Fine-tuning of amino sugar homeostasis by EIIANtr in Salmonella Typhimurium

The nitrogen-metabolic phosphotransferase system, PTSNtr, consists of the enzymes INtr, NPr and IIANtr that are encoded by ptsP, ptsO, and ptsN, respectively. Due to the proximity of ptsO and ptsN to rpoN, the PTSNtr system has been postulated to be closely related with nitrogen metabolism. To define the correlation between PTSNtr and nitrogen metabolism, we performed ligand fishing with EIIANtr as a bait and revealed that D-glucosamine-6-phosphate synthase (GlmS) directly interacted with EIIANtr. GlmS, which converts D-fructose-6-phosphate (Fru6P) into D-glucosamine-6-phosphate (GlcN6P), is a key enzyme producing amino sugars through glutamine hydrolysis. Amino sugar is an essential structural building block for bacterial peptidoglycan and LPS. We further verified that EIIANtr inhibited GlmS activity by direct interaction in a phosphorylation-state-dependent manner. EIIANtr was dephosphorylated in response to excessive nitrogen sources and was rapidly degraded by Lon protease upon amino sugar depletion. The regulation of GlmS activity by EIIANtr and the modulation of glmS translation by RapZ suggest that the genes comprising the rpoN operon play a key role in maintaining amino sugar homeostasis in response to nitrogen availability and the amino sugar concentration in the bacterial cytoplasm.


Materials and Methods for Supplementary information
Construction of bacterial strains 26 The phage λ-derived Red recombination system was used to delete genes in-frame or to fuse 27 genes/proteins with peptide tags 1 . The Km R cassette from pKD13 was amplified using the 28 ptsN-del-F and ptsN-del-R, and glmS-del-F and glmS-del-R primers, respectively, to 29 construct the SR7001 (ΔptsN) and SR7002 (ΔglmS) strains, and the resulting PCR products 30 were introduced into the SL1344 strain containing the pKD46 plasmid. Recombinant bacteria 31 containing the Km R cassette in place of the target genes were detected using kanamycin 32 resistance and diagnostic PCR. The Km R cassette was further removed using the pCP20 33 plasmid 1 . GlcNAc (0.2%) was added to the medium during ΔglmS selection to complement 34 the lethality of ΔglmS. lon was deleted and EIIA Ntr was tagged with the FLAG peptide at the 35 C-terminus using a phage λ-mediated recombination system 1,2 . The strain carrying a lacZ 36 fusion to ssaG was constructed similarly as described above. The Km R cassette that was 37 PCR-amplified from the pKD3 plasmid using the ssaG-lacZ-F and ssaG-lacZ-R primers was 38 introduced downstream of ssaG and then replaced with the lacZY genes of pCE70 via the 39 FRT site. The primers used to construct the bacterial strains are listed in Supplementary Table   40 S3.

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Plasmid construction 43 For the construction of pWJ04 producing EIIA Ntr under its putative intrinsic promoter, the 44 DNA containing the Salmonella ptsN gene was amplified by PCR using primers of ptsN-45 comple-F and ptsN-comple-R, and the PCR products were introduced between the HindIII 46 and SphI sites of pACYC184. To construct pWJ06 expressing EIIA Ntr under the lac promoter, 47 the ptsN gene was PCR amplified using the ptsN-F and ptsN-R primers and the purified PCR 48 3 fragments were inserted between the EcoRI and BamHI sites of the pUHE21-2lacI q vector 3 .

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The ptsN gene was also amplified using the ptsN-His-F and ptsN-His-R primers, and the PCR 50 products were introduced between the EcoRI and BamHI sites of the pUHE21-2lacI q vector 51 to construct pWJ07 producing EIIA Ntr tagged with six histidines at its C-terminus. The 52 pWJ10 plasmid expressing GlmS from the lac promoter was constructed by introducing PCR 53 fragments containing glmS between the BamHI and HindIII sites of the pUHE21-2lacI q 54 vector. The glmS-F and glmS-R primers were used to construct pWJ10. The pKT25-ptsN and 55 pUT18C-glmS plasmids were constructed for the bacterial two-hybrid analysis. The ptsN and 56 glmS genes were amplified using the ptsN-BTH-F and ptsN-BTH-R, and glmS-BTH-F and 57 glmS-BTH-R primers, respectively. The purified PCR products were inserted between the 58 BamHI and EcoRI sites of the pKT25 plasmid vector or between the BamHI and SacI sites of 59 the pUT18C plasmid vector 4 . The pWJ11, pWJ12, pWJ13, and pWJ14 plasmids were 60 constructed to extract and purify EI Ntr , NPr, EIIA Glc , and GlmS proteins, respectively. The 61 pET28a vector was used to purify soluble proteins containing a His 6 -tag at their N-termini.  Fig. S1). The pWJ15, pWJ16, and pWJ17 plasmids were constructed for the pull-down assay.

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The pETDuet-1 vector was used to express His 6 -GlmS and EIIA Ntr . The glmS and ptsN genes  with the E. coli BTH101 reporter strain, which lacks an endogenous functional cyaA gene.

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The proteins to be tested were fused to the T25-and T18-fragments of the CyaA protein from   Plasmids of pWJ07, pWJ08, and pWJ09 using pUHE21-2lacI q as a backbone were 156 introduced into the ΔptsN mutant strain harboring PssaG-lacZ. They were designed to express 157 EIIA Ntr -His 6 , EIIA Ntr -His 6 (H73A), and EIIA Ntr -His 6 (K75D), respectively, upon induction 158 with IPTG (0.1 mM). The β-galactosidase assay was performed in triplicate as described