An Orphan MbtH-Like Protein Interacts with Multiple Nonribosomal Peptide Synthetases in Myxococcus xanthus DK1622

Abstract

One mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 from Myxococcus xanthus DK1622 for characterization. The M. xanthus DK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination of in vivo and in vitro assays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping of M. xanthus DK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a "universal" MLP for generating functional hybrid NRPSs.IMPORTANCE MbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP from Myxococcus xanthus DK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a "universal" MLP during the construction of functional hybrid NRPSs.

Keywords: MbtH-like protein; Myxococcus xanthus; combinatorial biosynthesis; natural products; nonribosomal peptide synthetase.

FIG 1

Schematic of the genomic regions of Myxococcus species and strains surrounding the MXAN_3118 homolog (red arrows). Similar gray shading identifies homologs of MXAN_3115 to MXAN_3117 and MXAN_3119 to MXAN_3120. The 1-kb bar indicates the scale for all genomic regions except those show in shades of blue, the sizes of which are noted in parentheses. Locus tag abbreviations: M. xanthus DK1226 (MXAN_3115 to MXAN_3120), M. virescens DSM 2260 (ga0070493_12055 to ga0070493_12060), M. fulvus HW-1 (LILAB_23300 to LILAB_23325), M. hansupus DSM 436 (A176_04382 to A176_04387), M. macrosporus DSM 14697 (MYMAC_003060 to MYMAC_003065), M. stipitatus DSM 14675 (MYSTI_03587 to MYSTI_03603), M. fulvus DSM 16525 (Ga0131203_1181 to Ga0131203_1186 and Ga0131203_1071 to Ga0131203_1081). M. xanthus DK1622^a is representative of all other M. xanthus strains (DSM 16525, DZ2, and DF1). In M. stipitatus DSM 14675^b, the blue genes are an NRPS-associated gene cluster covering 65 kb; the NRPS-associated gene cluster has homolog ORFs in M. fulvus DSM 16525. M. fulvus DSM 16525^c genome sequence was incomplete and two contigs were identified, the first contig covers 24 kb terminating in the middle of the NRPS-encoding gene 1181 and the second contig is 26 kb starting in the middle of genes 1071 to 1079, missing the homolog for gene MYSTI_03592 in the M. fulvus DSM 16525 cluster.

FIG 2

Potential NRPS partners for the MXAN_3118 orphan MLP. (A) Schematic of the MYSTI_03590 to MYSTI_03598 NRPS/PKS hybrid megasynthase from M. stipitatus DSM 14675. (B) Schematic of the MXAN_3779 and MXAN_3634 to MXAN_3636 NRPS/PKS hybrid megasynthases from M. xanthus DK1622. Domain abbreviations: AL, acyl-CoA ligase; T, thiolation; C, condensation; ox, oxidation; KS, ketosynthase; AT, acyltransferase; E, epimerase; Te, thioesterase.

FIG 3

Myxoprincomide (Mpc) NRPS protein interactions with MXAN_3118. (A) Schematic of the MXAN_3779 NRPS/PKS hybrid assembly line with boxes highlighting the fourth (M5_Mpc) and tenth (M12_Mpc) modules investigated for MXAN_3118 interactions. Domain abbreviations: AL, acyl-CoA ligase; T, thiolation; C, condensation; ox, oxidation; MT, methyltransferase; KS, ketosynthase; AT, acyltransferase; Te, thioesterase. (B) Results from the B2H assay. Quantitative β-galactosidase assay of the α-subunit-A domain fusions with MXAN_3118-λcI fusion (+) or λcI alone (−). EntF (A domain) and YbdZ (MLP) were used as controls of MLP-NRPS positive interactions. Error bars show standard deviations from three independent assays. *, P ≤ 0.05; ****, P ≤ 0.0001 by Student's t test. (C) Tris-Tricine (16.8%) polyacrylamide gels and Coomassie blue staining of His-tagged M5_Mpc eluting from an Ni-NTA column after overproduction without (empty expression vector [EV]) or with (expression vector expressing MXAN_3118 [3118]) untagged MXAN_3118. Ten micrograms of protein was loaded in each lane. (D) Tris-Tricine (16.8%) polyacrylamide gels and Coomassie blue staining of His-tagged M12_Mpc eluting from an Ni-NTA column after overproduction without (EV) or with (3118) untagged MXAN_3118. Seventeen micrograms of protein was loaded in each lane.

FIG 4

MXAN_3634 to MXAN_3636 interactions with MXAN_3118. (A) Schematic of the MXAN_3634 to MXAN_3636 NRPS/PKS hybrid assembly line. Boxes highlight the third (M3), eighth (M8), eleventh (M11), and fifteenth (M15) modules investigated for MLP interactions. Domain abbreviations: AL, acyl-CoA ligase; T, thiolation; C, condensation; ox, oxidation; KS, ketosynthase; AT, acyltransferase; E, epimerase; Te, thioesterase. (B) Results from B2H assays. Quantitative β-galactosidase assay of the α-subunit-A domain fusions with MXAN_3118-λcI fusion (+) or λcI alone (−). Error bars show standard deviations from three independent cultures. **, P ≤ 0.01; ****, P ≤ 0.0001; ns, not significant by Student's t test.

FIG 5

Protein-protein interactions of M. xanthus DK1622 NRPS and MXAN_3118 using B2H assay. (A) Schematic of the NRPS proteins from the 13 NRPS encoding gene clusters that were tested. Boxes highlight the modules containing the A domain tested. Domain abbreviations: AL, acyl-CoA ligase; T, thiolation; C, condensation; ox, oxidation; KS, ketosynthase; AT, acyltransferase; E, epimerase; Te, thioesterase; R, reductase; KR, ketoreductase; DH, dehydratase; and AMT, aminotransferase. (B) Results of a quantitative β-galactosidase assay of the α-subunit-A domain fusions with MXAN_3118-λcI fusion (+) or λcI alone (−). Error bars show standard deviations from three independent cultures. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 by Student's t test.