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As an ideal carotenoid producer, Blakeslea trispora has gained much attention due to its large biomass and high production of β-carotene and lycopene. However, carotenogenesis regulation in B. trispora still needs to be clarified, as few investigations have been conducted at the molecular level in B. trispora. In this study, a gene homologous to carotenogenesis regulatory gene (crgA) was cloned from the mating type (-) of B. trispora, and the deduced CrgA protein was analyzed for its primary structure and domains. To clarify the crgA-mediated regulation in B. trispora, we used the strategies of gene knockout and complementation to investigate the effect of crgA expression on the phenotype of B. trispora. In contrast to the wild-type strain, the crgA null mutant (ΔcrgA) was defective in sporulation but accumulated much more β-carotene (31.2% improvement at the end) accompanied by enhanced transcription of three structural genes (hmgR, carB, and carRA) for carotenoids throughout the culture time. When the wild-type copy of crgA was complemented into the crgA null mutant, sporulation, transcription of structural genes, and carotenoid production were restored to those of the wild-type strain. A gas chromatography-mass spectrometry (GC-MS)- based metabolomic approach and multivariate statistical analyses were performed to investigate the intracellular metabolite profiles. The reduced levels of tricarboxylic acid (TCA) cycle components and some amino acids and enhanced levels of glycolysis intermediates and fatty acids indicate that more metabolic flux was driven into the mevalonate (MVA) pathway; thus, the increase of precursors and fat content contributes to the accumulation of carotenoids. [ABSTRACT FROM AUTHOR]

The invasion and colonization of host plants by the destructive pathogen Ralstonia solanacearum rely on its cell motility, which is controlled by multiple factors. Here, we report that the LysR-type transcriptional regulator CrgA (RS_RS16695) represses cell motility in R. solanacearum GMI1000. CrgA possesses common features of a LysR-type transcriptional regulator and contains an N-terminal helix-turn-helix motif as well as a C-terminal LysR substrate-binding domain. Deletion of crgA results in an enhanced swim ring and increased transcription of flhDC In addition, the Δ crgA mutant possesses more polar flagella than wild-type GMI1000 and exhibits higher expression of the flagellin gene fliC Despite these alterations, the Δ crgA mutant did not have a detectable growth defect in culture. Yeast one-hybrid and electrophoretic mobility shift assays revealed that CrgA interacts directly with the flhDC promoter. Expressing the β-glucuronidase (GUS) reporter under the control of the crgA promoter showed that crgA transcription is dependent on cell density. Soil-soaking inoculation with the crgA mutant caused wilt symptoms on tomato ( Solanum lycopersicum L. cv. Hong yangli) plants earlier than inoculation with the wild-type GMI1000 but resulted in lower disease severity. We conclude that the R. solanacearum regulator CrgA represses flhDC expression and consequently affects the expression of fliC to modulate cell motility, thereby conditioning disease development in host plants. IMPORTANCE Ralstonia solanacearum is a widely distributed soilborne plant pathogen that causes bacterial wilt disease on diverse plant species. Motility is a critical virulence attribute of R. solanacearum , motility-defective strains are markedly affected in pathogenicity, which is coregulated with multiple virulence factors. In this study, we identified a new LysR-type transcriptional regulator (LTTR), CrgA, that negatively regulates motility. The mutation of the corresponding gene leads to the precocious appearance of wilt symptoms on tomato plants when the pathogen is introduced using soil-soaking inoculation. This study indicates that the regulation of R. solanacearum , motility-defective strains are markedly affected in pathogenicity, which is coregulated with multiple virulence factors. In this study, we identified a new LysR-type transcriptional regulator (LTTR), CrgA, that negatively regulates motility. The mutation of the corresponding gene leads to the precocious appearance of wilt symptoms on tomato plants when the pathogen is introduced using soil-soaking inoculation. This study indicates that the regulation of R. solanacearum motility is more complex than previously thought and enhances our understanding of flagellum regulation in R. solanacearum ., (Copyright © 2020 American Society for Microbiology.)

As an ideal carotenoid producer, Blakeslea trispora has gained much attention due to its large biomass and high production of β-carotene and lycopene. However, carotenogenesis regulation in B. trispora still needs to be clarified, as few investigations have been conducted at the molecular level in B. trispora In this study, a gene homologous to carotenogenesis regulatory gene ( crgA ) was cloned from the mating type (-) of B. trispora , and the deduced CrgA protein was analyzed for its primary structure and domains. To clarify the crgA -mediated regulation in B. trispora , we used the strategies of gene knockout and complementation to investigate the effect of crgA expression on the phenotype of B. trispora In contrast to the wild-type strain, the crgA null mutant (Δ crgA ) was defective in sporulation but accumulated much more β-carotene (31.2% improvement at the end) accompanied by enhanced transcription of three structural genes ( hmgR , carB , and carRA ) for carotenoids throughout the culture time. When the wild-type copy of crgA was complemented into the crgA null mutant, sporulation, transcription of structural genes, and carotenoid production were restored to those of the wild-type strain. A gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and multivariate statistical analyses were performed to investigate the intracellular metabolite profiles. The reduced levels of tricarboxylic acid (TCA) cycle components and some amino acids and enhanced levels of glycolysis intermediates and fatty acids indicate that more metabolic flux was driven into the mevalonate (MVA) pathway; thus, the increase of precursors and fat content contributes to the accumulation of carotenoids. IMPORTANCE The zygomycete Blakeslea trispora is an important strain for the production of carotenoids on a large scale. However, the regulation mechanism of carotenoid biosynthesis is still not well understood in this filamentous fungus. In the present study, we sought to investigate how crgA influences the expression of structural genes for carotenoids, carotenoid biosynthesis, and other anabolic phenotypes. This will lead to a better understanding of the global regulation mechanism of carotenoid biosynthesis and facilitate engineering this strain in the future for enhanced production of carotenoids., (Copyright © 2020 American Society for Microbiology.)