When faced with increased osmolarity in the environment, many bacterial cells accumulate the comp... more When faced with increased osmolarity in the environment, many bacterial cells accumulate the compatible solute ectoine and its derivative 5-hydroxyectoine. Both compounds are not only potent osmostress protectants, but also serve as effective chemical chaperones stabilizing protein functionality. Ectoines are energy-rich nitrogen and carbon sources that have an ecological impact that shapes microbial communities. Although the biochemistry of ectoine and 5-hydroxyectoine biosynthesis is well understood, our understanding of their catabolism is only rudimentary. Here, we combined biochemical and structural approaches to unravel the core of ectoine and 5-hydroxyectoine catabolisms. We show that a conserved enzyme bimodule consisting of the EutD ectoine/5-hydroxyectoine hydrolase and the EutE deacetylase degrades both ectoines. We determined the high-resolution crystal structures of both enzymes, derived from the salt-tolerant bacteria Ruegeria pomeroyi and Halomonas elongata. These structures, either in their apo-forms or in forms capturing substrates or intermediates, provided detailed insights into the catalytic cores of the EutD and EutE enzymes. The combined biochemical and structural results indicate that the EutD homodimer opens the pyrimidine ring of ectoine through an unusual covalent intermediate, N-α-2 acetyl-L-2,4-diaminobutyrate (α-ADABA). We found that α-ADABA is then deacetylated by the zinc-dependent EutE monomer into diaminobutyric acid (DABA), which is further catabolized to L-aspartate. We observed that the EutD–EutE bimodule synthesizes exclusively the α-, but not the γ-isomers of ADABA or hydroxy-ADABA. Of note, α-ADABA is known to induce the MocR/GabR-type repressor EnuR, which controls the expression of many ectoine catabolic genes clusters. We conclude that hydroxy-α-ADABA might serve a similar function.
Biofilms can be viewed as tissue‐like structures in which microorganisms are organized in a spati... more Biofilms can be viewed as tissue‐like structures in which microorganisms are organized in a spatial and functional sophisticated manner. Biofilm formation requires the orchestration of a highly integrated network of regulatory proteins to establish cell differentiation and production of a complex extracellular matrix. Here, we discuss the role of the essential Bacillus subtilis biofilm activator RemA. Despite intense research on biofilms, RemA is a largely underappreciated regulatory protein. RemA forms donut‐shaped octamers with the potential to assemble into dimeric superstructures. The presumed DNA‐binding mode suggests that RemA organizes its target DNA into nucleosome‐like structures, which are the basis for its role as transcriptional activator. We discuss how RemA affects gene expression in the context of biofilm formation, and its regulatory interplay with established components of the biofilm regulatory network, such as SinR, SinI, SlrR, and SlrA. We emphasize the additional role of RemA played in nitrogen metabolism and osmotic‐stress adjustment.
Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrheas up to ... more Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrheas up to a toxic megacolon and are currently among the major causes of lethal bacterial infections. Successful bacterial propagation in the gut is strongly associated with the adaptation to changing nutrition-caused environmental conditions; e.g. environmental salt stresses. Concentrations of 350 mM NaCl, the prevailing salinity in the colon, led to significantly reduced growth of C. difficile. Metabolomics of salt- stressed bacteria revealed a major reduction of the central energy generation pathways, including the Stickland-fermentation reactions. No obvious synthesis of compatible solutes was observed up to 24 h of growth. The ensuing limited tolerance to high salinity and absence of compatible solute synthesis might result from an evolutionary adaptation to the exclusive life of C. difficile in the mammalian gut. Addition of the compatible solutes carnitine, glycine-betaine, γ-butyrobetaine, crotonobetaine, homobetaine, proline-betaine and dimethylsulfoniopropionate (DMSP) restored growth (choline and proline failed) under conditions of high salinity. A bioinformatically-identified OpuF-type ABC-transporter imported most of the used compatible solutes. A long-term adaptation after 48 h included a shift of the Stickland fermentation-based energy metabolism from the utilization to the accumulation of L-proline and resulted in restored growth. Surprisingly, salt stress resulted in the formation of coccoid C. difficile cells instead of the typical rod-shaped cells, a process reverted by the addition of several compatible solutes. Hence, compatible solute import via OpuF is the major immediate adaptation strategy of C. difficile to high salinity-incurred cellular stress. This article is protected by copyright. All rights reserved.
SummaryThe ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ‐ProA‐ProH... more SummaryThe ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ‐ProA‐ProH), osmostress‐adaptive synthesis of the compatible solute proline. The proA‐encoded gamma‐glutamyl phosphate reductase is also used for anabolic proline synthesis (ProB‐ProA‐ProI). Transcription of the proHJ operon is osmotically inducible whereas that of the proBA operon is not. Targeted and quantitative proteome analysis revealed that the amount of ProA is not limiting for the interconnected anabolic and osmostress‐responsive proline production routes. A key player for enhanced osmostress‐adaptive proline production is the osmotically regulated proHJ promoter. We used site‐directed mutagenesis to study the salient features of this stress‐responsive promoter. Two important features were identified: (i) deviations of the proHJ promoter from the consensus sequence of SigA‐type promoters serve to keep transcription low under non‐inducing growth conditions, while still allowing a finely tuned induction of transcriptional activity when the external osmolarity is increased and (ii) a suboptimal spacer length for SigA‐type promoters of either 16‐bp (the natural proHJ promoter), or 18‐bp (a synthetic promoter variant) is strictly required to allow regulation of promoter activity in proportion to the external salinity. Collectively, our data suggest that changes in the local DNA structure at the proHJ promoter are important determinants for osmostress‐inducibility of transcription.
