Dissecting the Arginine and Lysine Biosynthetic Pathways and Their Relationship in Haloarchaeon Natrinema gari J7-2 via Endogenous CRISPR-Cas System-Based Genome Editing

Abstract

The evolutionary relationship between arginine and lysine biosynthetic pathways has been well established in bacteria and hyperthermophilic archaea but remains largely unknown in haloarchaea. Here, the endogenous CRISPR-Cas system was harnessed to edit arginine and lysine biosynthesis-related genes in the haloarchaeon Natrinema gari J7-2. The ΔargW, ΔargX, ΔargB, and ΔargD mutant strains display an arginine auxotrophic phenotype, while the ΔdapB mutant shows a lysine auxotrophic phenotype, suggesting that strain J7-2 utilizes the ArgW-mediated pathway and the diaminopimelate (DAP) pathway to synthesize arginine and lysine, respectively. Unlike the ArgD in Escherichia coli acting as a bifunctional aminotransferase in both the arginine biosynthesis pathway and the DAP pathway, the ArgD in strain J7-2 participates only in arginine biosynthesis. Meanwhile, in strain J7-2, the function of argB cannot be compensated for by its evolutionary counterpart ask in the DAP pathway. Moreover, strain J7-2 cannot utilize α-aminoadipate (AAA) to synthesize lysine via the ArgW-mediated pathway, in contrast to hyperthermophilic archaea that employ a bifunctional LysW-mediated pathway to synthesize arginine (or ornithine) and lysine from glutamate and AAA, respectively. Additionally, the replacement of a 5-amino-acid signature motif responsible for substrate specificity of strain J7-2 ArgX with that of its hyperthermophilic archaeal homologs cannot endow the ΔdapB mutant with the ability to biosynthesize lysine from AAA. The in vitro analysis shows that strain J7-2 ArgX acts on glutamate rather than AAA. These results suggest that the arginine and lysine biosynthetic pathways of strain J7-2 are highly specialized during evolution. IMPORTANCE Due to their roles in amino acid metabolism and close evolutionary relationship, arginine and lysine biosynthetic pathways represent interesting models for probing functional specialization of metabolic routes. The current knowledge with respect to arginine and lysine biosynthesis is limited for haloarchaea compared to that for bacteria and hyperthermophilic archaea. Our results demonstrate that the haloarchaeon Natrinema gari J7-2 employs the ArgW-mediated pathway and the DAP pathway for arginine and lysine biosynthesis, respectively, and the two pathways are functionally independent of each other; meanwhile, ArgX is a key determinant of substrate specificity of the ArgW-mediated pathway in strain J7-2. This study provides new clues about haloarchaeal amino acid metabolism and confirms the convenience and efficiency of endogenous CRISPR-Cas system-based genome editing in haloarchaea.

Keywords: CRISPR; arginine biosynthesis; diaminopimelate pathway; genome editing; haloarchaea; lysine biosynthesis; α-aminoadipate pathway.

FIG 1

Lysine and arginine biosynthetic pathways in prokaryotes. The four subpathways of the DAP pathway are indicated (numbered 1 to 4). The proteins sharing the same color (cyan, red, blue, green, brown, or purple) in different pathways are homologs.

FIG 2

Challenging the CRISPR-Cas system of strain J7-2 with invader plasmids. (A) Schematic representation of the type I-B CRISPR-Cas system of strain J7-2. The Cas genes (cas1 to cas8) and CRISPR1 are drawn to scale as arrows. The accession numbers of the Cas genes are shown below the arrows. Rectangles and diamonds represent the spacers and direct repeats, respectively. (B) Schematic representation of the invader plasmid. The invader DNA fragment comprising the PAM and protospacer was inserted into the vector pSHS to construct the invader plasmid for transformation of strain J7-2. (C) Transformation efficiencies of strain J7-2 by invader plasmids carrying the same PAM (TTC) and different protospacers matching spacer1-36, spacer1-18, and spacer1-1 of CRISPR1, respectively; (D) Transformation efficiencies of invader plasmids carrying the same protospacer (p1-36) and different trinucleotides as PAMs. The values are expressed as means ± standard deviation (SD) from three independent experiments (***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., no significance; calculated by Student's t test) (C and D), and the raw data are listed in Table S4.

FIG 3

Knockout of crtB by endogenous CRISPR-Cas system in strain J7-2. (A) Schematic representation of pKC-mediated knockout of crtB in the strain J7-2 genome. The artificial mini-CRISPR on the plasmid pKC contains two repeats (green diamonds) separated by a spacer (red rectangle) matching a PAM (TTC [in blue])-preceded protospacer within crtB on strain J7-2 genome. The mini-CRISPR is driven by the sptA promoter (P_sptA). The donor (U1 and D1) and the positions of the primers (co-f, co-r, ci-f, ci-r, s1-f, and si-r) used for PCR analysis are shown. (B) Comparison of the transformation rates of strain J7-2 by plasmids pSHS and pKC. The transformants were grown on 18% MGM plates containing 5 μg/mL mevinolin, and the values are expressed as means ± SD from three independent experiments (***, P < 0.001; calculated by Student's t test). The raw data are listed in Table S5. (C) Percentages of white and red colonies obtained when strain J7-2 was transformed by pKC. The values are calculated based on 462 pKC transformants. (D) PCR analyses of 10 randomly selected white colonies of pKC transformants and the ΔcrtB mutant strain. The genomic DNAs of strain J7-2 and plasmid pKC were used as controls. (E) Color phenotypes of the J7-2 and ΔcrtB strains grown on an 18% MGM plate; (F) Representative of the chromatograph of the DNA sequencing result of the ΔcrtB strain.

