Escherichia coli dh5α

Table 1 lists the strains used in this study. Escherichia coli DH5α (Gibco Life Technologies, Gaithersburg, Md.), E. coli DH5α λpir, E. coli BW20767/pRL27 were cultured at 37°C on Luria-Bertani (LB) medium [36] . Pseudomonas species were cultured at 30°C on LB or 21C minimal medium (MM) [37] complemented with one of the following carbon sources: 0.5, 5, or 10 mM 3-chlorobenzoate (3CBA), 15 mM succinate or 10 mM fructose. Antibiotics were supplemented to the growth medium to select for maintenance of genetic constructions at the following concentrations: kanamycin (Km) 25 µg/ml, chloramphenicol (Cm) 20 µg/ml, rifampicin (Rif) 50 µg/ml, nalidixic acid (Nal) 50 µg/ml, gentamicin (Gm) 20 µg/ml, and ampicillin (Ap) 100 µg/ml.

The xynA gene from P. citrinum FERM P-15944 was synthesized with codon optimization for expression in P. pastoris based on the nucleotide database (GenBank: accession no. AB198065.1) [21 (link)] and introduced into the pUC57-Kan vector by GenScript (Piscataway, NJ, USA). The P. pastoris expression kit, including the P. pastoris strain X-33, constitutive expression vector (pGAPZα A), methanol-inducible expression vector (pPICZα A) and Zeocin were all from Invitrogen (Carlsbad, CA, USA). All restriction enzymes and Phusion High-Fidelity DNA polymerase were from New England BioLabs (Beverly, MA, USA). Birchwood xylan was from Sigma-Aldrich (St Louis, MO, USA), Azo-xylan was from Megazyme (Wicklow, Ireland).
Pichia pastoris was cultured in YPD medium 1% (w/v) yeast extract, 2% (w/v) bacteriological peptone and 2% (w/v) dextrose at 30 °C with shaking at 200 rpm. The transformants were selected on YPDS plates (YPD with 1 M sorbitol) containing 100–2000 µg/mL Zeocin. The pGAPZα A transformants were chosen for preliminary selection on YP (YPD without dextrose) with 0.2% (w/v) Azo-xylan.
Escherichia coli DH5α (Gibco) was used for vector propagation and was grown in a Luria-Bertani (LB) medium at 37 °C with either kanamycin (50 µg/mL) to select for pUC57-Kan or Zeocin (25 µg/mL) to select for pGAPZα A and pPICZα A transformants.

All strains used in the present study can be found in Supplementary Table 2. Escherichia coli DH5α (Gibco-BRL), MG1655(DE3) (James Imlay), BL21(DE3) (Novagen), JM109 (Stratagene), B834(DE3) (Novagen), and MC1061^{28 (link)} were grown in Luria Broth (LB), unless otherwise indicated, with aeration at 250 rpm. Media contained kanamycin (50 μg/mL), chloramphenicol (25 μg/mL), or carbenicillin (50 μg/mL) where appropriate. Plasmids were transformed into cells by electroporation using a Bio-Rad Gene Pulser and recovered for 1 h at 37 °C in SOC medium (0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, and 20 mM glucose). Transformants were selected by plating on LB agar with the appropriate antibiotic and incubated at 37 °C overnight.

Probe 45S rDNA was produced from plasmid pTa71, which contains the complete sequence of this Triticum aestivum rDNA [35 (link)]. In turn, arthropod telomere sequences and U2 snDNA were obtained by Polymerase Chain Reaction (PCR) using previously described primers [36 (link),37 (link)], respectively, and genomic DNA from B. amazonicus. The PCR reactions consisted of: 16.25 µL of sterile water, 2.5 µL of 10x Taq Polymerase buffer, 2 µL of DNTP (2 mM), 1 µL of genomic DNA (100 ng), 1 µL of MgCl2 (50 mM), 1 µL of each forward and reverse primer (10 mM), and 0.25 µL of 1U Taq Polymerase. The thermal configurations of the PCRs were: 1 cycle of 94 °C (5 min); 35 cycles of 94 °C (1 min), 53 to 55 °C (1 min, normally 53 °C for U2 snDNA and 55 °C for telomeric repeats) and 72 °C (1 min); 1 cycle of 72 °C (10 min); and 1 cycle 4 °C (hold). The U2 snDNA PCR product was cloned into the pGEM-T vector (Promega^®) and transformed into the bacterium Escherichia coli DH5α (Gibco GBRL^®). All probes were produced by nick and translated using digoxigenin-14-dUTP (Roche, Mannheim, Germany).

Escherichia coli DH5α (Gibco Life Technologies) and DH5α λpir [17 (link)] for plasmid constructions was routinely grown at 37°C on LB medium [41 ]. E. coli MG1655 [42 (link)], Cupriavidus necator H16 [43 (link)], and Pseudomonas putida UWC1 [44 (link)] were cultured at 30°C on LB or type 21C minimal medium (MM) [45 ] containing either 10 mM Fructose or 5 mM 3-chlorobenzoate (3CBA). If necessary, antibiotics were added at the following concentrations; kanamycin 25 μg mL^-1, gentamicin 20 μg mL^-1, ampicillin 100 μg mL^-1, and tetracycline 10 μg mL^-1 for E. coli, 25 μg mL^-1 for C. necator and 50 μg mL^-1 for P. putida.

The bacterial strains and plasmids used in this study are listed in S1 Table. Primers are listed in S2 Table. GBS ST17 clinical strains isolated from invasive infections (i.e. from blood culture, cerebro-spinal fluid or other sterile sites) were obtained from the French National Reference Center for Streptococci in Paris (http://www.cnr-strep.fr) [5 (link)]. GBS NEM316, capsular serotype III ST-23, A909, capsular serotype Ia ST-9 and GBS BM110 capsular serotype III ST17 are well-characterized isolates from human with invasive infections. Seven non-ST17 GBS isolates (HN016, WC135, YM001, GX064, LMG14608, LMG15083, BSU178) whose genome are available were previously described [10 (link), 26 (link)]. Escherichia coli DH5α (Gibco-BRL) was used for cloning experiments.
S. agalactiae strains were cultured in Todd Hewitt (TH) broth or agar (Difco Laboratories, Detroit, MI) at 37°C in standing filled flasks and E. coli in Luria-Bertani (LB) medium. Lactococcus lactis NZ9000 was grown in M17 medium supplemented with 1% glucose at 30°C or 37°C. Antibiotics were used at the following concentrations: for E. coli, erythromycin, 150 μg ml⁻¹, kanamycin, 50 μg ml⁻¹; for S. agalactiae, erythromycin, 10 μg; for L. lactis, erythromycin, 5 μg ml⁻¹.