Urease in acetogenic Lachnospiraceae drives urea carbon salvage in SCFA pools

Abstract

The gut microbiota produces short-chain fatty acids (SCFA) and acidifies the proximal colon which inhibits enteric pathogens. However, for many microbiota constituents, how they themselves resist these stresses is unknown. The anaerobic Lachnospiraceae family, which includes the acetogenic genus Blautia, produce SCFA, are genomically diverse, and vary in their capacity to acidify culture media. Here, we investigated how Lachnospiraceae tolerate pH stress and found that subunits of urease were associated with acidification in a random forest model. Urease cleaves urea into ammonia and carbon dioxide, however the role of urease in the physiology of Lachnospiraceae is unknown. We demonstrate that urease-encoding Blautia show urea-dependent changes in SCFA production, acidification, growth, and, strikingly, urease encoding Blautia directly incorporate the carbon from urea into SCFAs. In contrast, ureolytic Klebsiella pneumoniae or Proteus mirabilis do not show the same urea-dependency or carbon salvage. In agreement, the combination of urease and acetogenesis functions is rare in gut taxa. We find that Lachnospiraceae urease and acetogenesis genes can be co-expressed in healthy individuals and colonization of mice with a ureolytic Blautia reduces urea availability in colon contents demonstrating Blautia urease activity in vivo. In human and mouse microbial communities, the acetogenic recycling of urea carbon into acetate by Blautia leads to the incorporation of urea carbon into butyrate indicating carbon salvage into broader metabolite pools. Altogether, this shows that urea plays a central role in the physiology of health-associated Lachnospiraceae which use urea in a distinct manner that is different from that of ureolytic pathogens.

Keywords: Anaerostipes; Blautia; Lachnospiraceae; Urease; acetate; acetogenesis; acidification; butyrate; cross-feeding; microbiota; short-chain fatty acids; urea; urea-derived acetate production.

Figure 1.

Urease is associated with increased acidification in Lachnospiraceae. a) %increase mean square error of annotated genes in a random forest model of acidification showing importance of urease genes. b) Culture acidification data from 273 Lachnospiraceae isolates stratified by urease gene presence. Two-tailed ttest performed, p = 2.2e-16. c) Schematic of urease complex function. Structural subunits (orange) are assembled with the help of the accessory genes (blue), which also chaperone the Ni²⁺ ion into the active site of the enzyme. Urea can enter the cell through the urea channel UreI (red), where it is hydrolyzed by the urease enzyme. d) Percentage of isolates encoding urease genes across 273 Lachnospiraceae isolate biobank. e) Proportion of urease encoding isolates identified as Blautia and other genera in 4901 whole-genome sequenced gut isolates as described in methods.

Figure 2.

Blautia encode a urease gene cluster with similarity to Helicobacter. a) Urease gene clusters of Blautia isolates and other ureolytic bacteria. b) Alphafold predicted UreI structures of Blautia (blue), H. pylori (green), and S. salivarius (red). Highlighted regions show the loop domains in HpUreI and BlUreI that are absent in SsUreI. c) Alphafold predicted structure of the urease enzyme of Blautia. The urease enzyme is a trimer of oligomers, with each oligomer shown in a different color. d) Urease structure of Klebsiella aerogenes captured by X-ray diffraction (PDB ID: 1EJX). e) Amino acid sequence homology of concatenated urease structural proteins.

Figure 3.

Urease regulates viability and pH modulation in urease-positive Blautia isolates. a) viability and b) acidification of urease-encoding Blautia isolates in neutral or acidic starting pH conditions with indicated urea supplementation. c) Viability and acidification of urease positive blautia in neutral conditions with urea supplementation and urease inhibitor (flurofamide) or vehicle (DMSO). Data points represent individual samples from independent replicates. (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4.

Regulation of viability and acidification in urease-positive K. pneumoniae and P. mirabilis. a) Viability and b) Acidification of ureolytic pathogens in neutral or acidic starting pH conditions with 10 mM urea supplementation. Data points represent individual samples from independent replicates. (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5.

Blautia increase acetate production in response to urea. a) Production of acetate by urease encoding Blautia in neutral or acidic starting pH with various urea supplementations and b) Urease inhibitor. c) Production of acetate by urease-negative Blautia C supplemented with urea. d) Production of acetate by ureolytic pathogens supplemented with 20 mM urea. Data points represent individual values, lines connect samples within experimental replicates. The change in concentration (upper portion of graph) was calculated within each experimental replicate, between the treatment conditions. (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 6.

Blautia incorporate urea carbon into SCFA. a) Schematic of the Wood-Ljungdahl pathway of acetogenesis showing incorporation of urea-derived CO_2. schematic shows an absence of formate dehydrogenase which has been previously reported in Blautia and describes Blautia A and B. b) Relative abundance of heavy (+1) isotopes of acetate and succinate when supplemented with ¹³C-labeled urea. c) Relative abundance of ¹³C-labeled acetate from urease-positive Blautia a cultures supplemented with ¹³C-labeled urea, ¹³C-labeled formate, or both. Solid horizontal lines indicate the natural or baseline abundance of the given isotope. d) Schematic showing: i) Urease-encoding Blautia incorporate urea-derived carbon into acetate. ii) Urease-negative Blautia do not respond to urea. iii, iv)Putative pathways showing urease-negative Blautia incorporating urea carbon into acetate following release through urease activity in P. mirabilis (iii) or exogenous urease (iv). e) Relative abundance of heavy (+1) isotopes of acetate from cultures of urease-negative Blautia C with urease activity provided by purified Jackbean urease or P. mirabilis in coculture. Dashed horizontal lines indicate the average relative abundance of acetate urease-positive Blautia cultures with C-label urea calculated from values in B. Data points represent individual samples from independent replicates. (*p < 0.05, **p < 0.01, ***p < 0.001). f) Presence of acetogenesis pathway (acsC and acsD) and urease (structural subunits and accessory proteins) across species representatives in the UHGG collection. g) Transcriptomic reads from HMP healthy donors aligned with Bowtie2 to urease and acetogenesis genes from 4901 gut isolates as described in methods. All genera labelled are members of Lachnospiraceae. (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 7.

Urea-derived acetogenesis allows for cross-feeding to other SCFA in communities. a) Relative abundance of heavy (+1) isotopes of acetate and butyrate from ¹³C-urea supplemented cocultures of indicated strains with Anaerostipes hadrus. b) Relative abundance of heavy (+1) isotopes of acetate, butyrate and propionate from 6-hour ex vivo cultures of healthy human stool with or without the presence of urease-encoding Blautia. Solid horizontal lines indicate the baseline abundance of the given isotope. c) Schematic of mouse experiment. Created in BioRender. d) Normalized peak area of urea from mouse pellets harvested at sacrifice. e) Relative abundance of heavy (+1) isotopes of acetate, butyrate, propionate and succinate from ex vivo assays of mouse pellets. Solid horizontal lines indicate the natural or baseline abundance of the given isotope. (*p < 0.05, **p < 0.01, ***p < 0.001).