Methylation-Independent Chemotaxis Systems Are the Norm for Gastric-Colonizing Helicobacter Species

Abstract

Many bacteria and archaea rely on chemotaxis signal transduction systems for optimal fitness. These complex, multiprotein signaling systems have core components found in all chemotactic microbes, as well as variable proteins found in only some species. We do not yet understand why these variations exist or whether there are specific niches that favor particular chemotaxis signaling organization. One variation is in the presence/absence of the chemotaxis methylation adaptation enzymes CheB and CheR. Genes for CheB and CheR are missing in the gastric pathogen Helicobacter pylori but present in related Helicobacter that colonize the liver or intestine. In this work, we asked whether there was a general pattern of CheB/CheR across multiple Helicobacter species. Helicobacter spp. all possess chemotactic behavior, based on the presence of genes for core signaling proteins CheA, CheW, and chemoreceptors. Genes for the CheB and CheR proteins, in contrast, were variably present. Niche mapping supported the idea that these genes were present in enterohepatic Helicobacter species and absent in gastric ones. We then analyzed whether there were differences between gastric and enterohepatic species in the CheB/CheR chemoreceptor target methylation sites. Indeed, these sites were less conserved in gastric species that lack CheB/CheR. Lastly, we determined that cheB and cheR could serve as markers to indicate whether an unknown Helicobacter species was of enterohepatic or gastric origin. Overall, these findings suggest the interesting idea that methylation-based adaptation is not required in specific environments, particularly the stomach. IMPORTANCE Chemotaxis signal transduction systems are common in the archaeal and bacterial world, but not all systems contain the same components. The rationale for this system variation remains unknown. In this report, comparative genomics analysis showed that the presence/absence of CheR and CheB is one main variation within the Helicobacter genus, and it is strongly associated with the niche of Helicobacter species: gastric Helicobacter species, which infect animal stomachs, have lost their CheB and CheR, while enterohepatic Helicobacter species, which infect the liver and intestine, retain them. This study not only provides an example that a chemotaxis system variant is associated with particular niches but also proposes that CheB and CheR are new markers distinguishing gastric from enterohepatic Helicobacter species.

Keywords: CheB; CheR; Helicobacter; adaptation; chemotaxis; enterohepatic; gastric; methylation.

FIG 1

Phyletic distribution of five chemotaxis proteins in the Campylobacterales order: CheA (blue), CheW (orange), CheB (green), CheR (red), and CheV (purple). Helicobacteraceae are indicated by light purple shading. In the ring just inside the CheA ring, individual species are denoted by separating lines, with 35 species in the Helicobacteraceae family (purple area) and 26 in the Campylobacteraceae one. The figure was created using Aquerium.

FIG 2

Phylogenetic tree of Helicobacteraceae species combined with niche mapping and cheB/cheR presence/absence. The outer-most layer shows the cheB/cheR presence. Red dots indicate genomes without cheB/cheR, and green dots indicate genomes with cheB/cheR. The second layer indicates Helicobacter species with known gastric (orange) or enterohepatic (blue) niches, indicated by both dots and shading. The species name with RS GCF indicates the representative species (RS) number in Genome Taxonomy Database (GTDB), when it does not have a NCBI organism name. The tree was built using 120 single-copy marker proteins and was collected from the GTDB (28) and Annotree (81).

FIG 3

Protein sequence alignments of predicted CheB (A) and CheR (B) protein sequences from enterohepatic Helicobacter species. In both panels, predicted active sites residues are indicated with orange stars. In panel B, red dots indicate sites conserved in CheR from other non-Helicobacter bacteria; blue stars indicate interaction sites with AdoMet; and green dots indicate residues that form hydrogen bonds with AdoMet. GXX indicates the motif at the β-subdomain, which present only in CheR that interacts with pentapeptides. Multiple sequence alignments were performed by EBI-MAFFT (77), analyzed with Jalview (78), and visualized using WebLogo3 (80).

FIG 4

Analysis of conservation patterns in methylation sites 1, 2, and 3, comparing chemoreceptors from gastric (top) or enterohepatic (bottom) Helicobacter species. The seven-residue coiled coil repeat positions (a to g) are indicated at the top. The methylation sites are located at position c or d and indicated with red color. The consensus sequence for methylation sites from E. coli, S. enterica, and T. maritima are indicated as insets in the bottom panel. Multiple sequence alignments were performed by EBI-MAFFT (77) and visualized using WebLogo3 (80). Methylation sites were identified by aligning of Helicobacter species chemoreceptors with E. coli and C. jejuni chemoreceptors with known methylation sites.

FIG 5

Analysis of prevalence of different types of enterohepatic Helicobacter chemoreceptors with or without methylation sites. Chemoreceptor types were collected from the MiST database and named 24H through 44H according to the number of heptads (H). PAS, Cache, 4HB, CZB, NIT, etc., indicate the ligand-binding domain of cognate chemoreceptors and were also collected from MiST.

FIG 6

Mapping the genome size of Helicobacter species with or without CheB/CheR to the phylogenetic tree of Helicobacteraceae. Red squares and red highlighting indicate genomes without CheB/CheR, while the blue squares and blue highlighting indicate genomes with CheB/CheR. The inset shows the genome size in gastric or enterohepatic Helicobacter species, with each dot representing a species and the solid lines indicating the average. Comparison between these two groups was done using a Student’s t test, yielding a P value of <0.0001, indicated by ****.