Miniprep kit

The analysis of transcript abundance of the ldh gene of P. vulgatus strains was performed by RT-qPCR experiments. Total RNA from P. vulgatus was isolated from 50 ml cultures grown in the BHI medium to mid-exponential phase. The total RNA Miniprep Kit (New England Biolabs, Ipswich, USA) was used to purify RNA from cells following the manufacturer’s protocol. RNA samples were treated with DNase I to remove residual DNA, and RNA concentrations were measured spectrophotometrically using a BioSpectrometer® (Eppendorf, Hamburg, Germany). Additionally, control PCR experiments were performed to confirm that RNA samples did not contain DNA contaminations. Gene-specific primers used for RT-qPCR experiments were the same as for qPCR experiments. The gene l23, encoding the ribosomal protein L23, was chosen as a reference. RT-qPCR reactions were performed with a Luna® Universal One-Step RT-qPCR Kit (New England Biolabs, Ipswich, USA). Each PCR reaction contained 200 ng of purified RNA. For temperature cycling and fluorescence measurement, a cycler CFX Connect™ and a suitable software (BioRad, Munich, Germany) were used. Melting curve analysis resulted in single peaks for the respective PCR fragments, confirming specific products from PCR reactions. To determine transcript abundance, the ΔΔCt values and the fold change were evaluated as described above.

The bacterial strains and plasmids used in this study are listed in Tables S1 and S2, respectively. PCR was performed by KOD Hot Start DNA polymerase (National England BioLabs/NEB) or Q5 High-Fidelity 2× Master Mix (NEB) for cloning purposes according to the manufacturer’s instructions. Plasmid DNA and PCR fragments were purified using the plasmid miniprep kit (NEB) and DNA gel extraction kit (NEB). E. coli K12 chromosomal DNA was used as the template. E. coli DH5α stain was used for cloning, and E. coli BL21 (DE3) was used for recombinant protein purification. E. coli MG1655 strain and KO mutants were used for growth curve measurement, PG composition analysis, immunoblotting, and MIC study.
Unless otherwise specified, bacteria were cultured in LB [1% tryptone, 0.5% yeast extract, 0.5% NaCl] or LB-salt–free [1% tryptone, 0.5% yeast extract] medium at 37 °C. Agar 1.5% (w/v) was used in solid plates. Antibiotics were used at the following concentrations (per mL): 30 (chloramphenicol; Cm30), 50 (kanamycin; Kan50), or 100 (ampicillin; Amp100) where appropriate.

Plasmid DNA was extracted using the NEB miniprep kit (T1010L) and 400 ng of DNA was used as input for the rapid barcoding kit library prep (SQK-RBK004). Plasmids were then sequenced using R9.4.1 Flongle flow cells (FLO-FLG001) or R9.4.1 minION flow cells until approximately 200 $\times$ coverage was obtained for each barcode based on an expected plasmid size of 20 kilobases. Basecalling was perfomed using Guppy v4.2.2 in high-accuracy mode (Oxford Nanopore Technologies). Reads were filtered by retaining only those near the expected plasmid length. Reads were then assembled using miniasm^{58 (link)}. The assembly was then polished using minipolish^{59 (link)} and medaka (Oxford Nanopore Technologies). Polished assemblies were then compared to the expected sequence to determine if any mutations were present.

The gene replacement constructs, and ectopic expression (add back) constructs were purified using the Qiagen Miniprep kit, linearized by NotI (NEB) restriction digestion, precipitated and washed twice with 70% ethanol, and redissolved in sterile water. The precipitated linearized DNA was used to electroporate T. brucei PCF as described in (37 (link)). The generation of sKO and dKO (null) mutants was confirmed by Southern blot.

Genomic DNA (gDNA) purifications were performed using bacterial gDNA extraction kits from Sigma-Aldrich. Plasmid DNA was purified with miniprep kits from NEB. Screening PCRs were performed using DreamTaq polymerase (Thermo Fisher). Oligonucleotides were synthesized by Sigma-Aldrich. Sanger sequencing was performed by Source Bioscience.