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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.

SUBMITTER: Qian G

PROVIDER: S-EPMC6773520 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The twitching motility of bacteria is closely related to environmental adaptability and pathogenic behaviors. Lysobacter is a good genus in which to study twitching motility because of the complex social activities and distinct movement patterns of its members. Regardless, the mechanism that induces twitching motility is largely unknown. In this study, we found that the interspecies signal indole caused Lysobacter to have irregular, random twitching motility with significantly enhanced speed. Deletion of qseC or qseB from the two-component system for indole signaling perception resulted in the disappearance of rapid, random movements and significantly decreased twitching activity. Indole-induced, rapid, random twitching was achieved through upregulation of expression of gene cluster pilE1-pilY11-pilX1-pilW1-pilV1-fimT1 In addition, under conditions of extremely low bacterial density, individual Lysobacter cells grew and divided in a stable manner in situ without any movement. The intraspecies quorum-sensing signaling factor 13-methyltetradecanoic acid, designated L. enzymogenes diffusible signaling factor (LeDSF), was essential for Lysobacter to produce twitching motility through indirect regulation of gene clusters pilM-pilN-pilO-pilP-pilQ and pilS1-pilR-pilA-pilB-pilC These results demonstrate that the motility of Lysobacter is induced and regulated by indole and LeDSF, which reveals a novel theory for future studies of the mechanisms of bacterial twitching activities.IMPORTANCE The mechanism underlying bacterial twitching motility is an important research area because it is closely related to social and pathogenic behaviors. The mechanism mediating cell-to-cell perception of twitching motility is largely unknown. Using Lysobacter as a model, we found in this study that the interspecies signal indole caused Lysobacter to exhibit irregular, random twitching motility via activation of gene cluster pilE1-pilY11-pilX1-pilW1-pilV1-fimT1 In addition, population-dependent behavior induced by 13-methyltetradecanoic acid, a quorum-sensing signaling molecule designated LeDSF, was involved in twitching motility by indirectly regulating gene clusters pilM-pilN-pilO-pilP-pilQ and pilS1-pilR-pilA-pilB-pilC The results demonstrate that the twitching motility of Lysobacter is regulated by these two signaling molecules, offering novel clues for exploring the mechanisms of twitching motility and population-dependent behaviors of bacteria.

Project description:Lysobacter species are a group of environmental bacteria that are emerging as a new source of antibiotics. One characteristic of Lysobacter is intrinsic resistance to multiple antibiotics, which had not been studied. To understand the resistance mechanism, we tested the effect of blocking two-component regulatory systems (TCSs) on the antibiotic resistance of Lysobacter enzymogenes, a prolific producer of antibiotics. Upon treatment with LED209, an inhibitor of the widespread TCS QseC/QseB, L. enzymogenes produced a large amount of an unknown metabolite that was barely detectable in the untreated culture. Subsequent structural elucidation by nuclear magnetic resonance (NMR) unexpectedly revealed that the metabolite was indole. Indole production was also markedly induced by adrenaline, a known modulator of QseC/QseB. Next, we identified two TCS genes, L. enzymogenesqseC (Le-qseC) and Le-qseB, in L. enzymogenes and found that mutations of Le-qseC and Le-qseB also led to a dramatic increase in indole production. We then chemically synthesized a fluorescent indole probe that could label the cells. While the Le-qseB (cytoplasmic response regulator) mutant was clearly labeled by the probe, the Le-qseC (membrane sensor) mutant was not labeled. It was reported previously that indole can enhance antibiotic resistance in bacteria. Therefore, we tested if the dramatic increase in the level of indole production in L. enzymogenes upon blocking of Le-qseC and Le-qseB would lead to enhanced antibiotic resistance. Surprisingly, we found that indole caused the intrinsically multiantibiotic-resistant bacterium L. enzymogenes to become susceptible. Point mutations at conserved amino acids in Le-QseC also led to antibiotic susceptibility. Because indole is known as an interspecies signal, these findings may have implications.IMPORTANCE The environmental bacterium Lysobacter is a new source of antibiotic compounds and exhibits intrinsic antibiotic resistance. Here, we found that the inactivation of a two-component regulatory system (TCS) by an inhibitor or by gene deletion led to a remarkable increase in the level of production of a metabolite in L. enzymogenes, and this metabolite was identified to be indole. We chemically synthesized a fluorescent indole probe and found that it could label the wild type and a mutant of the TCS cytoplasmic response regulator but not a mutant of the TCS membrane sensor. Indole treatment caused the intrinsically multidrug-resistant bacterium L. enzymogenes to be susceptible to antibiotics. Mutations of the TCS sensor also led to antibiotic susceptibility. Because indole is known as an interspecies signal between gut microbiota and mammalian hosts, the observation that indole could render intrinsically resistant L. enzymogenes susceptible to common antibiotics may have implications.

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

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