In vivo functional analysis of L-rhamnose metabolic pathway in Aspergillus niger: a tool to identify the potential inducer of RhaR
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ABSTRACT: The genes of the non-phosphorylative L-rhamnose catabolic pathway have been identified for several yeast species. In Pichia stipitis, all L-rhamnose pathway genes are organized in a cluster, which is conserved in Aspergillus niger, except for the lra-4 ortholog (lraD). The A. niger cluster also contains the gene encoding the L-rhamnose responsive transcription factor (RhaR) that has been shown to control the expression of genes involved in L-rhamnose release and catabolism. In this paper, we confirmed the function of the first three putative L-rhamnose utilisation genes from A. niger through gene deletion. We explored the identity of the inducer of the pathway regulator (RhaR) through expression analysis of the deletion mutants grown in transfer experiments to L-rhamnose and L-rhamnonate. Reduced expression of L-rhamnose-induced genes on L-rhamnose in lraA and lraB deletion strains, but not on L-rhamnonate (the product of LraB), demonstrate that the inducer of the pathway is of L-rhamnonate or a compound downstream of it. Reduced expression of these genes in the lraC deletion strain on L-rhamnonate show that it is in fact a downstream product of L-rhamnonate. This work showed that the inducer of RhaR is beyond L-rhamnonate dehydratase (LraC) and is likely to be the 2-keto-3-L-deoxyrhamnonate.
Project description:In Scheffersomyces stipitis and related fungal species the genes for L-rhamnose catabolism RHA1, LRA2, LRA3 and LRA4 but not LADH are clustered. We find that located next to the cluster is a transcription factor, TRC1, which is conserved among related species.Our transcriptome analysis shows that all the catabolic genes and all genes of the cluster are up-regulated on L-rhamnose. Among the genes that were also up-regulated on L-rhamnose were two transcription factors including the TRC1. In addition, in 16 out of the 32 analysed fungal species only RHA1, LRA2 and LRA3 are in a cluster. The clustering of RHA1, LRA3 and TRC1 is also conserved in species not closely related to S. stipitis. Since the LRA4 is often not part of the cluster and it has several paralogs in L-rhamnose utilising yeasts we analysed the function of one of the paralogs, RHA41 by heterologous expression and biochemical characterization. Rha41p has similar catalytic properties but the transcript was not up-regulated on L-rhamnose. The RHA1, LRA2, LRA4 and LADH genes were previously characterized in Sheffersomyces (Pichia) stipitis. We expressed the L-rhamnonate dehydratase, Rha3p, in S. cerevisiae, estimated the kinetic constants of the protein and showed that it indeed has activity with L-rhamnonate.
Project description:In Scheffersomyces stipitis and related fungal species the genes for L-rhamnose catabolism RHA1, LRA2, LRA3 and LRA4 but not LADH are clustered. We find that located next to the cluster is a transcription factor, TRC1, which is conserved among related species.Our transcriptome analysis shows that all the catabolic genes and all genes of the cluster are up-regulated on L-rhamnose. Among the genes that were also up-regulated on L-rhamnose were two transcription factors including the TRC1. In addition, in 16 out of the 32 analysed fungal species only RHA1, LRA2 and LRA3 are in a cluster. The clustering of RHA1, LRA3 and TRC1 is also conserved in species not closely related to S. stipitis. Since the LRA4 is often not part of the cluster and it has several paralogs in L-rhamnose utilising yeasts we analysed the function of one of the paralogs, RHA41 by heterologous expression and biochemical characterization. Rha41p has similar catalytic properties but the transcript was not up-regulated on L-rhamnose. The RHA1, LRA2, LRA4 and LADH genes were previously characterized in Sheffersomyces (Pichia) stipitis. We expressed the L-rhamnonate dehydratase, Rha3p, in S. cerevisiae, estimated the kinetic constants of the protein and showed that it indeed has activity with L-rhamnonate. A six chip study using total RNA recovered from three separate cultures of S. stipitis CBS 6054 grown glucose and respectively three separate cultures grown on rhamnose
Project description:The genome of the filamentous fungus Aspergillus niger is rich in genes encoding pectinases, a broad class of enzymes that have been extensively studied due to their use in industrial applications. The sequencing of the A. niger genome provided more knowledge concerning the individual pectinolytic genes, but relatively little is still known about the regulatory genes involved in pectin degradation. Understanding regulation of the pectinolytic genes provides a tool to optimize the production of pectinases in this industrially important fungus. This study describes the identification and characterization of one of the activators of pectinase-encoding genes, RhaR. Inactivation of the gene encoding this regulator resulted in down-regulation of genes involved in the release and catabolism of L-rhamnose from the pectinolytic substructure rhamnogalacturonan I. We aim to discover differencial expressed genes in A.niger wild type strain N402 and M-NM-^TrhaR mutant while growing on rhamnose as carbon source. Biological duplicates were made for both strain at the growth of 2 hours, Affymetrix microarray experiments were performed on these samples.
