Transcriptome dynamics-based operon prediction and verification in Streptomyces coelicolor

Charaniya, Salim; Mehra, Sarika; Lian, Wei; Jayapal, Karthik P.; Karypis, George; Hu, Wei‐Shou

doi:10.1093/nar/gkm501

Cited by 32 publications

(40 citation statements)

References 59 publications

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“…The cotranscription of the cps genes in D39 was determined by intergenic RT-PCR essentially as described previously (51). The cDNA molecules (cDNAs) of D39 were prepared using total RNA preparation (described above) with an iScript cDNA synthesis kit (Bio-Rad, Beijing, China) as instructed by the supplier.…”

Section: Methodsmentioning

confidence: 99%

Sequence Elements Upstream of the Core Promoter Are Necessary for Full Transcription of the Capsule Gene Operon in Streptococcus pneumoniae Strain D39

et al. 2015

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e Streptococcus pneumoniae is a major bacterial pathogen in humans. Its polysaccharide capsule is a key virulence factor that promotes bacterial evasion of human phagocytic killing. While S. pneumoniae produces at least 94 antigenically different types of capsule, the genes for biosynthesis of almost all capsular types are arranged in the same locus. The transcription of the capsular polysaccharide (cps) locus is not well understood. This study determined the transcriptional features of the cps locus in the type 2 virulent strain D39. The initial analysis revealed that the cps genes are cotranscribed from a major transcription start site at the ؊25 nucleotide (G) upstream of cps2A, the first gene in the locus. Using unmarked chromosomal truncations and a luciferasebased transcriptional reporter, we showed that the full transcription of the cps genes not only depends on the core promoter immediately upstream of cps2A, but also requires additional elements upstream of the core promoter, particularly a 59-bp sequence immediately upstream of the core promoter. Unmarked deletions of these promoter elements in the D39 genome also led to significant reduction in CPS production and virulence in mice. Lastly, common cps gene (cps2ABCD) mutants did not show significant abnormality in cps transcription, although they produced significantly less CPS, indicating that the CpsABCD proteins are involved in the encapsulation of S. pneumoniae in a posttranscriptional manner. This study has yielded important information on the transcriptional characteristics of the cps locus in S. pneumoniae. Streptococcus pneumoniae (pneumococcus) is a major cause of bacterial infections in humans, including otitis media, pneumonia, bacteremia, and meningitis (1). As the outermost structure, the capsule is the major virulence factor of S. pneumoniae and protects the bacterium by interfering with host phagocytic killing (2). Virtually all of the clinical isolates are encapsulated, and mutations in the cps locus result in the loss of virulence (3). The capsule of S. pneumoniae is composed of capsular polysaccharides (CPSs). The CPSs are immunogenic and thus serve as the target antigens for the current pneumococcal vaccines (4). Due to host immune selection that targets the capsule, the CPSs have undergone extensive chemical and antigenic variation via genetic diversity in the CPS biosynthesis genes. At least 94 antigenically distinct capsular serotypes have been identified in S. pneumoniae (5, 6). While the CPSs of types 3 and 37 are produced as relatively simple repeats of polysaccharides by the synthase pathway (7, 8), all other capsule types are much more complex and are synthesized by WZY-dependent polymerization, a mechanism of capsule production that is widely found in other bacteria (9-11).Except for type 37, all of the CPS types in S. pneumoniae are determined by a set of capsule biosynthesis genes, which are historically designated cps or cap (12). Bentley et al. recently renamed the pneumococcal capsule genes on the basis of their overall...

