Two forms of phosphomannomutase in gammaproteobacteria: The overlooked membrane-bound form of AlgC is required for twitching motility of Lysobacter enzymogenes

Abstract

Lysobacter enzymogenes, a member of Xanthomonadaceae, is a promising tool to control crop-destroying fungal pathogens. One of its key antifungal virulence factors is the type IV pili that are required for twitching motility. Transposon mutagenesis of L. enzymogenes revealed that the production of type IV pili required the presence of the Le2152 gene, which encodes an AlgC-type phosphomannomutase/phosphoglucomutase (PMM). However, in addition to the cytoplasmic PMM domain, the Le2152 gene product contains a ~200-aa N-terminal periplasmic domain that is anchored in the membrane by two transmembrane segments and belongs to the dCache superfamily of periplasmic sensor domains. Sequence analysis identified similar membrane-anchored PMMs, encoded in conserved coaBC-dut-algC gene clusters, in a variety of gammaproteobacteria, either as the sole PMM gene in the entire genome or in addition to the gene encoding the stand-alone enzymatic domain. Previously overlooked N-terminal periplasmic sensor domains were detected in the well-characterized PMMs of Pseudomonas aeruginosa and Xanthomonas campestris, albeit not in the enzymes from Pseudomonas fluorescens, Pseudomonas putida or Azotobacter vinelandii. It appears that after the initial cloning of the enzymatically active soluble part of P. aeruginosa AlgC in 1991, all subsequent studies utilized N-terminally truncated open reading frames. The N-terminal dCache sensor domain of AlgC is predicted to modulate the PMM activity of the cytoplasmic domain in response to as yet unidentified environmental signal(s). AlgC-like membrane-bound PMMs appear to comprise yet another environmental signalling system that regulates the production of type IV pili and potentially other systems in certain gammaproteobacteria.

Figure 1.. Le2152 is required for twitching motility in Lysobacter enzymogenes OH11.

Loss of Le2152 resulted in the absence of motile cells on the margins of the mutant colonies. A. OH11, wild-type strain of L. enzymogenes. B. ΔLe2152, the Le2152 in-frame deletion mutant of OH11. C. ΔLe2152(2152), ΔLe2152 strain complemented with plasmid-borne Le2152 under its native promoter. D. ΔLe2152(pBBR), ΔLe2152 strain haboring an empty vector.

Figure 2.. Domain organization of the Lysobacter enzymogenes Le2152 protein.

Blue boxes indicate predicted transmembrane regions; PGM-PMM indicates the cytoplasmic phosphomannomutase domain, a combination of Pfam (El-Gebali et al., 2019) domains PGM_PMM_I (PF02878), PGM_PMM_II (PF02879), PGM_PMM_III (PF02880), and PGM_PMM_IV (PF00408). The transmembrane orientation and domain architecture of Le2152 were predicted using the TMHMM and SMART tools (Krogh et al., 2001; Letunic and Bork, 2018). The predicted periplasmic domain was identified as dCache using HHpred (Zimmermann et al., 2018), see Supporting Information Figure S2 for details.

Figure 3.. Genomic neighborhoods of phosphomannomutase genes in selected gammaproteobacteria.

Phosphomannomutase (PMM) genes are shown in red, their genomic locus tags and GenBank accession numbers are listed in Table 1. Members of the conserved neighborhoods are indicated with bright colors with same colors for all homologs; variable genes are in grey, uncharacterized genes are in white. Gene names are from the COG database (Galperin et al., 2015), the shapes are drawn approximately to size. A. Gene clusters of the algC (“long” PMM) genes. The coloring of algC reflects domain organization of its product: the periplasmic dCache domain is shown as red checkered box, two transmembrane segments as black checkered boxes, the flexible linker as red dotted box and the enzymatic domain is in red. B. Gene clusters of the “short” PMM genes. See text for details.

Figure 3.. Genomic neighborhoods of phosphomannomutase genes in selected gammaproteobacteria.

Phosphomannomutase (PMM) genes are shown in red, their genomic locus tags and GenBank accession numbers are listed in Table 1. Members of the conserved neighborhoods are indicated with bright colors with same colors for all homologs; variable genes are in grey, uncharacterized genes are in white. Gene names are from the COG database (Galperin et al., 2015), the shapes are drawn approximately to size. A. Gene clusters of the algC (“long” PMM) genes. The coloring of algC reflects domain organization of its product: the periplasmic dCache domain is shown as red checkered box, two transmembrane segments as black checkered boxes, the flexible linker as red dotted box and the enzymatic domain is in red. B. Gene clusters of the “short” PMM genes. See text for details.