Restricted distribution of the butyrate kinase pathway among butyrate-producing bacteria from the human colon

Abstract

The final steps in butyrate synthesis by anaerobic bacteria can occur via butyrate kinase and phosphotransbutyrylase or via butyryl-coenzyme A (CoA):acetate CoA-transferase. Degenerate PCR and enzymatic assays were used to assess the presence of butyrate kinase among 38 anaerobic butyrate-producing bacterial isolates from human feces that represent three different clostridial clusters (IV, XIVa, and XVI). Only four strains were found to possess detectable butyrate kinase activity. These were also the only strains to give PCR products (verifiable by sequencing) with degenerate primer pairs designed within the butyrate kinase gene or between the linked butyrate kinase/phosphotransbutyrylase genes. Further analysis of the butyrate kinase/phosphotransbutyrylase genes of one isolate, L2-50, revealed similar organization to that described previously from different groups of clostridia, along with differences in flanking sequences and phylogenetic relationships. Butyryl-CoA:acetate CoA-transferase activity was detected in all 38 strains examined, suggesting that it, rather than butyrate kinase, provides the dominant route for butyrate formation in the human colonic ecosystem that contains a constantly high concentration of acetate.

FIG. 1.

Arrangement of the genes for phosphotransbutyrylase and butyrate kinase (ptb and buk) and neighboring genes in C. acetobutylicum ATCC 924 (34), C. perfringens 13 (47), Clostridium tetani E88 (13), and strain L2-50. For the genes of strain L2-50, the closest relatives and their identity and similarity (shown as percentages) according to a BLASTP search are also shown. FAD, flavin adenine dinucleotide.

FIG. 2.

Phylogenetic tree of low mol% G+C-content gram-positive bacteria based on 16S rRNA sequence corresponding to positions 100 to 1447 of the E. coli numbering system (12). Strains examined in this study are shown in boldface, while the remaining strains served as reference sequences. Accession numbers for the sequences used are given in brackets. Bootstrap values greater than 95 (per 100 replications) are shown at branch points. Arrows indicate strains that carry the butyrate kinase pathway for butyrate formation (solid arrows, human fecal strains; open arrows, strains from other environments). Clostridial clusters (14) are indicated by roman numbers. The scale bar represents genetic distance (10 substitutions per 100 nucleotides).

FIG. 3.

Phylogenetic tree of part of the deduced protein sequence (amino acid positions 22 to 127 of strain L2-50) of the butyrate kinase gene (buk) from bacteria examined in this study (shown in boldface) and several clostridial sequences (13, 34, 35, 47). Accession numbers for the sequences used are given in brackets. Bootstrap values greater than 95 (per 100 replications) are shown at branch points. The scale bar represents genetic distance (10 substitutions per 100 nucleotides). *, no corresponding isolate available.

FIG. 4.

Enzyme activities for acetate kinase (solid gray bars), butyrate kinase (black bars), and butyryl-CoA:acetate CoA-transferase (light gray bars) from human colon bacteria. The butyryl-CoA:acetate CoA-transferase values are divided by 1,000.