B. subtilis is a well characterized facultative anaerobe which is considered as a Gram- positive model bacterium. In the bacteria, aerobic and anaerobic respiration are facilitated in the presence of oxygen and nitrate as final electron acceptors respectively. Electron transport from electron donors (NADH, FADH2) is conducted by ‘quinone’- a lipid soluble organic molecule located in the membrane. Under anaerobic condition, when nitrate is absent, B. subtilis undergoes fermentation to utilize electrons generated throughout the glycolysis and Krebs’s cycle. Studying the anaerobic growth modes of bacteria is essential to acquire insight of anaerobic growth strategies of other endospore former Gram-positive pathogenic bacteria such as B. cereus, B. anthracis etc. which are able to colonize host gastrointestinal (GI) tract.
Quinones are indispensable for respiration and B. subtilis is known to utilize menaquinone (MK), a naphthoquinone in respiration. In the MK biosynthetic pathway, in B. subtilis, two types of naphthoquinones such as menaquinone (MK) and demethylmenaquinone (DMK) are found. DMK is the precursor for MK synthesis by menG. Function of DMK is yet to be investigated. This study aspired to characterize the importance and preference of anaerobic respiration and fermentation under anaerobic condition. To answer this query, growth of the two quinone mutants under aerobic and anaerobic conditions were observed, moreover, the expression of anaerobic respiratory (nasD, hmp, narG, fnr) and fermentative genes (lctE, alsS) was determined as well. Function of the DMK in respiration of B. subtilis was also interrogated. For anaerobic growth specially through anaerobic respiration ResDE two component system is inevitable. Respiratory nitrate reductase (narGHJI) and assimilatory nitrite reductase (nasDE), and Fnr (fnr)are essential complexes in anaerobic nitrate respiration in B. subtilis. Expression of nasD, hmp and fnr is ResDE -dependent. In spite of known essential function of ResDE system in anaerobic respiration, the signal for the activation of the sensor domain ResE is unknown. Henceforth, another aim of the study was to determine the function of quinone (MK and DMK) in the activation of ResE. In this study, the expression of nasD, hmp, and fnr was considered as reporters for ResE activation.
In order to conduct the study, two quinone mutants were constructed to facilitate distinction between two anaerobic growth modes and those were derived from wild type B. subtilis 168. Among two the quinone mutants, ∆menA strain is unable to produce any of the quinones due to menA (DMK synthesis) deletion. In addition, another quinone mutant ∆menG strain accumulates only DMK due to menG deletion. To determine the essentiality of quinones in B. subtilis, quinone mutants were grown aerobically or anaerobically in media containing glucose or nitrate or with both; media with no glucose and nitrate was considered as basal media for comparison. Glucose and nitrate were provided to facilitate fermentation and nitrate respiration respectively. RNA samples collected from these cultures were analyzed to determine the expression of, hmp, narG, fnr, lctE, and alsS genes. When ∆ menA strain was grown in media containing both glucose and nitrate without MK-4 supplementation under anaerobic condition, the bacteria grew well and fermentative genes lctE and alsS were upregulated compared to that in media without MK-4 supplementation. In addition, expression of anaerobic respiratory genes nasD, hmp was downregulated. This result suggested that without MK the bacteria grows anaerobically via fermentation, moreover, MK is essential for anaerobic respiration. In terms of ResE activation, downregulation of nasD and hmp in the ∆ menA strain cultured without MK-4 compared to that with MK-4 suggested that MK pool could have function in ResE activation. Growth of ∆menG strain in media containing both glucose and nitrate without MK supplementation under aerobic and anaerobic condition suggested that DMK is functional in aerobic respiration, however, it is not functional under anaerobic respiration condition. In addition, in the presence of both MK and DMK, the ∆menG strain showed significantly slower growth speed than in WT when cultured both aerobically and anaerobically. This result suggested the possibility of inhibitory function of DMK in bacterial growth. In ∆menG strain the expression of nasD and hmp in presence of both MK, DMK and in the presence of only DMK were similar, which suggested possible involvement of DMK in the inactivation of ResE. Further studies are required to understand the functional mechanism of ResE activation or deactivation by MK and DMK.

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