1 UCLA School of Dentistry, Los Angeles, CA 90025, USA
2 UCLA Molecular Biology Institute, Los Angeles, CA 90025, USA
Correspondence
Fengxia Qi
fqi{at}dent.ucla.edu
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ABSTRACT |
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INTRODUCTION |
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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., 1999
, 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, mutA', 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 MutA' show strong similarity to each other. While mutA is the structural gene for prepromutacin I, mutA' 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 of premature mutacin. MutF, MutE and MutG are probable immunity proteins for mutacin I. The functions of OrfX, OrfY and OrfZ proteins in mutacin I production are unknown (Qi et al., 2000
).
Previous studies indicate that the proteins encoded by ciaH and luxS are required for mutacin I production (Merritt et al., 2005; Qi et al., 2004
). CiaH is the histidine kinase of a two-component signal-transduction system, CiaH/R (Giammarinaro et al., 1999
; Zähner et al., 2002
), while LuxS is responsible for the synthesis of the interspecies cell signalling molecule autoinducer 2 (AI-2) (Schauder et al., 2001
; Xavier & Bassler, 2003
). In addition to regulating mutacin I production, both CiaH and LuxS were also found to be involved in regulating other cellular functions in S. mutans such as biofilm formation and stress tolerance (Len et al., 2004
; Merritt et al., 2003
; Qi et al., 2004
). As a preliminary attempt to understand the control of mutacin I production and its relationships with these pleiotropic regulators, we employed a random insertional mutagenesis approach to obtain a global view of genes involved in mutacin I production. Our results indicate that mutacin I production seems to be affected by numerous global regulatory systems.
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METHODS |
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Construction of a random mutagenesis library.
Genomic DNA extracted from S. mutans strain UA159 was randomly cleaved by CviJI restriction enzyme digestion. Digested DNA fragments of 300500 bp were purified using the QIAquick gel extraction kit (Qiagen). The pZero-2 cloning vector (Invitrogen) was used to facilitate library construction in E. coli, and as a suicide vector in S. mutans. To adapt the use of this plasmid for the selection of positively transformed S. mutans, the kanamycin-resistance gene from the pZero-2 vector was excised by AvaI and BglII restriction enzymes and the pZero-2 backbone was ligated with a spectinomycin-resistance gene (aad9) (Podbielski et al., 1996). The modified vector, p0SC, was linearized by EcoRV digestion and purified by gel extraction. For ligation, the reaction contained S. mutans DNA fragments combined with the linearized p0SC vector in a 5 : 1 ratio and 2 U T4 DNA ligase (Promega). Competent E. coli DH5
cells were transformed with the ligation mixture by electroporation. Plasmids harvested from positive E. coli transformants were used to transform S. mutans strain UA140 for mutagenesis, using protocols described by Perry et al. (1983)
. Transformants were selected on BHI plates containing 800 µg spectinomycin ml1. The overall strategy for the construction of this library is summarized in Fig. 1
. Confirmation of DNA integration was performed by PCR and Southern blotting. PCRs were performed using primers specific to the spectinomycin-resistance gene: Scaad9Up (5'-ACTGCATTTCCCGCAATATC-3') and SCaad9Dn (5'-GGATCAGGAGTTGAGAGTGG-3'). Southern blotting was performed with digoxigenin (DIG)-labelled aad9 PCR product as a probe, synthesized and detected by the DIG DNA labelling and detection kit (Roche Diagnosis).
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Identification of mutated genes.
Chromosomal DNA from each mutacin-defective mutant was prepared from 10 ml cell culture at OD600 0·8 by a standard DNA extraction protocol (Sambrook et al., 1989). Chromosomal DNA from each mutant was digested with one of the following restriction enzymes: XmnI, ScaI, AclI or BstBI. All of these restriction enzymes do not cut within the p0SC plasmid sequence but excise the genome into fragments amenable to further manipulation. DNA fragments were circularized by self-ligation with T4 DNA ligase (Promega). The recircularization of the DNA fragments allowed the targeting inserts to be directly amplified by PCR using M13 primers flanking the multiple cloning site of the p0SC plasmid: M13F (5'-GTAAAACGACGGCCAG-3') and M13R (5'-CAGGAAACAGCTATGAC-3'). The PCR was performed with initial denaturation of DNA at 95 °C for 4 min, followed by 40 cycles of amplification consisting of 95 °C for 30 s, 55 °C for 1 min, 72 °C for 5 min and 1 cycle of final extension of 72 °C for 10 min. The recircularized DNA fragments were also used to transform E. coli DH5
cells. The rescued plasmids or PCR products were analysed by gel electrophoresis, purified with a Qiagen gel extraction kit and sequenced by the UCLA core DNA sequencing facility. The sequences obtained were compared to the genomic sequences of S. mutans UA159 available at the Los Alamos oral pathogen sequence databases (http://www.oralgen.lanl.gov) via BLAST.
