Introduction

To understand the putative function of a gene, one needs to either inactivate or selectively modify it. In vitro transposition mutagenesis and gene disruption by homologous recombination using an antibiotic resistance cassette are the most common techniques for gene inactivation. The classical strategy for gene disruption requires cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette, and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and other recombinational technologies can also be exploited to substantially accelerate virtually all steps involved in the gene disruption process. However, all these methods require utilization of an antibiotic resistance marker, which often causes polar effect on the downstream genes. Moreover, these disruption procedures cannot be used for insertion or deletion of single amino acids or modification of single or multiple amino acids. These modifications are important to study biological functions of a given protein.

The use of a thermosensitive plasmid such as pGhost4 (1) in gene disruption can circumvent the use of antibiotic resistance cassette for unmarked gene modification; however this method is labor-intensive and time-consuming (2). Genetic analysis of many oral strepto-cocci, including Streptococcus mutans, has thus far been limited in scope due to the lack of advanced genetic tools. Although some basic tools that work in other low G+C Gram positive bacteria can be used in oral streptococci, their effectiveness is very much limited. Here we develop a rapid and easy mutagenesis strategy for S. mutans without the use of selectable markers. This strategy exploits the fact that S. mutans is competent for genetic transformation and can be cotransformed (i.e., can uptake two different DNA molecules by a competent cell effectively). We show that this method is highly efficient for insertion or deletion modification of chromosomal genes.

Materials and Methods

Bacterial Strains and Growth Conditions

Escherichia coli strain DH5α and TG1Rep+ (3) were grown in LB medium. TG1Rep+ contains a chromosomal copy of the wild-type repA gene and was used as a host for propagation of pGhost4 at 37°C. Thereafter, 100 µg/mL ampicillin, 100 µg/mL kanamycin, 100 µg/mL spectinomycin, and/or 250 µg/mL erythromycin were added to the LB when needed. S. mutans strain UA159 was grown in Todd-Hewitt broth with 0.2% yeast extract (THY), and when necessary, 300 µg/mL kanamycin, 300 µg/mL spectinomycin, and/or 10 µg/mL erythromycin were added.

Transformation of S. mutans and Screening of Mutants

S. mutans cultures for transformations were grown in THY with 5% heat-inactivated horse serum. Bacterial cultures were grown at 37°C until the absorbance reached at 600 nm was approximately 0.2. At this point, competence-stimulating peptide (CSP) was added at a final concentration of 500 ng/ mL. The amino acid sequence of CSP is SGSLSTFFRLFNRSFTQALGK (4), and it was synthesized by Mimotopes Pvt. Ltd (Clayton, VIC, Australia). Transforming DNA was added to the culture and incubated for 60–90 min at 30°C. We routinely used 200 ng/mL plasmid DNA with or without 1 µg/mL second unselected target locus DNA. The transformed cells were plated on antibiotic-containing media and further incubated for 48 h at 30°C under microaerophilic conditions. Transformants that appeared on the selective media were checked for unselected chromosomal recombination events (Figure 1) either by patching on antibiotic plates or by quick lysis followed by PCR verification. Quick lysis PCR was performed using PrepMan® Ultra reagents (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. A small bacterial colony was resuspended in PrepMan Ultra reagent and heated for 10 min at 100°C. PCR was performed using an aliquot of this sample after removal of cell debris from lysed bacteria by centrifugation.

Figure 1. Diagram for markerless genetic exchange based on cotransformation. (Click to enlarge)

Plasmid and DNA Constructs

Plasmid pGhost4 is derived from the broad host range replicon pWV01 and contains a thermosensitive origin of replication (due to mutations in the repA gene, repA^Ts) and an erythromycin resistance (Em^R) marker (1). For modification of htrA locus, plasmid pIB102 was used as described previously (5). To inactivate Smu486 locus, two separate constructs were made. First, a 1.47-kb fragment spanning the entire Smu486 region was amplified by PCR from UA159 genomic DNA using primers Bam-Smu486-F1 (5′-cgcggatccTGTTGAT GTCTCAGTTAGTTTGG-3′) and Bam-Smu487-R1 (5′-cgcggatccTT TAGC AACCTGCTTC AATG AC AGG-3′), in which the lowercase sequences denote the incorporated restriction sites. This fragment was cloned into pGEM®-T-Easy vector (Promega, Madison, WI, USA) to create pIB38. A 0.87-kb spectinomycin resistance gene (aad9) was amplified from pUCSpec (6) using primers Spec-P-For (5′-TATCTAGAAT AAAAAATTTAGAAGCCCAATG-3′) and Spec-Rev (5′-TTTTATAATTTTT TTAATCTGTTATTTAAATAG-3′) and cloned into XhoI-XbaI-digested and T4 polymerase-blunted pIB38 to generate pIB74. In this plasmid, a 0.37-kb internal region of Smu486 is replaced with the aad9 gene. To generate pIB45, pIB38 was digested with XhoI and XbaI and blunt-ended with T4 polymerase followed by self-ligation. Thus, in pIB45, the same 0.37-kb region of Smu486 is deleted.