Project description:The genome of the filamentous fungus Aspergillus niger is rich in genes encoding pectinases, a broad class of enzymes that have been extensively studied due to their use in industrial applications. The sequencing of the A. niger genome provided more knowledge concerning the individual pectinolytic genes, but relatively little is still known about the regulatory genes involved in pectin degradation. Understanding regulation of the pectinolytic genes provides a tool to optimize the production of pectinases in this industrially important fungus. This study describes the identification and characterization of one of the activators of pectinase-encoding genes, RhaR. Inactivation of the gene encoding this regulator resulted in down-regulation of genes involved in the release and catabolism of L-rhamnose from the pectinolytic substructure rhamnogalacturonan I.
Project description:Among the three major genetic lineages of L. monocytogenes (i.e. LI, LII, and LIII), LI and LII are predominantly associated with foodborne listeriosis outbreaks, whereas LIII is rarely implicated in human infections. In a previous study, we identified a Crp/Fnr family transcription factor lmo0753 that was highly specific to outbreak-associated LI and LII but absent from LIII. Lmo0753 shares two conserved functional domains including a DNA-binding domain with the well-characterized master virulence regulator PrfA in L. monocytogenes. In this study, we constructed a lmo0753 deletion and complementation mutants of the fully sequenced L. monocytogenes LII strain EGDe. We found that deletion of lmo0753 led to the loss of L-rhamnose utilization in EGDe. Transcriptomic comparison of the EGDe lmo0753 deletion mutant and the wild type incubated in phenol-red medium containing L-rhamnose as the sole carbon source revealed 126 (4.5%) and 546 (19.5%) out of 2,798 genes in the EGDe genome that were up- and down-regulated for more than 2-fold, respectively. Genes involved in biotin biosynthesis, general stress response and rhamnose metabolism were shown to be differentially regulated by Lmo0753. Findings from this study may partially explain why LIII of L. monocytogenes is underrepresented in the environment and rarely associated with human listeriosis outbreaks due to the inability of rhamnose utilization. We report the transcriptomic profile of L. monocytogenes M-NM-^Tlmo0753 LII strain (EGDe) in broth media with L-rhamnose as the sole carbon source. Examination of deletion of Lmo0753 on L-rhamnose utilization in L. monocytogenes. Two biological replicates per WT and M-NM-^Tlmo0753.
