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. 2015 Aug;197(16):2622-30.
doi: 10.1128/JB.00035-15. Epub 2015 May 18.

An SOS Regulon Under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria

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An SOS Regulon Under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria

Neus Sanchez-Alberola et al. J Bacteriol. .
Free PMC article

Abstract

The SOS response is a transcriptional regulatory network governed by the LexA repressor that activates in response to DNA damage. In the Betaproteobacteria, LexA is known to target a palindromic sequence with the consensus sequence CTGT-N8-ACAG. We report the characterization of a LexA regulon in the iron-oxidizing betaproteobacterium Sideroxydans lithotrophicus. In silico and in vitro analyses show that LexA targets six genes by recognizing a binding motif with the consensus sequence GAACGaaCGTTC, which is strongly reminiscent of the Bacillus subtilis LexA-binding motif. We confirm that the closely related Gallionella capsiferriformans shares the same LexA-binding motif, and in silico analyses indicate that this motif is also conserved in the Nitrosomonadales and the Methylophilales. Phylogenetic analysis of LexA and the alpha subunit of DNA polymerase III (DnaE) reveal that the organisms harboring this noncanonical LexA form a compact taxonomic cluster within the Betaproteobacteria. However, their lexA gene is unrelated to the standard Betaproteobacteria lexA, and there is evidence of its spread through lateral gene transfer. In contrast to other reported cases of noncanonical LexA-binding motifs, the regulon of S. lithotrophicus is comparable in size and function to that of many other Betaproteobacteria, suggesting that a convergent SOS regulon has reevolved under the control of a new LexA protein. Analysis of the DNA-binding domain of S. lithotrophicus LexA reveals little sequence similarity with that of other LexA proteins targeting similar binding motifs, suggesting that network structure may limit site evolution or that structural constrains make the B. subtilis-type motif an optimal interface for multiple LexA sequences.

Importance: Understanding the evolution of transcriptional systems enables us to address important questions in microbiology, such as the emergence and transfer potential of different regulatory systems to regulate virulence or mediate responses to stress. The results reported here constitute the first characterization of a noncanonical LexA protein regulating a standard SOS regulon. This is significant because it illustrates how a complex transcriptional program can be put under the control of a novel transcriptional regulator. Our results also reveal a substantial degree of plasticity in the LexA recognition domain, raising intriguing questions about the space of protein-DNA interfaces and the specific evolutionary constrains faced by these elements.

Figures

FIG 1
FIG 1
Sequence alignment of the region immediately upstream of the S. lithotrophicus (Slit_2300) and G. capsiferriformans (Galf_0635) lexA genes. Predicted −35 and −10 promoter elements are boxed. The predicted translational start site is underlined. The conserved palindromic sequences corresponding to putative LexA-binding sites are shaded in gray.
FIG 2
FIG 2
EMSAs were performed with 61-bp fragments of G. capsiferriformans and S. lithotrophicus DNA centered on putative LexA-binding sites predicted in silico. The numbers between brackets denote the distance of the targeted site to the predicted TLS, as reported in Table 1. Locus tags for putative SOS genes are reported. Lanes E and B designate assays with E. coli and B. subtilis LexA proteins, respectively. For all other lanes, G. capsiferriformans LexA protein was used. The “+” and “−” symbols denote, respectively, the presence or absence of LexA protein. The molecular masses of these LexA proteins are as follows: E. coli (22.35 kDa), B. subtilis (22.85 kDa), and G. capsiferriformans (21.97 kDa).
FIG 3
FIG 3
Consensus tree of DnaE (top) and LexA (bottom) protein sequences. Branch support values are provided as Bayesian posterior probabilities. LexA-binding motifs for different clades are illustrated using sequence logos made with inferred binding sites (Gallionellales cluster) or aggregate experimental LexA-binding data downloaded from the CollecTF database. Different clades are shaded in alternating shades of gray. The cluster containing the Gallionellales DnaE and LexA proteins is boxed and lightly shaded. The scale bar on each tree denotes expected substitutions per residue. To enhance alignment quality, the DnaE tree was rooted using two Alphaproteobacteria LexA sequences as outgroup and the LexA tree was rooted with four Gram-positive sequences as outgroup. A DnaE tree rooted with Gram-positive sequences is provided in Fig. S7 in the supplemental material for a direct comparison. The protein accession numbers and full species names for all represented protein sequences are available in Table S3 in the supplemental material.
FIG 4
FIG 4
Sequence logo of aligned LexA α3 helix sequences and experimentally validated LexA-binding sites in different clades. Each α3 helix logo was generated using 8 to 10 aligned representative sequences from each clade (see Fig. S4 in the supplemental material). Clade aggregate LexA-binding data were obtained from the CollecTF database. The α3 helix and binding motif of Betaproteobacteria and Xanthomonadaceae (1) LexA are shown for comparison. The LexA protein targets a GAAC/GTAC half-site in all of the other depicted clades. Accession numbers and species names for all proteins used in the multiple-sequence alignment are available in Table S4 in the supplemental material.

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