Cloned gene encoding the delta subunit of Bacillus subtilis RNA polymerase

Lampe, Mary F.; Binnie, Craig; Schmidt, Ruth; Losick, Richard

doi:10.1016/0378-1119(88)90003-0

Cited by 29 publications

(24 citation statements)

References 20 publications

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“…S1 in the supplemental material), an additional RNAP subunit is present, termed the ␦ factor, or RpoE (2). In Bacillus subtilis, the 173-amino-acid delta subunit has been shown to reduce nonspecific binding of RNAP to DNA and lead to an elevated preference for DNA regions that include promoter sequences (3,4). In early experiments, it was shown that RpoE has a role specifically confined to promoter selection and influences the ability of RNAP to form open promoter complexes (5,6).…”

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confidence: 99%

The δ Subunit of RNA Polymerase Guides Promoter Selectivity and Virulence in Staphylococcus aureus

et al. 2014

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In Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the ␦ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureus and other species and demonstrated binding to core RNAP that is mediated by the ␤ and/or ␤= subunits. To identify the impact of the ␦ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage SA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the ␦ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection. Bacterial gene transcription is a complex, multifactorial process that involves several key enzymes and regulatory elements. It is driven by the activity of DNA-dependent RNA polymerase (RNAP) and its associated proteins, which form a multisubunit enzyme consisting of one ␤ subunit, one ␤= subunit, two identical ␣ subunits, and one subunit (reviewed in reference 1). Together these form the RNAP apoenzyme, which is able to perform RNA elongation and termination; however, initiation requires the involvement of a factor. Typically, most bacterial species harbor several different factors, a primary one ( A or 70 ) that mediates housekeeping gene transcription and a variety of alternative sigma factors, which aid in the response to unfavorable environmental conditions and stress.In certain Gram-positive species and particularly the Firmicutes (see Fig. S1 in the supplemental material), an additional RNAP subunit is present, termed the ␦ factor, or RpoE (2). In Bacillus subtilis, the 173-amino-acid delta subunit has been shown to reduce nonspecific binding of RNAP to DNA and lead to an elevated preference for DNA regions that include promoter sequences (3, 4). In early experiments, it was shown that RpoE has a role specifically confined to promoter selection and influences the ability of RNAP to form open promoter complexes (5, 6). Fulllength RpoE has also been described as displacing nucleic acids from RNAP-DNA or RNAP-RNA complexes in vitro, which may explain the decreased affinity of RNAP-RpoE for nonpromoter regions. Thi...

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The δ Subunit of RNA Polymerase Guides Promoter Selectivity and Virulence in Staphylococcus aureus

et al. 2014

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“…Initiation of transcription requires core RNAP and several different protein factors (18). The minimal core RNAP consists of four subunits (␤␤Ј␣ 2 ) and is sufficient to catalyze the polymerization of nucleoside triphosphates into RNA (16). Ancillary proteins modify RNAP during promoter binding, initiation, elongation, and termination steps to effect changes in gene expression (19).…”

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“…Core enzyme interacts with sigma factors that provide specificity, to form ␤␤Ј␣ 2 , which has the capacity to initiate transcription at promoters. Other proteins, including the delta protein encoded by rpoE, are frequently isolated as components of the purified RNAP, but their roles in transcription are less clear (2,16). On the basis of genome database sequence information, rpoE appears to be ubiquitous among gram-positive bacterial species.…”

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The Delta Subunit of RNA Polymerase Is Required for Virulence of Streptococcus agalactiae

2003

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Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia, and meningitis in the United States despite the chemoprophylaxis strategies for preventing infection recommended by the Centers for Disease Control and Prevention. Using signature-tagged transposon mutagenesis to screen for novel virulence factors, we identified the rpoE gene as essential for development of sepsis in a neonatal rat model of GBS infection. An rpoE allelic replacement mutant displayed attenuated virulence in the sepsis model of infection identical to that of the transposon mutant, confirming linkage of the phenotype to the mutation in rpoE. The rpoE mutants also displayed increased sensitivity to killing in whole-blood bactericidal assays, which may explain the attenuated virulence. The mutants were otherwise phenotypically identical to the wild-type strain, including growth rate in plasma, indicating that a growth defect is not responsible for the attenuated virulence. rpoE is found only in gram-positive bacterial species and encodes the delta peptide, a subunit of RNA polymerase. Previous in vitro studies in other bacteria suggest that the delta peptide plays a role in maintaining transcriptional fidelity by blocking RNA polymerase binding at all but the strongest promoters, thereby inhibiting initiation of transcription. Despite the availability of rpoE mutants for several gram-positive bacterial species, a role for the peptide in vivo has not been defined, though it has been postulated that the delta peptide may be important for long-term survival in vitro or during growth phase transitions. Our data represent the first report of a phenotype relevant to virulence for rpoE mutants.Streptococcus agalactiae (group B streptococci [GBS]) is the leading cause of neonatal sepsis, pneumonia, and meningitis in the United States and Western Europe and is an emerging pathogen in immunocompromised adults (31, 32). GBS associated with human disease are almost invariably encapsulated, belonging to one of the nine recognized capsular serotypes: Ia, Ib, or II to VIII. The type-specific capsule of GBS has been extensively studied and is an important determinant for both virulence and immunity (30,36). Other studies of GBS virulence have examined adherence to and invasion of epithelial and endothelial barriers and interactions with host immune pathways and have focused on a few specific factors, such as hemolysin, alpha and beta proteins, and C5a peptidase (for a review, see reference 24). Recognition of the prevalence and severity of human neonatal and adult disease emphasizes the significance of investigating the mechanisms important to the pathogenesis of GBS infections.We previously reported the use of signature-tagged transposon mutagenesis (STM) as a screening tool in a neonatal rat sepsis infection model to identify new virulence factors for GBS (13). The most attenuated mutant identified in the STM screen had a transposon insertion in the rpoE gene, encoding the delta subunit of RNA polymerase (...

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“…Each sub-genomic DNA library was transformed into a recA E. coli strain, DHSa, and spread directly onto Amersham nylon membranes. Preparation of filters with fixed colonies and colony blot hybridization of the filters were done essentially as described by Lampe et al (1988) with the modifications made by Bookstein et al (1990). Usually, 2000-3000 colonies were screened for each sub-genomic library, using lo7 c.p.m.…”

Section: Methodsmentioning

confidence: 99%

Bacillus licheniformis APase I gene promoter: a strong well-regulated promoter in B. subtilis

Lee

Edwards²,

Hulett³

1991

Journal of General Microbiology

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Cloned gene encoding the delta subunit of Bacillus subtilis RNA polymerase

Cited by 29 publications

References 20 publications

The δ Subunit of RNA Polymerase Guides Promoter Selectivity and Virulence in Staphylococcus aureus

The δ Subunit of RNA Polymerase Guides Promoter Selectivity and Virulence in Staphylococcus aureus

The Delta Subunit of RNA Polymerase Is Required for Virulence of Streptococcus agalactiae

Bacillus licheniformis APase I gene promoter: a strong well-regulated promoter in B. subtilis

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