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RESULTS AND DISCUSSION |
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To allow the use of pZero-2 in S. mutans the plasmid was adapted by replacing the kanamycin resistance gene with a spectinomycin-resistance gene (aad9) (Podbielski et al., 1996). The replacement of the antibiotic-resistant gene did not affect the lethal selection property of the plasmid. Analysis by PCR amplification using universal primers flanking the multiple cloning sites demonstrated that nearly 100 % of E. coli clones carried an insert (data not shown).
Overall, the utilization of the restriction enzyme CviJI and the p0SC plasmid greatly streamlined the preparation of the S. mutans random insertional library. A total of 11 000 E. coli clones were obtained to represent approximately 6 times the total number of ORFs (1963) in the S. mutans genome (Ajdi et al., 2002
). Plasmids extracted from these clones were used to transform competent S. mutans UA140. Approximately 11 000 S. mutans transformants were subjected to phenotypic screening.
Screening for mutacin-defective mutants and identification of the disrupted genes
All 11 000 clones were subjected to a mutacin-activity assay. Clones that did not inhibit the growth of an indicator strain were selected and subjected to a total of three rounds of screening. To exclude those mutants whose mutacin-defective phenotype might be a result of severe growth defects, only clones with apparent normal growth rates (overnight culture with OD600 0·8) were chosen for the second round of screening. Clones with a mutacin-negative phenotype in all three rounds of screening were selected for gene identification.
Digested chromosomal DNA fragments were recircularized and used to transform competent E. coli, or used as PCR templates (see Methods). The PCR products or the rescued plasmids were directly sequenced, and 25 unique genes were identified after a BLAST search of the UA159 genome sequence database (Tables 1 and 2). The protein sequences were also compared with other known sequences available in the NCBI database. The genes proposed functions and their frequencies of disruption in the mutagenesis library are summarized in Table 1
. These genes are classified by their functional classes as described in the Los Alamos National Laboratory oral pathogen sequence database: (1) two-component sensory systems (TCSs), (2) stress responses, (3) energy metabolism, (4) central cellular processes and (5) genes with unknown function.
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Stress-response genes
The ability of bacteria to adapt to stresses, such as pH, temperature and oxidative changes, is essential for their survival. Lactic acid producing bacteria like S. mutans are constantly bombarded with dissociated protons from acids passively diffusing through the cell membrane into the cytoplasm. The resulting decrease in intracellular pH drastically alters the proton motive force required for numerous transmembrane transport processes (Presser et al., 1997). The internal acidification also reduces the activity of acid-labile enzymes and damages proteins and DNA (van de Guchte et al., 2002
). To stabilize the transmembrane pH during an acidic challenge, the multimeric F0F1-ATPase is utilized to expel protons out of the cell (Kuhnert et al., 2004
; Suzuki et al., 2000
). Mutations in the
(AtpD) and
(UncG) subunits of the F0F1-ATPase resulted in mutacin I defective phenotypes. BLASTP analysis of AtpD and UncG in S. mutans revealed high sequence conservation with orthologues in other streptococci, with AtpD and UncG exhibiting approximately 8090 % similarity to other ATPase subunits. It is speculated that mutacin I production may be abolished due to protein denaturation resulting from intracellular pH change. Alternatively, a stress signal secondary to the disruption of the ATPase may affect mutacin I production. A stress-modulated mutacin I regulation pathway is also supported by the evidence that another stress-response regulatory gene, hrcA, was also detected in our assay. BLASTP analysis of HrcA in S. mutans shows approximately 80 % similarity with HrcA in other streptococci. HrcA is a transcriptional regulator of class I heat-shock proteins, GroEL and DnaK, which assist the folding of newly synthesized proteins or proteins denatured during environmental insults (Jayaraman et al., 1997
; Schulz & Schumann, 1996
). HrcA represses the stress-related proteins by binding to a DNA element called CIRCE (controlling inverted repeat of chaperone expression) located in the regulatory region of the stress genes (Zuber & Schumann, 1994
). Mutation of the HrcA/CIRCE system in S. mutans has been demonstrated to derepress groESL and dnaK gene expression, and render the mutant sensitive to low pH (Lemos et al., 2001
). It is unlikely that HrcA directly controls the production of mutacin I since no CIRCE element has been found in the mutacin I operon sequence. Further investigation is needed to decipher the relationship between HrcA and mutacin I production.