When faced with increased osmolarity in the environment, many bacterial cells accumulate the comp... more When faced with increased osmolarity in the environment, many bacterial cells accumulate the compatible solute ectoine and its derivative 5-hydroxyectoine. Both compounds are not only potent osmostress protectants, but also serve as effective chemical chaperones stabilizing protein functionality. Ectoines are energy-rich nitrogen and carbon sources that have an ecological impact that shapes microbial communities. Although the biochemistry of ectoine and 5-hydroxyectoine biosynthesis is well understood, our understanding of their catabolism is only rudimentary. Here, we combined biochemical and structural approaches to unravel the core of ectoine and 5-hydroxyectoine catabolisms. We show that a conserved enzyme bimodule consisting of the EutD ectoine/5-hydroxyectoine hydrolase and the EutE deacetylase degrades both ectoines. We determined the high-resolution crystal structures of both enzymes, derived from the salt-tolerant bacteria Ruegeria pomeroyi and Halomonas elongata. These structures, either in their apo-forms or in forms capturing substrates or intermediates, provided detailed insights into the catalytic cores of the EutD and EutE enzymes. The combined biochemical and structural results indicate that the EutD homodimer opens the pyrimidine ring of ectoine through an unusual covalent intermediate, N-α-2 acetyl-L-2,4-diaminobutyrate (α-ADABA). We found that α-ADABA is then deacetylated by the zinc-dependent EutE monomer into diaminobutyric acid (DABA), which is further catabolized to L-aspartate. We observed that the EutD–EutE bimodule synthesizes exclusively the α-, but not the γ-isomers of ADABA or hydroxy-ADABA. Of note, α-ADABA is known to induce the MocR/GabR-type repressor EnuR, which controls the expression of many ectoine catabolic genes clusters. We conclude that hydroxy-α-ADABA might serve a similar function.
Biofilms can be viewed as tissue‐like structures in which microorganisms are organized in a spati... more Biofilms can be viewed as tissue‐like structures in which microorganisms are organized in a spatial and functional sophisticated manner. Biofilm formation requires the orchestration of a highly integrated network of regulatory proteins to establish cell differentiation and production of a complex extracellular matrix. Here, we discuss the role of the essential Bacillus subtilis biofilm activator RemA. Despite intense research on biofilms, RemA is a largely underappreciated regulatory protein. RemA forms donut‐shaped octamers with the potential to assemble into dimeric superstructures. The presumed DNA‐binding mode suggests that RemA organizes its target DNA into nucleosome‐like structures, which are the basis for its role as transcriptional activator. We discuss how RemA affects gene expression in the context of biofilm formation, and its regulatory interplay with established components of the biofilm regulatory network, such as SinR, SinI, SlrR, and SlrA. We emphasize the additional role of RemA played in nitrogen metabolism and osmotic‐stress adjustment.
Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrheas up to ... more Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrheas up to a toxic megacolon and are currently among the major causes of lethal bacterial infections. Successful bacterial propagation in the gut is strongly associated with the adaptation to changing nutrition-caused environmental conditions; e.g. environmental salt stresses. Concentrations of 350 mM NaCl, the prevailing salinity in the colon, led to significantly reduced growth of C. difficile. Metabolomics of salt- stressed bacteria revealed a major reduction of the central energy generation pathways, including the Stickland-fermentation reactions. No obvious synthesis of compatible solutes was observed up to 24 h of growth. The ensuing limited tolerance to high salinity and absence of compatible solute synthesis might result from an evolutionary adaptation to the exclusive life of C. difficile in the mammalian gut. Addition of the compatible solutes carnitine, glycine-betaine, γ-butyrobetaine, crotonobetaine, homobetaine, proline-betaine and dimethylsulfoniopropionate (DMSP) restored growth (choline and proline failed) under conditions of high salinity. A bioinformatically-identified OpuF-type ABC-transporter imported most of the used compatible solutes. A long-term adaptation after 48 h included a shift of the Stickland fermentation-based energy metabolism from the utilization to the accumulation of L-proline and resulted in restored growth. Surprisingly, salt stress resulted in the formation of coccoid C. difficile cells instead of the typical rod-shaped cells, a process reverted by the addition of several compatible solutes. Hence, compatible solute import via OpuF is the major immediate adaptation strategy of C. difficile to high salinity-incurred cellular stress. This article is protected by copyright. All rights reserved.
SummaryThe ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ‐ProA‐ProH... more SummaryThe ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ‐ProA‐ProH), osmostress‐adaptive synthesis of the compatible solute proline. The proA‐encoded gamma‐glutamyl phosphate reductase is also used for anabolic proline synthesis (ProB‐ProA‐ProI). Transcription of the proHJ operon is osmotically inducible whereas that of the proBA operon is not. Targeted and quantitative proteome analysis revealed that the amount of ProA is not limiting for the interconnected anabolic and osmostress‐responsive proline production routes. A key player for enhanced osmostress‐adaptive proline production is the osmotically regulated proHJ promoter. We used site‐directed mutagenesis to study the salient features of this stress‐responsive promoter. Two important features were identified: (i) deviations of the proHJ promoter from the consensus sequence of SigA‐type promoters serve to keep transcription low under non‐inducing growth conditions, while still allowing a finely tuned induction of transcriptional activity when the external osmolarity is increased and (ii) a suboptimal spacer length for SigA‐type promoters of either 16‐bp (the natural proHJ promoter), or 18‐bp (a synthetic promoter variant) is strictly required to allow regulation of promoter activity in proportion to the external salinity. Collectively, our data suggest that changes in the local DNA structure at the proHJ promoter are important determinants for osmostress‐inducibility of transcription.
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