Project description:Among the three major genetic lineages of L. monocytogenes (i.e. LI, LII, and LIII), LI and LII are predominantly associated with foodborne listeriosis outbreaks, whereas LIII is rarely implicated in human infections. In a previous study, we identified a Crp/Fnr family transcription factor lmo0753 that was highly specific to outbreak-associated LI and LII but absent from LIII. Lmo0753 shares two conserved functional domains including a DNA-binding domain with the well-characterized master virulence regulator PrfA in L. monocytogenes. In this study, we constructed a lmo0753 deletion and complementation mutants of the fully sequenced L. monocytogenes LII strain EGDe. We found that deletion of lmo0753 led to the loss of L-rhamnose utilization in EGDe. Transcriptomic comparison of the EGDe lmo0753 deletion mutant and the wild type incubated in phenol-red medium containing L-rhamnose as the sole carbon source revealed 126 (4.5%) and 546 (19.5%) out of 2,798 genes in the EGDe genome that were up- and down-regulated for more than 2-fold, respectively. Genes involved in biotin biosynthesis, general stress response and rhamnose metabolism were shown to be differentially regulated by Lmo0753. Findings from this study may partially explain why LIII of L. monocytogenes is underrepresented in the environment and rarely associated with human listeriosis outbreaks due to the inability of rhamnose utilization. We report the transcriptomic profile of L. monocytogenes Δlmo0753 LII strain (EGDe) in broth media with L-rhamnose as the sole carbon source.
Project description:L-rhamnose, a naturally abundant sugar, plays diverse biological roles in bacteria, influencing biofilm formation and pathogenesis. This study investigates the global impact of L-rhamnose on the transcriptome and biofilm formation of PHL628 E. coli under various experimental conditions. We compared growth in planktonic and biofilm states in rich (LB) and minimal (M9) media at 28 °C and 37 °C, with varying concentrations of L-rhamnose or D-glucose as a control. Our results reveal that L-rhamnose significantly affects growth kinetics and biofilm formation, particularly reducing biofilm growth in rich media at 37 °C. Transcriptomic analysis through RNA-seq showed that L-rhamnose modulates gene expression differently depending on the temperature and media conditions, promoting a planktonic state by upregulating genes involved in rhamnose transport and metabolism and downregulating genes related to adhesion and biofilm formation. These findings highlight the nuanced role of L-rhamnose in bacterial adaptation and survival, providing insights for potential applications in controlling biofilm-associated infections and industrial biofilm management.
Project description:This experiment aim was to characterize the catabolism of L-rhamnose of Clostridium beijerinckii DSM 6423 by transcriptomic analysis, generating new insights and knowledge on utilization of L-rhamnose for production of chemicals, including Isopropanol, Butanol, Ethanol (IBE) and 1,2-propandiol. These analysis on cultures grown on L-rhamnose compared to D-glucose grown cultures showed upregulation of the L-rhamnose-related clusters and genes, and lower expression of the solventogenic genes, which was reflected in the products formed.
Project description:In Aspergillus niger, the enzymes encoded by gaaA, gaaB, gaaC and gaaD catabolize D-galacturonic acid (GA) consecutively into L-galactonate, 2-keto-3-deoxy-L-galactonate, pyruvate and L-glyceraldehyde, and glycerol. We show here that deletion of gaaB or gaaC results in severely impaired growth on GA and accumulation of pathway intermediates L-galactonate and 2-keto-3-deoxy-L-galactonate, respectively. Expression levels of several GA-induced genes were specifically elevated in the ∆gaaC mutant on GA as compared to the reference strain or other GA catabolic pathway deletion mutants. The hyper-induction of GA-induced genes in ∆gaaC indicates that 2-keto-3-deoxy-L-galactonate is the inducer of genes required for GA utilization.
Project description:Burkholderia cenocepacia small RNA ncS27 was identified by transcriptome profiling. To investigate the function of this small RNA, ncS27 was overexpressed and silenced in trans and the effect of this on the transcriptome was studied. To overexpress ncS27 it was cloned into a vector with a rhamnose inducible promoter. To silence ncS27 it was cloned into the same vector, but in reverse orientation. The vectors were then transformed into B. cenocepacia J2315, creating two mutants. The third mutant in this experiment is a vector control mutant. All three mutants were cultivated in Lennox broth to OD 0.5, then the inducer rhamnose was added. After 30 min of incubation in presence of the inducer the cells were harvested, RNA was extracted and RNA-sequencing performed.