Todd hewitt medium

P. aeruginosa strains ATCC 39324 and ATCC 27318 used in this study were purchased from the American Type Culture Collection (Manassas, VA). Cation-adjusted Mueller Hinton broth (CA-MHB; Becton Dickson, Franklin Lakes, NJ) was used to grow the bacteria for determination of in-vitro antimicrobial activity and time-kill assays. Luria-Bertani medium (LB; Becton Dickson) was used for the membrane-depolarization assay and quantitative real time-polymerase chain reaction (qRT-PCR) analysis. Todd-Hewitt medium (Becton Dickson) supplemented with yeast extract (THY) was used to grow the bacteria for scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Gentamicin sulphate and polymyxin B were purchased from Fisher Scientific (Pittsburgh, PA).

All PCR primers used in this study were synthesized in Genery Co., Ltd. (Shanghai, China) and listed in Additional file 1, Table S6. Common DNA sequencing and genome sequencing of S. mutans B30 was performed by Novogene (Beijing, China). The genes encoding different MucF homologous proteins were codon optimized and synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). PCR reactions were carried out with Taq DNA polymerase (TransGene, Beijing, China) or Phusion® HF DNA polymerase (Thermo Fisher scientific, MA) according to the manufacturers’ instructions. Golden Gate cloning and overlapping PCR were performed following the literature procedures [45 (link)]. Preparation of genomic DNAs of different S. mutans strains was performed as described [22 (link)]. For natural transformation experiments, cells were maintained in Todd-Hewitt medium (Becton–Dickinson, Sparks, MD) supplemented with 0.3% yeast extract. A detailed natural transformation procedure was described previously [46 (link)].

Bacterial strains and plasmids used in this study are listed in Supplementary Data 1. For preparation of genomic DNAs, all Streptococcus strains and Staphylococcus strains were grown in brain heart infusion broth (BHI; OXOID LTD., Basingstoke, England) or on BHI agar plates; Clostridium strains were grown in reinforced medium for Clostridia (Hopebio Co., Qingdao, China). For transformation experiments, cells were maintained in Todd-Hewitt medium (Becton-Dickinson, Sparks, MD) supplemented with 0.3% yeast extract (THYE). The chemically defined medium ASS (Artificial Saliva Substitutes) was modified from the published ASS medium^{55 (link)} by adding extra ingredients: L-Val (0.12 g/L), L-Phe (0.17 g/L), L-Asp (0.13 g/L), L-Asn (0.13 g/L), L-Ala (0.09 g/L), and peptone (0.1%). For the selection of antibiotic-resistant colonies, BHI plates were supplemented with erythromycin (12.5 μg/mL), spectinomycin (1 mg/mL), kanamycin (800 μg/mL) and tetracycline (15 μg/mL). LB plates were supplemented with spectinomycin (100 μg/mL) and kanamycin (50 μg/mL).

Organs were homogenized in 2 mL (brain, spleen, intestine, Peyer’s patches, MLN) or 7 mL tubes (liver) containing a mix of 1.4 and 2.8 mm glass beads and sterile water using the Precellys system (Bertin Technologies). For quantification of intratissular bacteria, small intestine and Peyer’s patches were treated with 200 mg/L gentamicin for 2 hr at room temperature to kill extratissular bacteria before being washed three times in PBS and homogenized. Cultured cells were lysed with ice-cold water.
Serial dilutions were plated on Todd Hewitt medium (Becton Dickinson), and on Granada medium (bioMérieux) for intestine, Peyer’s patches, and MLN. Plates were incubated 24 hr at 37°C in aerobic (Todd Hewitt medium) or anaerobic (Granada medium) atmosphere.

S. mutans strain UA159 was used as the wild type, and its isogenic ΔclpE (strain IBSJ5) and ΔclpP (strain IBS512) mutants are described elsewhere (28 (link), 29 (link)). Cells were routinely grown in Todd-Hewitt medium (Becton, Dickinson) containing 0.2% yeast extract (THY) at 37°C under microaerophilic conditions (in a candle jar or statically) and in the presence of appropriate antibiotics, such as erythromycin (5 μg/ml), when needed. For some experiments, Bacto brain heart infusion (BHI; Becton, Dickinson) broth was also used. For growth kinetics assays, the effects of dithiothreitol (DTT; reducing agent), hydrogen peroxide (H₂O₂; oxidizing agent), and high temperature (42°C) on growth were also measured. Cells from overnight cultures were diluted in BHI medium to adjust the initial optical density at 600 nm (OD₆₀₀) to 0.05. Cells were then grown in the presence or absence of 1 mM H₂O₂ or 5 mM DTT at 37°C in a 96-well plate covered with a lid. The OD₆₀₀ was measured at 15-min intervals for 6 h using a microplate reader (Tecan Spark). In a different set, cells were also grown at 42°C to study the effect of high temperature on growth. Experiments were repeated three times or more.