Streamlining marker-less allelic replacement in Streptococcus pneumoniae through a single transformation step strategy: easyJanus

Abstract

ABSTRACT The ability to genetically manipulate bacteria is a staple of modern molecular microbiology. Since the 2000s, marker-less mutants of Streptococcus pneumoniae ( Spn ) have been made by allelic exchange predominantly using the kan R -rpsL cassette known as “Janus.” The conventional Janus protocol involves two transformation steps using multiple PCR-assembled products containing the Janus cassette and the target gene’s flanking DNA. We present an innovative strategy to achieve marker-less allelic replacement through a single transformation step. Our strategy involves integrating an additional copy of the target’s downstream region before the Janus cassette, leading to a modified genetic arrangement. This single modification reduced the number of required PCR fragments from five to four, lowered the number of assembly reactions from two to one, and simplified the transformation process to a single step. To validate the efficacy of our approach, we implemented this strategy to delete in Spn serotype 4 strain TIGR4 the virulence gene pspA, the entire capsular polysaccharide synthesis locus cps4 , and to introduce a single-nucleotide replacement into the chromosome. Notably, beyond streamlining the procedure, our method markedly reduced false positives typically encountered during negative selection with streptomycin when employing the traditional Janus protocol. Furthermore, and as consequence of reducing the amount of exogenous DNA required for construct synthesis, we show that our new method is amendable to the use of commercially available synthetic DNA for construct creation, further reducing the work needed to obtain a mutant. Our streamlined strategy, termed easyJanus, substantially expedites the genetic manipulation of Spn facilitating future research endeavors. IMPORTANCE We introduce a new strategy aimed at streamlining the process for marker-less allelic replacement in Streptococcus pneumoniae , a Gram-positive bacterium and leading cause of pneumonia, meningitis, and ear infections. Our approach involves a modified genetic arrangement of the Janus cassette to facilitate self-excision during the segregation step. Since this new method reduces the amount of exogenous DNA required, it is highly amendable to the use of synthetic DNA for construction of the mutagenic construct. Our streamlined strategy, called easyJanus, offers significant time and cost savings while concurrently enhancing the efficiency of obtaining marker-less allelic replacement in S. pneumoniae .