Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress

Abstract

The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, t o tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of 5. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS1 7, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology t o the y/xM-ffh operon of Bacillus subtik The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an 5. mutans genomic library. Sequencing of the complementing clone identified the intact yIxM and #%i genes as well as a partial ORF with homology t o the proU/opuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat=l::Tn917 mutation on ffh expression. Upon acid shock (pH 5)# wild-type ffh mRNA levels were found t o increase approximately four-to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptiveholerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acidAshocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable t o ferment sorbitol although it was able t o grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans t o changing environmental conditions. Abbreviations: ATR, acid tolerance response; GSP, general secretory pathway; SRP, signal recognition particle. KeywordsThe CenBank accession number for the DNA sequence of the 3 kb DNA fragment containing the y/xM-ffh genes is U88582. INTRODUCTIONStreptococcus mutans plays a major role in the aetiology of human dental caries. This bacterium is able to demineralize dental enamel by generation of a localized acidic environment in plaque by rapid glycolysis of dietary sugars to lactic acid. In addition to its acidogenicity, S. mutans is able to tolerate exposure to continual and rapid cycles of acid shock. In vivo pH measurements have demonstrated that the pH of plaque can drop from pH 7.0 to values ranging from 3.0 to 4.0 in less than 20 min following the intake of carbohydrates (Imfeld & Lutz, 1980;Yamada et al., 1980;Mormann & Muhlemann, 1981 ; Schachtele & Harlander, 1984;Jensen & Wefel, 1989). E...

Acid-resistant genes of oral plaque microbiome from the functional metagenomics

“…Both the inserts orf1 and ffh are known aciduric-related genes. The former encodes RNA-binding protein and a separate functional metagenomics study of the environmental sample confirmed its function [17], the latter comes from the S. mutans strain [11]. The purpose of construction of the strain E. coli DH10B/pSL-ffh and its functional verification was to detect whether the acid-resistant genes from oral bacteria could be heterologously expressed in E. coli .…”

“…After acid exposure, the intracellular pH decreases, and DNA is damaged. The cells can resist the acid stress through a variety of defence mechanisms, such as regulating the flow of protons inside and outside cells [11,13] and neutralizing intracellular protons through producing alkaline compounds [40]. Further, the DNA repair enzyme system can also assist cells to resist acid stress through repair of the damaged DNA [12].…”

“…In addition, we used two known acid-resistant genes, orf1 and ffh , to construct two positive controls, E. coli DH10B/pSL-orf1 and pSL-ffh, the former from the environmental microbes [17], the latter from S. mutans [11]. The detailed protocol is described in the Supplementary File.…”

“…In the mid-1980s, Loesche et al [5] reported that Streptococcus mutans is a principal causative agent of dental caries, and its prevalence was 70–100% of individual cases of caries. Subsequently, a large number of studies on the acid production and acid-resistant properties of Streptococcus were performed [9,10], and some relevant acid-resistant genes, such as ffh, uvrA, and dnaK , were discovered and studied [11–13]. These achievements can be partially attributed to the ease in propagating this strain in the laboratory.…”

Streptococcal viability and diminished stress tolerance in mutants lacking the signal recognition particle pathway or YidC2

“…This rapid acidification is followed by expression of numerous proteins, including chaperones and membrane proteins, in a process necessary for cellular viability referred to as the acidtolerance response (12,13). Transposon mutagenesis in S. mutans resulted in several acid-sensitive mutants (14) and one mutation mapped to ffh within the sat (secretion and acid tolerance) operon (15). Surprisingly, interruption of ffh was not lethal (16), in contrast to what was reported for E. coli and B. subtilis (3,17), but did render S. mutans sensitive to acidic pH.…”