Restriction endonuclease

All chemical reagents were of analytical grade and purchased from Sigma-Aldrich (St Louis, MO, USA). Primerstar Taq polymerase was purchased from Takara and restriction endonuclease, T4 ligase and their corresponding buffers were purchased from New England Biolabs (NEB). Ninety-six-well microplates were purchased from Nunc. Ni-NTA agarose resins were supplied by GE Healthcare for His-tagged protein purification.
All polymerase chain reactions (PCR) were performed using a thermal cycler (DNA Engine; Bio-Rad, Hercules, CA, USA). Colorimetric assay were measured by a microplate reader (SpectraMax M2e, Molecular Devices, Sunnyvale, CA, USA). HPLC analysis was performed by Agilent 1260 (Agilent Technologies, Waldbronn, Germany).

Restriction endonuclease and DNA modifying enzymes were purchased from New England Biolabs (NEB). PCRs were carried out by using Vent (NEB) or Blend Taq DNA polymerase (Toyobo). Primers used in this study are listed in S1 Table.
Total cellular RNA was isolated from streptococcal strains and further purified by using an RNeasy minikit (Qiagen). First-strand complementary DNA (cDNA) was generated from 2 μg of total cellular RNA with random hexamer primers. qPCR was used to evaluate the expression level of genes in the GlnR regulon. The reactions were carried out by using a Kapa SYBR fast kit (Kapa Biosystems) and a 7500 Fast real-time PCR system (Applied Biosystem). All reactions were run in triplicate, and at least three samples were analyzed. A melting curve analysis was performed with all pairs of primers to ensure the reaction efficiency. To be noted, the reaction efficiency of primers for glnA, glnQ, gdhA, citB, nrgA and glnP is 98.9%, 100%, 92.4%, 102.6%, 98.1% and 91.4%, respectively. The data were analyzed by using 7500 software v2.0.5. The change in the quantification cycle (ΔCq) of each sample was normalized with 16S RNA. The ΔCq derived from wild-type GS5 grown at neutral pH was used as a reference. The relative quantity of each sample was calculated as the ΔCq of the sample compared to the ΔCq of the reference using the formula 2^-ΔΔCq.

B. pumilus MK001, B. subtilis RCK and different E. coli strains, BL21(DE3), BL21(DE3) containing pTUM4 plasmid (bears genes for proteins assisting in refolding) and E. coli BL21(DE3) arctic expression host, were obtained from Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, New Delhi. B. subtilis RCK and B. pumilus MK001 were maintained on Horikoshi agar media containing 0.5% glucose, 0.5% peptone, 0.5% yeast extract, 0.15% KH₂PO₄, 0.01% MgSO₄.7H₂O and 2.0% agar, pH 9.0. Fusion DNA polymerase, restriction endonuclease and T₄ DNA ligase were procured from New England Biolabs Inc. (UK). Ampicillin and kanamycin antibiotics were obtained from HiMedia, India. Trypsin and Tris base were purchased from Sigma-Aldrich, USA, while remaining chemicals and media components of analytical grade were purchased locally.

All chemical reagents were of analytical grade and purchased from Sigma-Aldrich (St Louis, MO, USA). Primerstar Taq polymerase was purchased from Takara, restriction endonuclease, T4 DNA ligase and their corresponding buffers were purchased from New England Biolabs (USA). 96-well microtiterplates were purchased from Nunc (Denmark). Ni-NTA agarose resins were supplied by GE Healthcare (USA) for His-tagged protein purification.
All polymerase chain reactions (PCR) were performed using a thermal cycler (DNA Engine; Bio-Rad, Hercules, CA, USA). Colorimetric assays were measured using a microtiterplate reader (SpectraMax M2e, Molecular Devices, Sunnyvale, CA, USA). HPLC analysis was performed using an Agilent 1260 (Agilent Technologies, Waldbronn, Germany).

All the plasmids used in this study are listed in Supplementary Table S1. Restriction endonuclease and other enzymes used for cloning were from New England Biolabs (NEB). Complete EDTA-free protease inhibitor cocktail tablets were from Roche Diagnostics, Germany. Oligonucleotides were from MWG Biotech, Germany. Succinyl-CoA was prepared as in27 . meso-DAP was purified according to28 (link). NADP⁺ was from Melford, U.K. All other chemicals used in this study were purchased from Sigma Aldrich. Cellulose TLC plates were from Merck, Darmstadt, Germany.
E. coli BL21 (DE3) (Novagen) and C41 (DE3)29 (link) was used for expression. The construct of N-terminal His-tagged EcDapD in pFO430 (link) was used in this study. Expression constructs of Corynebacterium glutamicum meso-DAP dehydrogenase (Cgmeso-DAP dehydrogenase) in pET28b31 (link) and Bacillus anthracis DapF (BaDapF) in pET23a32 were kind gifts from Dr. David Roper, University of Warwick, Coventry, U.K.