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Section: Methodsmentioning

confidence: 99%

Sequence Elements Upstream of the Core Promoter Are Necessary for Full Transcription of the Capsule Gene Operon in Streptococcus pneumoniae Strain D39

et al. 2015

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“…Approximately 1 µg of RNA was converted to cDNA, and primers amplifying a region of the xdhB gene were chosen for semiquantitative PCR. The rpoA gene ( SCO4729 ), encoding the α‐subunit of RNA polymerase, was also used as a control; rpoA is transcribed as part of an operon containing ribosomal protein genes (Charaniya et al ., ) and its expression is expected to decrease on exposure to serine hydroxamate. Relative to hrdB , expression of xdhB was increased ∼2‐fold, whereas rpoA expression was reduced after 30 min incubation with serine hydroxamate (Fig.…”

Section: Resultsmentioning

confidence: 99%

Streptomyces coelicolor XdhR is a direct target of (p)ppGpp that controls expression of genes encoding xanthine dehydrogenase to promote purine salvage

Sivapragasam

Grove

2016

Molecular Microbiology

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The gene encoding Streptomyces coelicolor xanthine dehydrogenase regulator (XdhR) is divergently oriented from xdhABC, which encodes xanthine dehydrogenase (Xdh). Xdh is required for purine salvage pathways. XdhR was previously shown to repress xdhABC expression. We show that XdhR binds the xdhABC-xdhR intergenic region with high affinity (Kd ∼ 0.5 nM). DNaseI footprinting reveals that this complex formation corresponds to XdhR binding the xdhR gene promoter at two adjacent sites; at higher protein concentrations, protection expands to a region that overlaps the transcriptional and translational start sites of xdhABC. While substrates for Xdh have little effect on DNA binding, GTP and ppGpp dissociate the DNA-XdhR complex. Progression of cells to stationary phase, a condition associated with increased (p)ppGpp production, leads to elevated xdhB expression; in contrast, inhibition of Xdh by allopurinol results in xdhB repression. We propose that XdhR is a direct target of (p)ppGpp, and that expression of xdhABC is upregulated during the stringent response to promote purine salvage pathways, maintain GTP homeostasis and ensure continued (p)ppGpp synthesis. During exponential phase growth, basal levels of xdhABC expression may be achieved by GTP serving as a lower-affinity XdhR ligand.

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“…The differential expression of genes within the abeABCD operon is not unprecedented in S. coelicolor (9), but it also is not considered to be the norm. Microarray studies of S. coelicolor gene expression have shown that the first gene of an operon is typically the most highly expressed, with expression levels then decreasing throughout the length of the operon (26).…”

Section: Discussionmentioning

confidence: 99%

Regulation of a Novel Gene Cluster Involved in Secondary Metabolite Production in Streptomyces coelicolor

2010

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Antibiotic biosynthesis in the streptomycetes is a complex and highly regulated process. Here, we provide evidence for the contribution of a novel genetic locus to antibiotic production in Streptomyces coelicolor. The overexpression of a gene cluster comprising four protein-encoding genes (abeABCD) and an antisense RNAencoding gene (␣-abeA) stimulated the production of the blue-pigmented metabolite actinorhodin on solid medium. Actinorhodin production also was enhanced by the overexpression of an adjacent gene (abeR) encoding a predicted Streptomyces antibiotic regulatory protein (SARP), while the deletion of this gene impaired actinorhodin production. We found the abe genes to be differentially regulated and controlled at multiple levels. Upstream of abeA was a promoter that directed the transcription of abeABCD at a low but constitutive level. The expression of abeBCD was, however, significantly upregulated at a time that coincided with the initiation of aerial development and the onset of secondary metabolism; this expression was activated by the binding of AbeR to four heptameric repeats upstream of a promoter within abeA. Expressed divergently to the abeBCD promoter was ␣-abeA, whose expression mirrored that of abeBCD but did not require activation by AbeR. Instead, ␣-abeA transcript levels were subject to negative control by the double-strand-specific RNase, RNase III.

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Transcriptome dynamics-based operon prediction and verification in Streptomyces coelicolor

Cited by 32 publications

References 59 publications

Sequence Elements Upstream of the Core Promoter Are Necessary for Full Transcription of the Capsule Gene Operon in Streptococcus pneumoniae Strain D39

Sequence Elements Upstream of the Core Promoter Are Necessary for Full Transcription of the Capsule Gene Operon in Streptococcus pneumoniae Strain D39

Streptomyces coelicolor XdhR is a direct target of (p)ppGpp that controls expression of genes encoding xanthine dehydrogenase to promote purine salvage

Regulation of a Novel Gene Cluster Involved in Secondary Metabolite Production in Streptomyces coelicolor

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