Genes involved in energy metabolism
In S. mutans, pyruvate generated from glycolysis can be converted into a variety of acidic end products (Carlsson et al., 1985). Under anaerobic conditions and limited glucose, pyruvate is converted into formate and acetate or ethanol through the pyruvate formate-lyase pathway. In one branch of this pathway, pyruvate becomes acetyl-CoA, which is in turn converted to ethanol by a bifunctional enzyme, alcohol-acetaldehyde dehydrogenase (AdhE), in a two-step process (Carlsson & Griffith, 1974
; Yamada & Carlsson, 1975
). Interestingly, among all of the mutacin-defective mutants selected from our library, adhE had the highest frequency of disruption. A BLASTP search revealed numerous matches to AdhE, with the best match to the enzyme from Lactococcus lactis (82 % similarity). The dysfunction of AdhE may cause shunting of pyruvate towards the production of formate. Formic acid is a strong acid, which may contribute to a greater production of intracellular H+ (Len et al., 2004
). This possible internal acidification may create a severe stress signal leading to the abrogation of mutacin production. Another gene involving metabolism, pgm, encodes a phosphoglycerate mutase-like protein. Pgm participates in the final conversion of 3-phosphoglycerate to pyruvate in the glycolysis pathway. Pgm matches phosphoglycerate mutases in other species, with the best match exhibiting 76 % similarity to a predicted enzyme from Listeria innocua. Pgm mutation may alter the metabolic balance and affect mutacin I production similarly to AdhE mutation.
Another disruption was located in pttB, which encodes a transmembrane trehalose-specific IIBC component in the phosphoenolpyruvate : sugar phosphotransferase systems (PTS). PttB appears to be conserved among both Gram-positive and Gram-negative species. The S. mutans PttB is 85 % similar to S. pneumoniae EIIBC-tre and 66 % similar to E. coli EIIBC-tre. S. mutans contains numerous PTS systems (Ajdi et al., 2002
), and is remarkably adaptable in the types of carbohydrates it can utilize as an energy source (Gauthier et al., 1984
; Schachtele, 1975
; Vadeboncoeur & Pelletier, 1997
). Besides their roles in sugar transportation, PTS components have been demonstrated to influence urease expression (Weaver et al., 2000
), haemolysin production (Lun & Willson, 2005
) and biofilm formation (Abranches et al., 2003
) in various streptococci. Furthermore, reports have shown that sensitivity to bacteriocin is related to the presence of the mannose-specific phosphotransferase enzyme IIAB in Enterococcus faecalis and Listeria monocytogenes (Dalet et al., 2001
; Héchard et al., 2001
; Ramnath et al., 2000
). However, there is currently no data, to our knowledge, that supports regulation of bacteriocin production by the PTS.
Genes involved in central cellular processes
Mutations in rexB, priA, ligA, 23S/16S rRNA and tRNA sequences were all predicted to affect central cellular processes such as DNA replication, repair and recombination as well as translation. RexB is 78 % similar to RexB in Streptococcus agalactiae, and 72 % similar to the B subunit of a putative ATP-dependent exonuclease in S. pyogenes. PriA is approximately 80 % similar to the putative primosomal replication factor Y in S. pyogenes and S. pneumoniae. LigA is approximately 85 % similar to putative DNA ligases in other streptococci.
