Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis

Abstract

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (P bio ) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative P bio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminatorlike sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis.Biotin biosynthesis in Escherichia coli is regulated at the level of transcription by a classical repressor-operator mechanism (5,10,12). The repressor is encoded by the birA gene, and when complexed with its corepressor, biotinoyl-5Ј-AMP, BirA binds to an operator that overlaps the promoters for divergent bioA and bioBCDF operons and represses transcription from both promoters (reviewed in reference 10). Binding is cooperative and involves two holorepressor monomers binding to the two palindromic half sites of the operator (1). Transcription of the bio genes is controlled from this bidirectional promoteroperator sequence, and the detection of additional, internal promoters has not been reported. This regulatory model has been confirmed by several methods, including isolation of mutations in the operator region or the birA gene that deregulated biotin production (4,5,9,15,17,21), DNA binding studies of purified BirA to the E. coli bio operon, and DNA protection (5,11,23).In gram-positive bacteria, biotin synthesis has been studied extensively in two species, Bacillus sphaericus (14,16,22) and, more recently, Bacillus subtilis (8,6,7). In B. sphaericus, the genes are located in two separate operons, bioXWF and bioDAYB. Characterization of the regulatory apparatus controlling expression of these genes included the isolation of constitutive biotin-producing mutations that map either to a conserved 15-bp inverted repeat preceding the two operons (i.e., possible operator mutants) or to another site(s) on the chromosome unlinked to either operon (25). Recently, our research group has cloned and sequenced the biotin biosynthetic genes of B. subtilis (7,8). B. subtilis contains at least six bio genes that are organized in a single operon, bioWAFDB Iorf2; a seventh open reading frame (orf2) of unknown function is located at the end of the bio operon. Four of the genes, bioA, -B, -D, and -F, show strong similarity to genes of the same name from B. sphaericus and E. coli, and bioW shows strong similarity to bioW of B. sphaericus. The bioI gene encodes a cytochrome P-450-like enzyme that appears to be involved ...

RNA Expression Analysis Using an Antisense Bacillus subtilis Genome Array

“…Transcription of the ribC gene remained unchanged; transcripts of ribR, a ribC paralog (31), were not detected. Collectively, these results were in good agreement with past Northern blot and lacZ reporter gene fusion studies (1,26,29,44,45; K. Eichler, S. Taylor, C. Vockler, Y. Zhang, V. Delague, T. P. Begley, and A. P. G. M. van Loon, unpublished results).…”

“…Transcription of the biotin operon is tightly regulated by a repressor or operator mechanism involving biotin and the B. subtilis BirA-like repressor (3,4). Location of a rho-independent transcription terminator between the fifth and sixth genes results in the synthesis of two polycistronic transcripts: a fulllength transcript of 7.2 kb covering the entire operon (bio-WAFDBI-ytbQ) and a shorter 5.2-kb mRNA species that cover the first five genes (bioWAFDB) (26). Northern blots indicated that abundance of the shorter transcript is ϳ8-fold higher than the full-length transcript.…”

Defining the structure of the general stress regulon of Bacillus subtilis using targeted microarray analysis and random forest classification

“…For 20 of the 37 genes, additional regulation by secondary regulators has already been proven by previous studies. These 20 genes are: bioB, bmrR, purK, radA, yacK, yacM, ydaF, ykrT, yocL, ypuD, yraA, ysnF, ywhH, yxjG and the operon yceC-yceD-yceE-yceF-yceG-yceH (Au et al, 2005;Banse et al, 2008;Baranova et al, 1999;Bower et al, 1996; Cao et al, 2002;Chu et al, 2006;Chumsakul et al, 2011;Comella & Grossman, 2005;Derré et al, 1999;Drzewiecki et al, 1998;Ebbole & Zalkin, 1987;Eiamphungporn & Helmann, 2008;Erwin et al, 2005;Grundy & Henkin, 1998; Höper et al, 2005;Jervis et al, 2007;Kearns et al, 2005; Krüger et al, 1996;Kumaraswami et al, 2010;Leelakriangsak et al, 2007;Lei et al, 2009;Marvasi et al, 2010;Nakano et al, 2003;Nguyen et al, 2009;Perkins et al, 1996; Petersohn et al, 2001;Sekowska & Danchin, 2002;Wang et al, 2006;Weng et al, 1995; You et al, 2008). Details about their secondary regulators are included in Supplementary Table S4.…”

Engineering of a Bacillus subtilis Strain with Adjustable Levels of Intracellular Biotin for Secretory Production of Functional Streptavidin

“…In E. coli, release of intracellular biotin to the external environment has been demonstrated to be an energy-independent event, although the biotin uptake process is energy dependent (27). In B. subtilis, overproduction of biotin can result in an accumulation of biotin in the culture medium as high as 250 g/liter in certain engineered strains (25). Since WB800BIO(pSSAV-Tcry) cultivated in 0.2% gluconate grew even better than WB800(pSSAV-Tcry) given 40 M (9.76 mg/liter) biotin (Fig.…”