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Genetics of Group A Streptococci

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  • Authors: Kyu Hong Cho1, Gary C. Port2, Michael Caparon3,4
  • Editors: Vincent A. Fischetti5, Richard P. Novick6, Joseph J. Ferretti7, Daniel A. Portnoy8, Miriam Braunstein9, Julian I. Rood10
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Department of Biology, Indiana State University, Terre Haute, IN 47809; 2: Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO 63110; 3: Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO 63110; 4: Current address: Elanco Animal Health, Natural Products Fermentation, Eli Lilly and Company, Indianapolis, IN 46285; 5: The Rockefeller University, New York, NY; 6: Skirball Institute for Molecular Medicine, NYU Medical Center, New York, NY; 7: Department of Microbiology & Immunology, University of Oklahoma Health Science Center, Oklahoma City, OK; 8: Department of Molecular and Cellular Microbiology, University of California, Berkeley, Berkeley, CA; 9: Department of Microbiology and Immunology, University of North Carolina-Chapel Hill, Chapel Hill, NC; 10: Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia
  • Source: microbiolspec March 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.GPP3-0056-2018
  • Received 04 December 2018 Accepted 08 January 2019 Published 01 March 2019
  • Michael Caparone, [email protected]
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  • Abstract:

    (group A streptococcus) is remarkable in terms of the large number of diseases it can cause in humans and for the large number of streptococcal factors that have been identified as potential virulence determinants for these diseases. A challenge is to link the function of potential virulence factors to the pathogenesis of specific diseases. An exciting advance has been the development of sophisticated genetic systems for the construction of loss-of-function, conditional, hypomorphic, and gain-of-function mutations in targeted genes that can be used to test specific hypotheses regarding these genes in pathogenesis. This will facilitate a mechanistic understanding of how a specific gene function contributes to the pathogenesis of each streptococcal disease. Since the first genome was completed in 2001, hundreds of complete and draft genome sequences have been deposited. We now know that the average genome is approximately 1.85 Mb and encodes ∼1,800 genes and that the function of most of those genes in pathogenesis remains to be elucidated. However, advances in the development of a variety of genetic tools for manipulation of the genome now provide a platform for the interrogation of gene/phenotype relationships for individual diseases, which may lead to the development of more sophisticated and targeted therapeutic interventions. This article presents an overview of these genetic tools, including the methods of genetic modification and their applications.

  • Citation: Cho K, Port G, Caparon M. 2019. Genetics of Group A Streptococci. Microbiol Spectrum 7(2):GPP3-0056-2018. doi:10.1128/microbiolspec.GPP3-0056-2018.

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/content/journal/microbiolspec/10.1128/microbiolspec.GPP3-0056-2018
2019-03-01
2019-10-15

Abstract:

(group A streptococcus) is remarkable in terms of the large number of diseases it can cause in humans and for the large number of streptococcal factors that have been identified as potential virulence determinants for these diseases. A challenge is to link the function of potential virulence factors to the pathogenesis of specific diseases. An exciting advance has been the development of sophisticated genetic systems for the construction of loss-of-function, conditional, hypomorphic, and gain-of-function mutations in targeted genes that can be used to test specific hypotheses regarding these genes in pathogenesis. This will facilitate a mechanistic understanding of how a specific gene function contributes to the pathogenesis of each streptococcal disease. Since the first genome was completed in 2001, hundreds of complete and draft genome sequences have been deposited. We now know that the average genome is approximately 1.85 Mb and encodes ∼1,800 genes and that the function of most of those genes in pathogenesis remains to be elucidated. However, advances in the development of a variety of genetic tools for manipulation of the genome now provide a platform for the interrogation of gene/phenotype relationships for individual diseases, which may lead to the development of more sophisticated and targeted therapeutic interventions. This article presents an overview of these genetic tools, including the methods of genetic modification and their applications.

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Image of FIGURE 1
FIGURE 1

A chimeric secreted alkaline phosphatase reporter gene. The plasmid pABG5 is a pWV01-based -streptococcal shuttle vector which encodes resistance to chloramphenicol () and kanamycin (). The plasmid contains a promoterless reporter gene formed by the fusion of the N-terminal region of the cell wall-associated protein F () to the enzymatic domain of the enterococcal alkaline phosphatase (*). Since the chimeric protein (secreted protein F-PhoZ reporter, or PhoZF) lacks the C-terminal cell wall attachment domain of protein F and the N-terminal lipoprotein tethering domain of PhoZ, it is freely secreted from the cell. The PhoZF chimera retains the enzymatic activity of PhoZ and can be easily quantitated in culture supernatants. Restriction sites for II or HI and RI can be used to place the promoter of interest in an orientation to direct transcription of , which is then translated using the ribosome-binding site of (RBS). Insertion of an open reading frame of interest between the RI and either the I or I site places the open reading frame under the control of the strong promoter for ectopic expression and/or complementation analyses.