Recently, emerging evidence has suggested alternative mechanisms to those of classical transcriptional regulators, such as those involving termination/antitermination factors or regulatory RNA elements participating in gene expression (Anderson & Schneewind, 1997; Henkin, 2000
; Johansson & Cossart, 2003
; Ma et al., 2001
; Novick et al., 1993
; Shimizu et al., 2002
). It is interesting to find that a possible polar mutation in the nusAinfB operon, and alterations to rRNA and tRNA sequences in our mutants, have eliminated the production of mutacin I. It is arguable that mutacin I reduction in these mutants is not a specific phenomenon, but a part of the global physiologic stress due to major disruptions in cellular pathways. Nonetheless, the possible role of transcription termination and regulatory RNA elements in mutacin I regulation warrants further exploration. In pseudomonas, mutation of the DNA repair pathway has been shown to be associated with a reduced production of bacteriocin LlpA (de Los Santos et al., 2005
). Similarly, mutations in rexB, priA and ligA in our study represent major insults to the cells DNA replication, repair and recombination systems, and may create a negative effect on mutacin I gene expression through stress-response pathways.
Genes with unknown functions
Conserved hypothetical proteins and their homologies with other proteins are outlined in Table 2. Smu0274 is a protein that is 80 % similar to hypothetical proteins in S. pyogenes and S. agalactiae. Protein-domain analysis assigned this protein to the metallo-
-lactamase superfamily. Smu0378 is a conserved hypothetical protein immediately upstream of the putative transcription termination/antitermination factor nusA. This protein is 80 % similar to unknown proteins in other streptococci. Smu0530 is 85 % similar to putative haemolysins in S. pyogenes and S. pneumoniae. Smu0755 is a conserved hypothetical protein that has 11 transmembrane domains, and is linked to the cell-wall localization and side-chain formation of rhamnose-glucose polysaccharides in S. mutans (Yamashita et al., 1998
). It shares 48 % identity with a hypothetical protein in S. pyogenes from residues 53882. Smu1019 is predicted to have one transmembrane domain and is 49 % similar to another hypothetical protein in S. agalactiae. Smu1272 does not have any significant match with proteins in other species. It is unlikely that the mutacin I negative phenotype was a result of insertional mutation of Smu1272 due to its small size (42 aa). The phenotype could be a result of promoter-sequence interruption to the downstream gene, lepA. Smu1281 is a large protein (1345 aa) with unknown function, and is also found in the species of Streptococcus, Listeria, Neisseria and Fusobacterium. It does not have any significant paralogue in the S. mutans genome and its protein structure does not match with any known protein domain. Smu1292 shares 50 % identity with a putative acyl-acyl carrier protein (ACP) thioesterase in S. pneumoniae, and is predicted to belong to the fatty acid biosynthesis pathway. Smu1616 (59 aa) and Smu1617 (81 aa) were identified in the same mutant. Due to the small sizes of the genes, the phenotype observed in this mutant was probably due to duplication of these genes rather than insertional inactivation. Residues 177 of Smu1617 are assigned to the RelB family, which is a component of the RelE/RelB toxinantitoxin system. RelE represses translation, probably through binding ribosomes, whereas RelB stably binds RelE, presumably deactivating it (Christensen et al., 2001
; Galvani et al., 2001
; Pedersen et al., 2002
). Smu1713 is predicted to be a transmembrane protein with 70 % similarity to a putative membranous protein in S. agalactiae.
Limitations of this study
As an alternative to insertional inactivation, mutacin I defective phenotypes could have resulted from polar mutations to genes downstream of the disruptions. Among the 25 mutants, 18 were probably a consequence of polar mutations, which are outlined in Table 3. The random mutagenesis approach effectively identified the potential loci involved in mutacin I regulation. Nonetheless, in order to pinpoint the candidates responsible for mutacin regulation, experiments are in progress to distinguish between polar and non-polar mutational effects of these mutations.
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The data presented in this study indicate that mutacin I production is stringently controlled by complex regulatory pathways. S. mutans may utilize the TCS or PTS to survey environmental changes and fine tune mutacin I expression accordingly. During stressful situations, such as extreme nutrient deprivation, temperature changes or pH changes, S. mutans may minimize energy expenditure by exerting a negative feedback on mutacin I production via stress-regulatory networks. Further experiments are under way to explore the mechanisms involving the proteins affected in these mutants of mutacin I production.
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ACKNOWLEDGEMENTS |
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Received 24 May 2005;
revised 8 August 2005;
accepted 29 August 2005.
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