Roles of <scp>d</scp> -Lactate Dehydrogenases in the Anaerobic Growth of <i>Shewanella oneidensis</i> MR-1 on Sugars

Abstract

<i>Shewanella oneidensis</i> MR-1 is a facultative anaerobe that respires using a variety of electron acceptors. Although this organism is incapable of fermentative growth in the absence of electron acceptors, its genome encodes LdhA (a putative fermentative NADH-dependent d-lactate dehydrogenase [d-LDH]) and Dld (a respiratory quinone-dependent d-LDH). However, the physiological roles of LdhA in MR-1 are unclear. Here, we examined the activity, transcriptional regulation, and traits of deletion mutants to gain insight into the roles of LdhA in the anaerobic growth of MR-1. Analyses of d-LDH activity in MR-1 and the <i>ldhA</i> deletion mutant confirmed that LdhA functions as an NADH-dependent d-LDH that catalyzes the reduction of pyruvate to d-lactate. <i>In vivo</i> and <i>in vitro</i> assays revealed that <i>ldhA</i> expression was positively regulated by the cyclic-AMP receptor protein, a global transcription factor that regulates anaerobic respiratory pathways in MR-1, suggesting that LdhA functions in coordination with anaerobic respiration. Notably, we found that a deletion mutant of all four NADH dehydrogenases (NDHs) in MR-1 (ΔNDH mutant) retained the ability to grow on <i>N</i>-acetylglucosamine under fumarate-respiring conditions, while an additional deletion of <i>ldhA</i> or <i>dld</i> deprived the ΔNDH mutant of this growth ability. These results indicate that LdhA-Dld serves as a bypass of NDH in electron transfer from NADH to quinones. Our findings suggest that the LdhA-Dld system manages intracellular redox balance by utilizing d-lactate as a temporal electron sink under electron acceptor-limited conditions.<b>IMPORTANCE</b> NADH-dependent LDHs are conserved among diverse organisms and contribute to NAD⁺ regeneration in lactic acid fermentation. However, this type of LDH is also present in nonfermentative bacteria, including members of the genus <i>Shewanella</i>, while their physiological roles in these bacteria remain unknown. Here, we show that LdhA (an NADH-dependent d-LDH) works in concert with Dld (a quinone-dependent d-LDH) to transfer electrons from NADH to quinones during sugar catabolism in <i>S. oneidensis</i> MR-1. Our results indicate that d-lactate acts as an intracellular electron mediator to transfer electrons from NADH to membrane quinones. In addition, d-lactate serves as a temporal electron sink when respiratory electron acceptors are not available. Our study suggests novel physiological roles for d-LDHs in providing nonfermentative bacteria with catabolic flexibility under electron acceptor-limited conditions.