Source: microbiolspec March 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.GPP3-0056-2018
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FIGURE 2

p7INT and p7INT*. The plasmids p7INT and p7INT* are pUC18-based vectors that encode resistance to erythromycin (). Restriction sites for HI, , , and RI within can be used to insert any promoter and gene of interest for ectopic expression and/or complementation analyses. The plasmids also contain a phage integrase (INT) and an sequence which targets integration into the chromosome at the 3′ end of the tmRNA locus within the 96-bp “O” site of POP′ and BOB′ of and , respectively. With respect to O sites, has been found to contain one of two alleles with either an A or G (see text). Plasmid p7INT contains an A, whereas p7INT* has been mutated to contain a G, and the appropriate plasmid must be used depending on the allele found within the strain of interest. Following introduction of the appropriate plasmid into , selection for the antibiotic-resistant determinant of the plasmid () selects for chromosomes in which the plasmid has integrated into the chromosome at the site. The distribution of tmRNA and tmRNA* between a selection of commonly used strains is shown, with GenBank accession numbers as follows: HSC5, CP006366.1; SF370, AE004092.2; Manfredo, AM295007.1; MGAS10270, CP000260.1; MGAS5005, CP000017.2; MGAS6180, CP000056.1; MGAS10750, CP000262.1; MGAS2096, CP000261.1; MGAS9429, CP000259.1; NZ131, CP000829.1; JRS4, CP011414.1; MGAS8232, AE009949.1; MGAS10394, CP000003.1; MAGS315, AE014074.1; SSI-1, BA000034.2.

Source: microbiolspec March 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.GPP3-0056-2018
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FIGURE 3

Strategy for allelic replacement mutagenesis using linear DNA. Segments of DNA homologous to the 5′ and 3′ ends of the target gene are placed flanking a gene encoding a selectable antibiotic resistance gene, typically on a standard plasmid vector using -based molecular cloning technology. The resulting plasmid vector is converted to a linear molecule by PCR or by digestion using restriction enzymes with sites outside of the cloned streptococcal gene (vector DNA is represented by the curved lines) and introduced into with selection for resistance to the antibiotic encoded by the introduced resistance gene. Recombination between the two homologous sequences (indicated by the lines between the introduced DNA and chromosome) results in the replacement of the insertionally inactivated allele for the native chromosomal allele (shown below the arrow).

Source: microbiolspec March 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.GPP3-0056-2018
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FIGURE 4

Directed insertional mutagenesis of targeted genes using circular DNA. Insertional inactivation of the target gene. A DNA segment internal to the target gene (shown by the gray box enclosed by wavy lines) is cloned onto an plasmid which cannot replicate in . The plasmid is introduced into an host as a circular molecule (top of figure) with selection for a resistance marker on the plasmid. A single homologous recombination event between the chromosome and the circular molecule (shown by the “X”) results in the integration of the plasmid into the chromosome, a partial duplication of the gene (the two regions shaded in gray) in which neither of the two copies are complete, resulting in gene inactivation. Generation of a polar insertion 3′ to the target gene. If the segment of DNA cloned on the integrational plasmid includes the 3′ terminus and sequences downstream of the target gene (shown by the regions shaded in gray and the thick bar, respectively), homologous recombination (shown by the “X”) results in a partial gene duplication in which the 5′ copy is intact but the 3′ copy is truncated, producing a polar effect on any downstream gene(s) which may share a promoter in common with the target gene. When compared with an insertion mutation that inactivates the target gene ( Fig. 1 or Fig. 2 ), this strategy tests whether a phenotype is due to the loss of target gene function or to a polar effect on a distal gene. Mapping the -acting control regions of the target gene. The technique is modified by constructing several plasmids representing a nested set of the chromosomal region extending various distances 5′ of the target gene. The plasmids are integrated into the target locus as described above, and the end-products are a partial duplication of the target gene in which the proximal copy is truncated and inactive. If the duplicated region includes the -acting control regions (represented by the broken arrow and the closed circle labeled “P”), the distal copy of the target gene will be expressed (scenario 1). In contrast, if the cloned segment does not include the -acting control regions, then the distal copy will not be expressed (scenario 2).

