A new function for the Bacillus PbuE purine base efflux pump: efflux of purine nucleosides

Zakataeva, Natalia P.; Gronskiy, Sergey V.; Sheremet, Anastasia S.; Kutukova, Ekaterina A.; Novikova, Anna E.; Livshits, Vitaliy A.

doi:10.1016/j.resmic.2007.08.003

Cited by 21 publications

(13 citation statements)

References 18 publications

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“…In one study, the pbuE gene, including its leader sequence, from Bacillus amyloliquefaciens was cloned into E. coli and shown to be induced by the addition of adenine to the medium or by the disruption of the terminator structure by a point mutation (20). This behavior was in agreement with observations of the pbuE riboswitch (previously annotated as ydhL) in its native context (2).…”

Section: Validation Of Function Of An Adenine-responsivesupporting

confidence: 66%

Structure-guided Mutational Analysis of Gene Regulation by the Bacillus subtilis pbuE Adenine-responsive Riboswitch in a Cellular Context

Marcano-Velázquez

Batey

2015

Journal of Biological Chemistry

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Background: Riboswitches regulate purine biosynthesis and transport in many bacteria. Results: Mutagenic analysis of an adenine-responsive riboswitch revealed features important for efficient co-transcriptional regulation. Conclusion: Adenine binding to the riboswitch results in a local barrier to strand exchange by the transcriptional terminator, whose formation is facilitated by a stem-loop element. Significance: This is the first study comprehensively analyzing an RNA structural switch in a cellular context.Riboswitches are a broadly distributed form of RNA-based gene regulation in Bacteria and, more rarely, Archaea and Eukarya. Most often found in the 5-leader sequence of bacterial mRNAs, they are generally composed of two functional domains: a receptor (aptamer) domain that binds an effector molecule and a regulatory domain (or expression platform) that instructs the expression machinery. One of the most studied riboswitches is the Bacillus subtilis adenine-responsive pbuE riboswitch, which regulates gene expression at the transcriptional level, up-regulating expression in response to increased intracellular effector concentrations. In this work, we analyzed sequence and structural elements that contribute to efficient ligand-dependent regulatory activity in a co-transcriptional and cellular context. Unexpectedly, we found that the P1 helix, which acts as the antitermination element of the switch in this RNA, supported ligand-dependent activation of a reporter gene over a broad spectrum of lengths from 3 to 10 bp. This same trend was also observed using a minimal in vitro single-turnover transcription assay, revealing that this behavior is intrinsic to the RNA sequence. We also found that the sequences at the distal tip of the terminator not directly involved in alternative secondary structure formation are highly important for efficient regulation. These data strongly support a model in which the switch is highly localized to the P1 helix adjacent to the ligandbinding pocket that likely presents a local kinetic block to invasion of the aptamer by the terminator.Since their discovery and structural characterization, purineresponsive riboswitches have emerged as an important model system for exploring small molecule-RNA interactions, RNA structure, in vitro and in vivo folding, and conformational dynamics (reviewed in Ref. 1). Purine riboswitches present themselves as an ideal model system to explore various aspects of RNA chemistry and biology because of their small size and simple architecture of the ligand-binding domain, very well behaved folding properties, and multiple activities (small-molecule recognition and regulation of gene expression). However, the majority of studies of riboswitches have focused solely upon the ligand-binding (aptamer) domain in isolation using in vitro biochemical, biophysical and structural approaches. Thus, the wealth of information about the structure and function of the aptamer domain is poorly linked to an understanding of regulatory activity, as is the case for most clas...

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Section: Validation Of Function Of An Adenine-responsivesupporting

confidence: 66%

Structure-guided Mutational Analysis of Gene Regulation by the Bacillus subtilis pbuE Adenine-responsive Riboswitch in a Cellular Context

Marcano-Velázquez

Batey

2015

Journal of Biological Chemistry

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“…AJ1991 is able to produce simultaneously about 3 g/l inosine and 2 g/l guanosine due to mutations useful for de novo inosine and guanosine oversynthesis, namely adenine auxotrophy and a mutation conferring resistance to the 8-azaguanine purine analog [21].…”

Section: Resultsmentioning

confidence: 99%

“…To deliver the pbuE T!C mutation, plasmid pNZT1-pbuEm was constructed as follows. First, the pYDHL2 plasmid containing the B. amyloliquefaciens pbuE gene [21] was cut with PvuII, and the DNA fragment containing 0.4-kb pbuE upstream region and 0.1 kb of the gene coding sequence was ligated to Eco32I-digested pNZT1, giving pNZT1-pbuE. To generate the pbuE T!C mutation, site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis method (Stratagene) and Pfu DNA polymerase (Fermentas) with the Z3/Z4 primer pair and pNZT1-pbuE as a template, yielding pNZT1-pbuEm.…”

Section: Construction Of Plasmids Containing Gene Replacement Constructsmentioning

confidence: 99%

“…It was found that Escherichia coli NepI (YicM) and its homologue from Bacillus, PbuE (YdhL), which was previously described as a purine base exporter, are involved in export of purine nucleosides [7,9,16,21]. A low-copy plasmid containing the B. amyloliquefaciens pbuE gene was shown to increase the production of extracellular purine nucleosides by E. coli and B. amyloliquefaciens strains [21].…”

