A Counterselectable Sucrose Sensitivity Marker Permits Efficient and Flexible Mutagenesis in Streptococcus agalactiae

Abstract

Streptococcus agalactiae (group B Streptococcus [GBS]) is a cause of severe infections, particularly during the newborn period. While methods exist for generating chromosomal mutations in GBS, they are cumbersome and inefficient and present significant challenges if the goal is to study subtle mutations, such as single-base-pair polymorphisms. To address this problem, we have developed an efficient and flexible GBS mutagenesis protocol based on sucrose counterselection against levansucrase (SacB) expressed from a temperature-selective shuttle vector. GBS containing the SacB expression cassette demonstrates lethal sensitivity to supplemental sucrose whether the plasmid DNA is replicating outside of the chromosome or has been integrated during a crossover event. Transmission electron microscopy shows that SacB-mediated lethal sucrose sensitivity results from the accumulation of inclusion bodies that eventually lead to complete degradation of normal cellular architecture and subsequent lysis. We used this new mutagenesis technique to generate an in-frame, allelic exchange knockout of the GBS sortase gene srtA, demonstrating that >99% of colonies that emerge from our protocol had the expected knockout phenotype and that among a subset tested by sequencing, 100% had the correct genotype. We also generated barcoded nonsense mutations in the cylE gene in two GBS strains, showing that the approach can be used to make small, precise chromosomal mutations. IMPORTANCE The ability to generate chromosomal mutations is fundamental to microbiology. Historically, however, GBS pathogenesis research has been made challenging by the relative genetic intractability of the organism. Generating a single knockout in GBS using traditional techniques can take many months, with highly variable success rates. Furthermore, traditional methods do not offer a straightforward way to generate single-base-pair polymorphisms or other subtle changes, especially to noncoding regions of the chromosome. We have developed a new sucrose counterselection-based method that permits rapid, efficient, and flexible GBS mutagenesis. Our technique requires no additional equipment beyond what is needed for traditional approaches. We believe that it will catalyze rapid advances in GBS genetics research by significantly easing the path to generating mutants.

Genome-Wide fitness analysis of group B Streptococcus in human amniotic fluid reveals a transcription factor that controls multiple virulence traits

“…pyogenes [ 70 ]. We used a previously described sucrose-counterselectable mutagenesis plasmid, pMBsacB, to generate point mutations within the coding sequence of the cas9 gene in GBS strain CNCTC 10/84 (serotype V) [ 88 ], which is the strain with which our animal models of vaginal colonization and ascending chorioamnionitis have been optimized.…”

“…To generate GBS expressing dCas9, we used the mutagenesis plasmid pMBsacB in GBS strain 10/84 as previously described [ 88 ]. Two separate synthetic, double-stranded DNA fragments, 800 to 1000 nt in length, were designed and ordered from Genscript (Piscataway, NJ, USA).…”

“…We confirmed proper cloning by Sanger sequencing of miniprepped plasmid DNA, which we then used to transform electrocompetent GBS as previously described [ 88 , 136 , 137 ]. We performed the C-terminal (H840A) mutagenesis first, following the protocol we have described previously [ 88 ]. Once this mutation was generated and confirmed by Sanger sequencing, we made those GBS competent and created the second mutation using identical techniques.…”

“…Lopez-Sanchez et al have previously shown that the protospacer adjacent motif (PAM) necessary for GBS Cas9 genomic target recognition follows the same NGG pattern as in S. pyogenes [70]. We used a previously described sucrose-counterselectable mutagenesis plasmid, pMBsacB, to generate point mutations within the coding sequence of the cas9 gene in GBS strain CNCTC 10/84 (serotype V) [88], which is the strain with which our animal models of vaginal colonization and ascending chorioamnionitis have been optimized. , and those for which baseline fitness cannot be determined (white; see reference [41]).…”

“…These fragments were cloned into pMBsacB using Gibson assembly and used to transform chemically competent DH5α E. coli (New England Biolabs, Ipswich, MA, USA) according to manufacturer instructions. We confirmed proper cloning by Sanger sequencing of miniprepped plasmid DNA, which we then used to transform electrocompetent GBS as previously described [88,136,137]. We performed the C-terminal (H840A) mutagenesis first, following the protocol we have described previously [88].…”