Source: microbiolspec March 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.GPP3-0056-2018
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FIGURE 5

Construction of an in-frame deletion. Standard PCR-based methods are used to generate a deletion of the internal region of a copy of the target gene that has been cloned on an -streptococcal shuttle vector that is temperature-sensitive for replication (indicated by “ts ori”). The deletion is constructed so as to maintain the reading frame of the gene (represented as the bent line labeled “R,” connecting the 5′ region labeled “A” and the 3′ region labeled “B”). Following its introduction into , growth at a temperature that is nonpermissive for replication of the plasmid with selection for the antibiotic-resistant determinant of the plasmid () selects for chromosomes in which the plasmid has integrated by homologous recombination (indicated by the “X”). The two regions of homology flanking the deletion are represented by the solid and gray bars labeled “A” and “B.” Recombination between the A bars or, alternatively, between the B bars (shown by the brackets and arrows) results in excision of the plasmid and either the restoration of the wild-type structure or allelic exchange for the deletion allele (these products are illustrated below the second set of arrows). Growth at a temperature permissive for replication of the plasmid enriches for excisant chromosomes, presumably because if integrated, the plasmid origin of replication becomes active, resulting in misregulation of chromosomal replication. Presence of the wild-type or deletion allele in any isolate is easily determined by assay for the unique restriction site engineered into the deletion allele (indicated by the “R” above the bent line) or by PCR with primers flanking the region of interest.

Source: microbiolspec March 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.GPP3-0056-2018
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FIGURE 6

PheS**, a counterselectable marker. The phenylalanyl tRNA synthetase (alpha subunit) PheS catalyzes the attachment of phenylalanine onto its cognate transfer RNA. Introduction of a single amino acid substitution (PheS*, A314G) confers relaxed substrate specificity and allows for the misincorporation of a toxic derivative of phenylalanine, 4-chloro-phenylalanine (4CP), into proteins. Addition of a second substitution at position 260 (PheS**, either T260A or T260S in combination with A314G) facilitates increased misincorporation of 4CP. containing a multicopy plasmid (pABG5) either as an empty vector (WT) or engineered to express PheS*, or PheS** was serial diluted and plated onto solid media in the absence (top panel) or in the presence (bottom panel) of 5 mM 4CP. Strains expressing PheS* show moderate sensitivity to 4CP toxicity, whereas strains expressing PheS** show enhanced sensitivity.

Source: microbiolspec March 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.GPP3-0056-2018
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FIGURE 7

Transposon mutagenesis of . The several transposons that have been used for mutagenesis of are shown. Antibiotic resistance genes are represented by the gray bars, transposon ends and/or terminal inverted repeats are shown in black, genes that are essential for transposition are shown by white bars, and genes that are nonessential for transposition are represented by the stripped bars. Tn (GenBank accession no. U09422) is the prototype conjugative transposon and contains at least 24 different open reading frames. Of these, one is required for resistance to tetracycline, two are essential for transposition, and the rest are likely involved in conjugal transfer. Tn-LTV3 is a highly engineered derivative of the Tn-like transposon Tn. This element transposes via a replicative mechanism and has been modified to include a promoterless reporter gene to generate random transcriptional fusions and an ColE1 plasmid origin of replication to facilitate the cloning and analysis of inactivated loci. TnSpc is a derivative of the Tn-like transposon Tn, which transposes via a cut and paste mechanism and consists of the left and right inverted repeats and transposase of IS and a spectinomycin resistance gene. TnFuZ introduces the gene for the alkaline phosphatase () altered by removal of the region encoding its signal sequence (broken line at the 5′ end of *). TnFuZ acts as an “export signal sequence trap.” Insertions into genes that encode a protein export sequence promote the secretion of PhoZ* enzymatic activity, which can be detected by a number of high-throughput assays for alkaline phosphatase activity. TmErm is an engineered element that contains the right and left mosaic ends (ME) of Tn flanking an erythromycin resistance marker (Ω) that includes strong transcription and translation stop sites (). To apply transposome mutagenesis to , the DNA fragment containing TmErm is purified, such that the resulting preparation does not include the ampicillin resistance determinant contained on the pMOD-2::Ω vector backbone. The purified DNA fragment containing TmErm is then reacted with transposase (EZ::TN, Epicentre Technologies) to form the transposome that is then used to transform using electroporation. pOSKAR and pKRMIT are transposon systems incorporated into the ts pWV01-based -streptococcal shuttle vector that utilize the highly active transposable element (MarC9) under control of the P23 promoter from . Both transposon systems include a kanamycin resistance gene () flanked by long terminal repeats (LTR) whose promiscuous target insertion site requires only a TA dinucleotide sequence, leading to a highly random library of insertions. pOSKAR can be utilized for TraSH analysis through the use of outward-facing T7 promoters, while pKRMIT also includes an outward-cutting site for use with TnSeq analysis.

Source: microbiolspec March 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.GPP3-0056-2018
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Tables

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TABLE 1

Common plasmids utilized for genome engineering

Source: microbiolspec March 2019 vol. 7 no. 2 doi:10.1128/microbiolspec.GPP3-0056-2018

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