Section: Introductionmentioning

confidence: 99%

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Enhancement of extracellular purine nucleoside accumulation by Bacillus strains through genetic modifications of genes involved in nucleoside export

Sheremet

Gronskiy

Ахмадышин

et al. 2010

J Ind Microbiol Biotechnol

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Using a simple method to introduce genetic modifications into the chromosome of naturally nontransformable Bacillus, a set of marker-free inosine-producing and 5-aminoimidazole-4-carboxamide (AICA) ribonucleoside-producing Bacillus amyloliquefaciens strains has been constructed. These strains differ in expression levels of the genes responsible for nucleoside export. Overexpression of B. amyloliquefaciens pbuE and heterologous expression of Escherichia coli nepI, which encode nucleoside efflux transporters, each notably enhanced inosine production by a B. amyloliquefaciens nucleoside-producing strain. pbuE overexpression was found to increase AICA ribonucleoside accumulation, indicating that the substrate specificity of the PbuE pump extends to this nucleoside. These results demonstrate that identifying genes whose products facilitate transport of a desired nucleoside out of cells and enhancing their expression can improve the performance of strains used for industrial production.

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“…Bacillus amyloliquefaciens has been widely used as an industrial producer of primary metabolites, including vitamins and purine nucleosides such as inosine and guanosine (14,17). Purine nucleosides are important intermediates in the food and pharmaceutical industries, where they can be used to synthesize chicken essence (16) and nucleoside antiviral drugs (6,12) such as ribavirin and acyclovir.…”

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confidence: 99%

Complete Genome Sequence of Bacillus amyloliquefaciens XH7, Which Exhibits Production of Purine Nucleosides

et al. 2011

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Here, we report the complete annotated genome sequence of Bacillus amyloliquefaciens XH7, which is used to produce purine nucleosides in industry. The genome sequence will allow for the characterization of the molecular mechanisms underlying its beneficial properties.Bacillus amyloliquefaciens has been widely used as an industrial producer of primary metabolites, including vitamins and purine nucleosides such as inosine and guanosine (14,17). Purine nucleosides are important intermediates in the food and pharmaceutical industries, where they can be used to synthesize chicken essence (16) and nucleoside antiviral drugs (6, 12) such as ribavirin and acyclovir. Bacillus amyloliquefaciens XH7 is a high-yield purine nucleoside guanosine producer by traditional mutation breeding and has potential for industrial production of purine nucleoside inosine after deletion of the guaB gene. In order to further strain improvement, we report here the complete genome sequence of XH7.The genome was sequenced using the Illumina Solexa GA instrument at Beijing Genomics Institute (BGI) (Shenzhen, China). A library containing 500-bp inserts was constructed. Sequencing was performed with the pair-end strategy of 75-bp reads to produce 629 Mb of filtered sequences, representing a 160-fold coverage of the genome. The sequences were assembled into 76 contigs and 23 scaffolds using the SOAPdenovo package (9).The location and orientation of each scaffold were determined by mapping to the finished reference sequence of B. amyloliquefaciens DSM7 (13) using Mummer 3 (7). Gaps were closed by custom primer walks and long-distance PCR amplification followed by DNA sequencing with an ABI 3730 sequencer. Open reading frames (ORF) were identified by Glimmer 3.02 (4). The resulting translations were used for a BLASTP (1) search against the GenBank NR database, as well as the KEGG (5) and COG (15) databases. tRNA and rRNA genes were identified by tRNAscan-SE (10) and RNAmmer (8), respectively.The complete genomic information of B. amyloliquefaciens XH7 is contained on a single circular chromosome of 3,939,203 bp with an average GC content of 45.82%. A total of 4,204 protein coding genes, 75 tRNA genes, and 7 rrn operons were identified. A total of 25 pseudogenes were also identified, which suggested that B. amyloliquefaciens XH7 was selected by mutagenesis treatment.In performing a comparative analysis of B. amyloliquefaciens XH7 with reference genomes, those of B. amyloliquefaciens strains FZB42 (3) and DSM7 (13), we found some important mutations which may explain the molecular mechanisms for the production of purine nucleosides. Adenylosuccinate synthase gene purA, which blocked the pathway of IMP to AMP, was truncated. The nonsense mutation in GMP reductase gene guaC blocked the pathway of GMP to IMP. Comparative analysis of B. amyloliquefaciens XH7 with the reference genome of Escherichia coli K-12 (2) revealed that the homolog of E. coli purine nucleoside phosphorylase gene gsk was not found in XH7. This feature could make B. amyloliquefaciens ...

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A new function for the Bacillus PbuE purine base efflux pump: efflux of purine nucleosides

Cited by 21 publications

References 18 publications

Structure-guided Mutational Analysis of Gene Regulation by the Bacillus subtilis pbuE Adenine-responsive Riboswitch in a Cellular Context

Structure-guided Mutational Analysis of Gene Regulation by the Bacillus subtilis pbuE Adenine-responsive Riboswitch in a Cellular Context

Enhancement of extracellular purine nucleoside accumulation by Bacillus strains through genetic modifications of genes involved in nucleoside export

Complete Genome Sequence of Bacillus amyloliquefaciens XH7, Which Exhibits Production of Purine Nucleosides

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