Genome-wide detection and analysis of cell wall-bound proteins with LPxTG-like sorting motifs

Abstract

Surface proteins of gram-positive bacteria often play a role in adherence of the bacteria to host tissue and are frequently required for virulence. A specific subgroup of extracellular proteins contains the cell wall-sorting motif LPxTG, which is the target for cleavage and covalent coupling to the peptidoglycan by enzymes called sortases. A comprehensive set of putative sortase substrates was identified by in silico analysis of 199 completely sequenced prokaryote genomes. A combination of detection methods was used, including secondary structure prediction, pattern recognition, sequence homology, and genome context information. With the hframe algorithm, putative substrates were identified that could not be detected by other methods due to errors in open reading frame calling, frameshifts, or sequencing errors. In total, 732 putative sortase substrates encoded in 49 prokaryote genomes were identified. We found striking species-specific variation for the LPxTG motif. A hidden Markov model (HMM) based on putative sortase substrates was created, which was subsequently used for the automatic detection of sortase substrates in recently completed genomes. A database was constructed, LPxTG-DB (http://bamics3.cmbi.kun.nl/sortase_substrates), containing for each genome a list of putative sortase substrates, sequence information of these substrates, the organism-specific HMMs based on the consensus sequence of the sortase recognition motif, and a graphic representation of this consensus.

FIG. 1.

Detecting sortase substrates. The steps shown in the dashed rectangle were carried out for each of the 154 genomes individually. Gray arrows indicate that all proteins meeting the selection criteria described in the box were taken to the next step. Black arrows indicate that the proteins had to meet additional criteria, as follows. (i) Proteins should have a transmembrane helix following the sortase recognition motif LPxTG. (ii) This helix should be followed by positively charged amino acid residues. (iii) Proteins should have three or fewer transmembrane helices in their complete precursor sequence. (iv) Proteins should not have a predicted function indicating intracellular localization.

FIG. 2.

Organism-specific cleavage motifs. The consensus sortase cleavage sites of L. plantarum (LPQTxE, found in 23 of 27 predicted sortase substrates), Lactobacillus johnsonii (LPQTG, found in 12 of 16 substrates), L. monocytogenes (LPxTGD, found in 33 of 42 substrates), and S. coelicolor (LAxTG, found in 15 of 17 substrates) are organism-specific variations on the generic LPxTG consensus. The overall height of each stack indicates the sequence conservation at that position (measured in bits), whereas the height of symbols within the stack reflects the relative frequency of the corresponding amino acid at that position. The Weblogo software (11) was used to visualize the motifs.