Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics.

Morgan N Price,Judy D Wall,Jennifer V Kuehl,Adam P Arkin,Ryan A Melnyk,Grant M Zane,Adam M Deutschbauer,Josep Casadesús

doi:10.1371/journal.pgen.1007147

Morgan N Price, Judy D Wall + Show 6 more

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https://doi.org/10.1371/journal.pgen.1007147

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Abstract

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.

Highlights

It has been known for decades how the model bacterium Escherichia coli synthesizes all 20 of the standard amino acids, novel pathways for amino acid biosynthesis continue to be discovered in other bacteria and in archaea
We found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway
We studied 10 bacteria with predicted gaps in amino acid biosynthesis that grow in minimal media

Summary

Introduction

It has been known for decades how the model bacterium Escherichia coli synthesizes all 20 of the standard amino acids, novel pathways for amino acid biosynthesis continue to be discovered in other bacteria and in archaea. Mee and colleagues [6] and D’Souza and colleagues [7] both used comparative genomics to predict that most bacteria cannot synthesize all 20 of the standard amino acids. Both groups suggested that most free-living bacteria are reliant on cross-feeding of amino acids that are released by other bacteria. If we used the automated method that Mee and colleagues relied on [9] and excluded Escherichia coli, we had predictions for bacteria from 12 genera that grow in defined media All of these bacteria were incorrectly predicted to be auxotrophic for multiple amino acids. There were many putative gaps in the biosynthetic pathways—enzymatic steps that are required for biosynthesis and were missing from the genome annotation—but these gaps were misleading

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