Dataset Information

In vitro DNA Inversions Mediated by the PsrA Site-Specific Tyrosine Recombinase of Streptococcus pneumoniae.

ABSTRACT: Site-specific recombination is a DNA breaking and reconstructing process that plays important roles in various cellular pathways for both prokaryotes and eukaryotes. This process requires a site-specific recombinase and direct or inverted repeats. Some tyrosine site-specific recombinases catalyze DNA inversions and regulate subpopulation diversity and phase variation in many bacterial species. In Streptococcus pneumoniae, the PsrA tyrosine recombinase was shown to control DNA inversions in the three DNA methyltransferase hsdS genes of the type I restriction-modification cod locus. Such DNA inversions are mediated by three inverted repeats (IR1, IR2, and IR3). In this work, we purified an untagged form of the PsrA protein and studied its DNA-binding and catalytic features. Gel retardation assays showed that PsrA binds to linear and supercoiled DNAs, containing or not inverted repeats. Nevertheless, DNase I footprinting assays showed that, on linear DNAs, PsrA has a preference for sites that include an IR1 sequence (IR1.1 or IR1.2) and its boundary sequences. Furthermore, on supercoiled DNAs, PsrA was able to generate DNA inversions between specific inverted repeats (IR1, IR2, and IR3), which supports its ability to locate specific target sites. Unlike other site-specific recombinases, PsrA showed reliance on magnesium ions for efficient catalysis of IR1-mediated DNA inversions. We discuss that PsrA might find its specific binding sites on the bacterial genome by a mechanism that involves transitory non-specific interactions between protein and DNA.

SUBMITTER: Li J

PROVIDER: S-EPMC7096588 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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In vitro DNA Inversions Mediated by the PsrA Site-Specific Tyrosine Recombinase of Streptococcus pneumoniae.

Li Jingwen J Wang Juanjuan J Ruiz-Cruz Sofía S Espinosa Manuel M Zhang Jing-Ren JR Bravo Alicia A

Frontiers in molecular biosciences 20200319

Site-specific recombination is a DNA breaking and reconstructing process that plays important roles in various cellular pathways for both prokaryotes and eukaryotes. This process requires a site-specific recombinase and direct or inverted repeats. Some tyrosine site-specific recombinases catalyze DNA inversions and regulate subpopulation diversity and phase variation in many bacterial species. In Streptococcus pneumoniae, the PsrA tyrosine recombinase was shown to control DNA inversions i ...[more]

PMID: 32266289

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Project description:Streptococcus pneumoniae (the pneumococcus) is a formidable human pathogen that is capable of asymptomatically colonizing the nasopharynx. Progression from colonization to invasive disease involves adaptation to distinct host niches, which vary markedly in the availability of key nutrients such as sugars. We previously reported that cell-cell signaling via the autoinducer 2 (AI-2)/LuxS quorum-sensing system boosts the capacity of S. pneumoniae to utilize galactose as a carbon source by upregulation of the Leloir pathway. This resulted in increased capsular polysaccharide production and a hypervirulent phenotype. We hypothesized that this effect was mediated by phosphorylation of GalR, the transcriptional activator of the Leloir pathway. GalR is known to possess three putative phosphorylation sites, S317, T319, and T323. In the present study, derivatives of S. pneumoniae D39 with putative phosphorylation-blocking alanine substitution mutations at each of these GalR sites (singly or in combination) were constructed. Growth assays and transcriptional analyses revealed complex phenotypes for these GalR mutants, with impacts on the regulation of both the Leloir and tagatose 6-phosphate pathways. The alanine substitution mutations significantly reduced the capacity of pneumococci to colonize the nasopharynx, middle ear, and lungs in a murine intranasal challenge model.IMPORTANCE Pneumococcal survival in the host and capacity to transition from a commensal to a pathogenic lifestyle are closely linked to the organism's ability to utilize specific nutrients in distinct niches. Galactose is a major carbon source for pneumococci in the upper respiratory tract. We have shown that both the Leloir and tagatose 6-phosphate pathways are necessary for pneumococcal growth in galactose and demonstrated GalR-mediated interplay between the two pathways. Moreover, the three putative phosphorylation sites in the transcriptional regulator GalR play a critical role in galactose metabolism and are important for pneumococcal colonization of the nasopharynx, middle ear, and lungs.

Project description:Streptococcus pneumoniae (pneumococcus), a major human pathogen, is well known for its adaptation to various host environments. Multiple DNA inversions in the three DNA methyltransferase hsdS genes (hsdSA, hsdSB, and hsdSC) of the colony opacity determinant (cod) locus generate extensive epigenetic and phenotypic diversity. However, it is unclear whether all three hsdS genes are functional and how the inversions mechanistically occur. In this work, our transcriptional analysis revealed active expression of hsdSA but not hsdSB and hsdSC, indicating that hsdSB and hsdSC do not produce functional proteins and instead act as sources for altering the sequence of hsdSA by DNA inversions. Consistent with our previous finding that the hsdS inversions are mediated by three pairs of inverted repeats (IR1, IR2, and IR3), this study showed that the 15-bp IR1 and its upstream sequence are strictly required for the inversion between hsdSA and hsdSB Furthermore, a single tyrosine recombinase PsrA catalyzes the inversions mediated by IR1, IR2, and IR3, based on the dramatic loss of these inversions in the psrA mutant. Surprisingly, PsrA-independent inversions were also detected in the hsdS sequences flanked by the IR2 (298 bp) and IR3 (85 bp) long inverted repeats, which appear to occur spontaneously in the absence of site-specific or RecA-mediated recombination. Because the HsdS subunit is responsible for the sequence specificity of type I restriction modification DNA methyltransferase, these results have revealed that S. pneumoniae varies the methylation patterns of the genome DNA (epigenetic status) by employing multiple mechanisms of DNA inversion in the cod locus.IMPORTANCEStreptococcus pneumoniae is a major pathogen of human infections with the capacity for adaptation to host environments, but the molecular mechanisms behind this phenomenon remain unclear. Previous studies reveal that pneumococcus extends epigenetic and phenotypic diversity by DNA inversions in three methyltransferase hsdS genes of the cod locus. This work revealed that only the hsdS gene that is in the same orientation as hsdM is actively transcribed, but the other two are silent, serving as DNA sources for inversions. While most of the hsdS inversions are catalyzed by PsrA recombinase, the sequences bound by long inverted repeats also undergo inversions via an unknown mechanism. Our results revealed that S. pneumoniae switches the methylation patterns of the genome (epigenetics) by employing multiple mechanisms of DNA inversion.

Dataset Information

In vitro DNA Inversions Mediated by the PsrA Site-Specific Tyrosine Recombinase of Streptococcus pneumoniae.

Publications

<i>In vitro</i> DNA Inversions Mediated by the PsrA Site-Specific Tyrosine Recombinase of <i>Streptococcus pneumoniae</i>.

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