Transient MutS-Based Hypermutation System for Adaptive Evolution of Lactobacillus casei to Low pH

Abstract

This study explored transient inactivation of the gene encoding the DNA mismatch repair enzyme MutS as a tool for adaptive evolution of <i>Lactobacillus casei</i> MutS deletion derivatives of <i>L. casei</i> 12A and ATCC 334 were constructed and subjected to a 100-day adaptive evolution process to increase lactic acid resistance at low pH. Wild-type parental strains were also subjected to this treatment. At the end of the process, the Δ<i>mutS</i> lesion was repaired in representative <i>L. casei</i> 12A and ATCC 334 Δ<i>mutS</i> mutant isolates. Growth studies in broth at pH 4.0 (titrated with lactic acid) showed that all four adapted strains grew more rapidly, to higher cell densities, and produced significantly more lactic acid than untreated wild-type cells. However, the adapted Δ<i>mutS</i> derivative mutants showed the greatest increases in growth and lactic acid production. Further characterization of the <i>L. casei</i> 12A-adapted Δ<i>mutS</i> derivative revealed that it had a significantly smaller cell volume, a rougher cell surface, and significantly better survival at pH 2.5 than parental <i>L. casei</i> 12A. Genome sequence analysis confirmed that transient <i>mutS</i> inactivation decreased DNA replication fidelity in both <i>L. casei</i> strains, and it identified genetic changes that might contribute to the lactic acid-resistant phenotypes of adapted cells. Targeted inactivation of three genes that had acquired nonsense mutations in the adapted <i>L. casei</i> 12A Δ<i>mutS</i> mutant derivative showed that NADH dehydrogenase (<i>ndh</i>), phosphate transport ATP-binding protein PstB (<i>pstB</i>), and two-component signal transduction system (TCS) quorum-sensing histidine protein kinase (<i>hpk</i>) genes act in combination to increase lactic acid resistance in <i>L. casei</i> 12A.<b>IMPORTANCE</b> Adaptive evolution has been applied to microorganisms to increase industrially desirable phenotypes, including acid resistance. We developed a method to increase the adaptability of <i>Lactobacillus casei</i> 12A and ATCC 334 through transient inactivation of the DNA mismatch repair enzyme MutS. Here, we show this method was effective in increasing the resistance of <i>L. casei</i> to lactic acid at low pH. Additionally, we identified three genes that contribute to increased acid resistance in <i>L. casei</i> 12A. These results provide valuable insight on methods to enhance an organism's fitness to complex phenotypes through adaptive evolution and targeted gene inactivation.