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. 2017 Jul 11;199(15):e00272-17.
doi: 10.1128/JB.00272-17. Print 2017 Aug 1.

A Conserved Metal Binding Motif in the Bacillus Subtilis Competence Protein ComFA Enhances Transformation

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Free PMC article

A Conserved Metal Binding Motif in the Bacillus Subtilis Competence Protein ComFA Enhances Transformation

Scott S Chilton et al. J Bacteriol. .
Free PMC article

Abstract

Genetic competence is a process in which cells are able to take up DNA from their environment, resulting in horizontal gene transfer, a major mechanism for generating diversity in bacteria. Many bacteria carry homologs of the central DNA uptake machinery that has been well characterized in Bacillus subtilis It has been postulated that the B. subtilis competence helicase ComFA belongs to the DEAD box family of helicases/translocases. Here, we made a series of mutants to analyze conserved amino acid motifs in several regions of B. subtilis ComFA. First, we confirmed that ComFA activity requires amino acid residues conserved among the DEAD box helicases, and second, we show that a zinc finger-like motif consisting of four cysteines is required for efficient transformation. Each cysteine in the motif is important, and mutation of at least two of the cysteines dramatically reduces transformation efficiency. Further, combining multiple cysteine mutations with the helicase mutations shows an additive phenotype. Our results suggest that the helicase and metal binding functions are two distinct activities important for ComFA function during transformation.IMPORTANCE ComFA is a highly conserved protein that has a role in DNA uptake during natural competence, a mechanism for horizontal gene transfer observed in many bacteria. Investigation of the details of the DNA uptake mechanism is important for understanding the ways in which bacteria gain new traits from their environment, such as drug resistance. To dissect the role of ComFA in the DNA uptake machinery, we introduced point mutations into several motifs in the protein sequence. We demonstrate that several amino acid motifs conserved among ComFA proteins are important for efficient transformation. This report is the first to demonstrate the functional requirement of an amino-terminal cysteine motif in ComFA.

Keywords: ATPase; Bacillus subtilis; DEXD/DEXH box; genetic competence; helicase; natural transformation systems; transformation.

Figures

FIG 1
FIG 1
Schematics of ComFA and relevant motifs. (A) DEAD box and ATPase motifs are green. Within ComFA, the zinc finger motif is silver. Asterisks denote residues subjected to mutational analysis. (B) Schematic of ComFA amino acid sequence ΔS1 region enlarged to show the primary sequence and the motifs comprising the region. (C) Alignment of N-terminal regions of 15 ComFA homologs divided into groups based on the numbers of cysteine residues in their zinc finger motifs, i.e., group I with four conserved cysteines and group II with nine conserved cysteines. B. subtilis falls between the groups with five cysteines, four in the zinc finger domain and one just before the Walker A-I motif. The cysteine residues are highlighted in green, and the Q and Walker A-I motifs are highlighted in blue. Strain names are on the right with bullets indicating the strains listed as naturally transformable in reference . Though naturally competent, S. aureus ComFA notably lacks this zinc finger motif. See Fig. S1 in the supplemental material for the sequence alignments of this region from 68 different ComFA proteins.
FIG 2
FIG 2
The DEAD box and C4 motifs are required for efficient transformation and DNA uptake. (A) Transformation efficiency of strains carrying unmarked mutations of ComFA canonical DEAD box helicase motifs. All transformation efficiency values are normalized to the WT. The ΔS1 strain is included as a comFA mutant control (8, 9). (B) Transformation efficiency for unmarked mutations of the ComFA putative C4 zinc finger motif. All transformation efficiency values are normalized to the WT. WT and ΔS1 values are the same in panels A and B. The relative efficiency axis is on a log10 scale. Error bars show standard errors. WT, n = 35; all mutants, n = 5. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
FIG 3
FIG 3
The cysteines in the C4 motif are required for metal binding. (A) Transformation efficiency of a malE-comFA fusion relative to that of WT comFA. Both constructs were expressed from the yvbJ ectopic locus under the control of the PcomF promoter. The comF locus has been replaced with a cat cassette. Error bars show standard errors. WT, n = 2; malE-comFA mutant, n = 4. (B) SDS-PAGE of 3C protease cleavage of translational fusion constructs. Lanes M, molecular mass markers; lanes P, precleavage elution from Sepharose-dextrin column; lanes C, cleaved after a 2-h incubation with 1:100 (wt/wt) 3C protease. (C) SDS-PAGE of fractions from zinc-IMAC analysis. Lanes FT, flowthrough from the column; lanes E, eluate from the column following addition of 250 mM imidazole-HCl. Asterisks in panels B and C: *, MBP-ComFA; **, ComFA; ***, MBP.
FIG 4
FIG 4
The C4 motif requirement is independent of the DEAD box motif requirement. Transformation efficiency for mutations of ComFA putative C4 zinc finger motif combined with canonical DEAD box helicase motifs. All transformation efficiency values are normalized to the WT. WT, K152E, and ΔS1 values are those reported in Fig. 2A, and the 4CS value is that reported in Fig. 2B. The relative efficiency axis is on a log10 scale. Error bars show standard errors. WT, n = 35; all mutants, n = 5. *, P < 0.001; **, P < 0.0001.
FIG 5
FIG 5
Stability and transformation efficiency of WT and mutant GFP-ComFA proteins. (A, B) Immunoblot assay showing WT, 4CS, K152A, E234Q, R419K, S264A, and T266A GFP-ComFA fusions in the presence (A) or absence (B) of the endogenous comF operon. Note that GFP-ComFA4CS is less stable, showing less signal, as well as a 50-kDa immunoreactive degradation product, but all of the other mutants are stable. SigmaA was used as a loading control. M, molecular mass markers in kilodaltons. (C) Relative transformation efficiencies of the GFP-ComFA fusions in the presence or absence of the comF operon. The absolute efficiency of the GFP-WT ComFA fusion ectopically expressed at yvbJ (comF::cat [SC054]) is the same as that of WT ComFA (comF::cat [SC140]) also expressed at yvbJ (shown in Fig. 3A). The relative efficiency axis is on a log10 scale. Error bars show standard errors. WT, n = 14; GFP-ComFA, n = 10; comF::cat GFP-ComFA, n = 7; GFP-ComFA4CS, n = 6; all other mutant GFP fusions, n = 3 or 4. *, P < 0.05; **, P < 0.01 (log-transformed data).

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