Mechanisms In The Mutualism Between Bacillus Megaterium And Ketogulonigenium Vulgare

In this dissertation, the mechanism in mutualism between Bacillus megateriumWSH-002and Ketogulonigenium vulgare WSH-001was discussed and validated by specificgene deletion, microbial physiology, transcriptomics, transposon mutagenesis and highthroughput screening method research. Furthermore, the method for identifying key geneassociated with mutualism on genome scale was constructed. Which provides a novel routefor molecular mechanism research in mutualism of B. megaterium and K. vulgare. The mainresults were described as follows:1) B. megaterium sporulation and spore stability are two important factors for mutualism ofB. megaterium and K. vulgare. The mutants of sporulation deficient and unstable sporewere constructed by depletion of the master regulate gene spo0A and dipicolinic acidsynthase subunit A coding gene spoVFA, respectively. Afterwards, the mutants wereco-cultured with K. vulgare. Comparing with the wild type co-culture system, thesorbose conversion ratio of sporeless mutant co-culture system and unstable sporemutant co-culture system decreased by33%and70%, respectively. Meanwhile, themaxium cell number of K. vulgare declined by15%and49%; The sorbosedehydrogenase activity of specific cell number was about74%and11%of that inwild-type co-culture system. Furthermore, the study results indicated that sorboseconversion ratio was linearly correlated with sorbose dehydrogenase activity (adjustedR2=0.922). These results indicated B. megaterium sporulation process and stable sporeplayed important roles in mutual interaction between B. megaterium and K. vulgare, andexplained the phenomenon that many Bacillus species act as effective companions.2) The differences in fermentation process triggerd by B. megaterium were studied throughanalysis of extracellular nutrients factors, oxidation-reduction potential (ORP) andrelative expression level of key genes in many important metabolic pathway. The resultsshowed that abundant of metabolites were released to medium from B. megateriumduring sporulation. Many of them are necessary nutrient factors which can not bebiosynthesized de novo by K. vulgare or are deficient in fermentation medium, such asglycine, phenylalanine, isoleucine, leucine, threonine, asparagine, folic acid andriboflavin. Many upregulated key genes, which may be induced by the released nutrientfactors, indicated that amino acid metabolism, tricarboxylic acid cycle (TCA) andcofactor synthesis were stimulated by the companion.3) The K. vulgare genomic transcriptomics data of mid-exponential phase of fermentation showed95and81genes were remarkably upregulated and downregulated (foldchange>2.0and p<0.01) in co-culture system, respectively. The clusters of orthologousgroup of proteins (COG) analysis indicated that these remarkable changed genes mainlyassociated with amino acid transport and metabolism, ion transport and metabolism andenergy production and conversion. The analysis integrated with metabolic pathwaysuggested that the factors relaeased by B. megaterium induced numerous amnio acid oroligopeptide transporter encoding genes expression, as well as many rate-limit enzymesencoding genes in amino acid metabolism pathway. As a result, amino acids andoligopeptides were facilated to be uptaken and assimilated by K. vulgare in co-culturemode. On the contrary, in mono-culture mode, the down-regulation of pykA (codingpyruvate kinase) led to the flux increase of pentose phosphate pathway (PPP), NADPHsynthesis enhancement and TCA flux decrease. In addition, the whole siderophoreuptake pathway and Fe-S cluster assembly were upregulated. These results made thehypothesis was proposed that K. vulgare underwent oxidative stress duringfermentation.4) The hypothesis that K. vulgare underwent oxidative stress was validated by analysis oftotal reactive oxygen species (ROS) level, lipid peroxidation, total antioxidationcapacity (TAC), etc. The results indicated that extracellular ROS level in mono-culturesystem was stimulated and increased to2-5times as much as in co-culture system. Atthe mid-stage and late-stage of mono-culture fermentation, lipid peroxidation was7.3-fold and6.4-fold as much as in co-culture, respectively. The data suggested K.vulgare underwent oxidative stress triggered by ROS. In addition, the intracellular ratioof protein combined thiol to total thiol declined to13.8%at late-stage of mono-culture,which was about one seventh of that in co-culture. It indicated protein cysteine residuewas modified by ROS, resulting with enzymes inactivated. On the contraty, extracellularTAC in co-culture was1.5-6.0times as much as that in mono-culture, intracellular TACwas2.9-7.9times as well. Furthermore, superoxide dismutase (SOD) activity inco-culture was22,14.6and10.2times as high as that in mono-culture, respectively.Which indicated superoxide anion scavenge capability was much stronger in co-culturecells, resulting with Fe-S cluster avoid of been attacted by superoxide anion. Theseresults suggested that B. megaterium scavenging the ROS generated during metabolismcomposed part of mechanisms in mutualism of B. megaterium and K. vulgare.5) To screen key genes on genome-scale, a high throughput screen method based on24-well microtiter plates (MTP) and transposon mutgenesis technology was developedand validated on an integrated library of450transposon random insertional mutants and two sporulation-defective mutants. The results indicated2-keto-L-gulonic acid (2-KLG)accumulation accompanied with brown water-soluble pigments generation. Theproposed high throughput method based on24-well MTP and OD450was validated bequalified to screen co-culture system effectively. A mutant coded as m71was screenedout from1087insertional mutants, the m71co-culture conversion ratio was decreasedby about38%compared with that of the wild-type co-culture process. The insertion sitelocates B. megaterium WSH-002chromosome site of3330114, where locates inside ofthe CDS encoding a alpha/beta fold family hydrolase. The high throughput methodprovides a bridge that links transposon insertional technology and interactionmechanism research on genomic scale.