Identification of genes associated with mutacin I production in Streptococcus mutans using random insertional mutagenesis

Abstract

Streptococcus mutans is a major pathogen implicated in dental caries. Its virulence is enhanced by its ability to produce bacteriocins, called mutacins, which inhibit the growth of other Gram-positive bacteria. The goal of this study is to use a random insertional mutagenesis approach to search for genes that are associated with mutacin I production in the virulent strain UA140. A random insertional mutagenesis library consisting of 11 000 clones was constructed and screened for a mutacin-defective phenotype. Mutacin-defective clones were isolated, and their insertion sites were determined by PCR amplification or plasmid rescue followed by sequencing. A total of twenty-five unique genes were identified. These genes can be categorized into the following functional classes: two-component sensory systems, stress responses, energy metabolism and central cellular processes. Several conserved hypothetical proteins with unknown functions were also identified. These results suggest that mutacin I production is stringently controlled by diverse and complex regulatory pathways. INTRODUCTIONDental caries is a chronic condition affecting the teeth. Bacteria adhering to tooth surfaces produce acids from carbohydrate fermentation, which can demineralize tooth structures, resulting in cavitation on tooth surfaces. One of the major pathogens implicated in dental caries is Streptococcus mutans (Loesche, 1986). In addition to its various virulence attributes, such as glucan synthesis, acid production and acid tolerance, the production of bacteriocins, called mutacins, may also play an important role in the persistence of S. mutans in the dental-plaque community (Gronroos et al., 1998;Hillman, 2002).Bacteriocins produced by Gram-positive bacteria are peptide antibiotics classified as class I (lantibiotics) or class II (non-lantibiotics), based on their post-translational modifications (Guder et al., 2000;Riley & Wertz, 2002). In S. mutans, both classes of bacteriocins are produced. The lantibiotic mutacins have a wide spectrum of activity against Gram-positive bacteria, including multiple-drug-resistant pathogens (Novak et al., 1994;Qi et al., 1999Qi et al., , 2000, while the non-lantibiotic mutacins are more specific to closely related streptococcal species such as the mitis group streptococci and group A streptococci (Qi et al., 2001). Mutacin I, the focus of this study, is a 24 aa lantibiotic with a molecular mass of 2364 Da. The mutacin I biosynthesis gene locus consists of 14 genes in the order of mutR, mutA, mutA9, mutB, mutC, mutD, mutP, mutT, mutF, mutE, mutG, orfX, orfY, orfZ (Qi et al., 2000). MutR is thought to be the positive regulator for the expression of the mutacin I operon. MutA and MutA9 show strong similarity to each other. While mutA is the structural gene for prepromutacin I, mutA9 is not required for mutacin I activity. MutB, MutC and MutD constitute the modification apparatus for the premature peptide, and MutT and MutP are the ABC transporter and protease, respectively, for the transporting and processing...

Regulation of Bacteriocin Production and Cell Death by the VicRK Signaling System in Streptococcus mutans

“…A role for VicK in mutacin I production was also demonstrated by Tsang et al using a random insertional mutagenesis approach in the virulent strain UA140 (53). In that study, disruption of the VicRK was shown to abolish mutacin I production.…”

“…In that study, disruption of the VicRK was shown to abolish mutacin I production. It was later discovered that while both VicK and a stress response regulator called HrcA were needed to produce mutacin I, VicK's influence on mutacin I synthesis was exerted by controlling transcription of a putative stressresponsive regulator designated irvA (inducible repressor of virulence A) for its role as a repressor for mutacin I synthesis in S. mutans (53,54). In our microarray experiments, we observed that loss of VicK reduced the transcription of hrcA by Ͼ2.5-fold (see Table S1 in the supplemental material).…”

Interspecies Interactions within Oral Microbial Communities

“…Some recent molecular and genetic studies have revealed that bacteriocin production is a well-regulated event which is affected by both environmental conditions, such as cell density, nutritional availability, and pH, and genetic factors (100,101,136,162,203). These regulatory systems ensure that bacteriocins are produced at the right time and place so that they can be effectively used for "war and peace" activities within an oral microbial community.…”

SmbFT, a Putative ABC Transporter Complex, Confers Protection against the Lantibiotic Smb in Streptococci

“…A majority of the mutacins characterized to date belong to lantibiotics, such as the mutacins I, II, III, K8, B-Ny266, Smb, and 1140 (30)(31)(32)(33)(34)(35)(36)(37). Based on the primary amino acid sequences, mutacins belonging to lantibiotics are further subdivided into two subclasses: AI and AII (38)(39)(40).…”