FIG 4

Genes and some enzymes involved in arginine and lysine biosynthesis of strain J7-2 and their homologs. (A) Predicted arginine and lysine biosynthetic gene clusters in strain J7-2 and some haloarchaea. Gene names and their locus tag numbers are shown, and “hyp” represents hypothetical genes. (B) Predicted arginine and lysine biosynthetic pathways in strain J7-2. The missing DapC in the lysine biosynthetic pathway and the missing enzymes for α-aminoadipate (AAA) biosynthesis are boxed by dotted lines. The amino acid sequence identity shared by ArgB and Ask, ArgC and Asd, as well as ArgE and DapE, respectively, are shown. (C) Amino acid sequence alignment of strain J7-2 ArgW (NgArgW [AFO55519]) with its homologs from S. acidocaldarius (SaLysW [ALU31683]), T. thermophilus (TtLysW [BCZ92879]), and T. kodakarensis (TkLysW [WP_011249234]). The conserved Zn²⁺-binding Cys residues and C-terminal Glu residue are indicated by arrowheads. The C-terminal EDWGE motif is underlined. (D) Amino acid sequence alignment of strain J7-2 ArgX (NgArgX [AFO55518]) with its homologs from S. acidocaldarius (SaArgX [WP_011278435] and SaLysX [WP_015385490]), T. kodakarensis (TkLysX [WP_011249233]), and T. thermophilus (TtLysX [WP_011173917]). The conserved residues of the active site are indicated by asterisks. The 5-amino-acid signature motif and two highly conserved residues involved in substrate recognition are boxed in red and green, respectively. The C-terminal GSWGR motif is underlined. (E) Superimpositions of structure models of NgArgW (left panel) and NgArgX (right panel) predicted by RoseTTAFold and SWISS-MODEL with the crystal structures of TkLysW (5K2M) and TkLysX (5K2M), respectively. The C-terminal Glu residue (E54) of NgArgW is indicated. The crystal structures of ArgW (3VPB) and ArgX (3VPB) of Sulfolobus tokodaii were used as the templates for homology modeling of NgArgW and NgArgX by SWISS-MODEL, respectively.

FIG 5

Roles of NgArgW and NgArgX in arginine biosynthesis in strain J7-2. (A) Schematic representation of the in situ mutation of argW in strain J7-2. The self-targeting plasmid pWE45A carries a spacer matching a PAM (CTC [in blue])-preceded protospacer corresponding to the 3′-end region of argW on strain J7-2 genome, and the donor DNA (U5 and D5) in pWE45A contains a mutation (CGC [in red]) leading to the replacement of the Glu codon (GAG) by the Ala codon (GCG). (B) Representatives of chromatographs of DNA sequencing results of target mutant strains. (C) Amino acid requirements of strain J7-2 and its derivatives. Cells of each strain were streaked on SM plates without (−) or with (+) 0.1 mM lysine and/or arginine and grown at 37°C. Photographs were taken after 6 days. (D) Growth curves of target strains. Each strain was cultivated in liquid SM at 37°C. The values are expressed as means ± SD from three independent experiments. (E) Detection of recombinant proteins in complementary strains. The cell extracts of the indicated strains grown in liquid SM were subjected to anti-His tag immunoblot analysis after SDS-PAGE using a Tris-tricine buffer system. The cell extracts of strain J7-2 and the ΔargW/ArgW complementary strain were also subjected to Ni-NTA affinity chromatography, and the elution fractions were used as concentrated samples for immunoblot analysis. (F) SDS-PAGE and immunoblot analyses of purified recombinant NgArgW produced in E. coli. Purified NgArgW was subjected to urea-SDS-PAGE using a Tris-tricine buffer system in the absence (−) or presence (+) of 1% β-ME, followed by anti-His tag immunoblot analysis, with the cell extract of E. coli carrying a blank vector as the control (ec). Blotted proteins were also visualized and photographed by using ECL (lanes indicated by asterisks) with an exposure time of 1 or 5 min. Schematic representation of promoter (P₀₅₆₆ or P_T7)-preceded recombinant genes with His tag (H) coding sequence on expression plasmids are shown (E and F, bottom).

FIG 6

Functional analyses of selected genes in arginine and lysine biosynthetic pathways of strain J7-2. (A) Representative chromatographs of DNA sequencing results of target mutant strains; (B, C, E, and G) amino acid requirements of strain J7-2 and its derivatives. Cells of each strain were streaked on SM plates without (−) or with (+) lysine, arginine, or AAA and grown at 37°C. Photographs were taken after 6 days. (D) Detection of recombinant proteins in complementary strains. The cell extracts of the indicated strains grown in liquid SM or 18% MGM were subjected to anti-His tag immunoblot analysis after SDS-PAGE using a Tris-glycine buffer system. (F) Alignment of amino acid sequences around the 5-amino-acid signature motifs of NgArgX and its variants, SaLysX, and TkLysX. The signature motif is indicated by arrowheads. The mutated residues in the variants of NgArgX are marked in red.

FIG 7

LC-MS/MS analysis of trypsin-digested NgArgW derivatives. (A to D) Mass spectra of trypsin-digested NgArgW derivatives in different reaction mixtures as indicated. The MS/MS spectrum of the ion at m/z = 1,303.54 in panel A or C is boxed to the right, and the glutamate covalently linked to the C-terminal residue Glu⁵⁴ of NgArgW is marked in green.