Supplemental Material for: Nutritional control of antibiotic resistance via an interface between the phosphotransferase system and

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1 Supplemental Material for: Nutritional control of antibiotic resistance via an interface between the phosphotransferase system and a two-component signaling system Holly Snyder, Stephanie L Kellogg, Laura M Skarda, Jaime L Little, and Christopher J Kristich* 1

2 Table S1. Median minimal inhibitory concentrations a for antibiotics (μg/ml) in MHB. Strain b ceftazidime (cephalosphorin) chloramphenicol (ribosomal inhibitor) Wild-type ΔptsH a MICs were determined after 24 h at 37 C in MHB from a minimum of 3 independent experiments. b Strains used were: Wild-type, OG1; ΔptsH2, JL395. 2

3 Table S2. E. faecalis HPr mutants exhibiting impaired in vivo association with CroR but not HPr Mutation E2G Secondary structure element in HPr β strand A F29L loop 2 S31T loop 2 V55D loop 5 G67V loop 6 E70G M74T M74R M74K Q82H helix C helix C helix C helix C helix C 3

4 Table S3. Strains and plasmids used in this study Strain or plasmid Relevant genotype or description Source or reference Strains E. coli TOP10 routine cloning host Invitrogen Mach1-T1 R cloning host during Gateway constructions Invitrogen DH5α cloning host for pcjk218-based plasmids lab stock E. faecalis OG1 Wild-type reference strain (MLST 1) (1) JL395 OG1 ΔptsH2 This work JL422 OG1 ptsh E2G This work JL425 OG1 ptsh E70G This work SB23 OG1 ΔcroR2 This work SB27 JL425 ΔcroR2 This work SB31 JL422 ΔcroR2 This work SB39 JL395 ΔcroR2 This work OG1RF Spontaneous rifampicin and fusidic acid-resistant derivative of (2) OG1 T1 (SS498) Wild-type (MLST 21), CDC reference strain (3) E. faecium Com12 Fecal isolate (4) 1,141,733 Clinical isolate (4) Plasmids pcjk106 Plasmid carrying cror -lacz fusion (Em R ) C. Kristich, pcjk218 E. faecalis allelic exchange vector (Cm R ) (5) pjll80 ΔptsH2 (ΔH15-L81) in pcjk218 This work pjll85 ptsh E2G in pcjk218 This work pjll88 ptsh E70G in pcjk218 This work pslb38 ΔcroR2 (ΔI46-Q217) in pcjk218 This work pjrg8 E. faecalis expression vector w/ constitutive P23s promoter (6) (Em R ) pjll92 P ptsh -ptsh in pjrg8 (contains 275 nt upstream of ptsh) This work pjll100 P ptsh -ptsh H15A in pjrg8 This work pjll101 P ptsh -ptsh S46A in pjrg8 This work pjll102 P ptsh -ptsh H15A S46A in pjrg8 This work plms154 ptsh-f [1,2] in pjrg8 This work plms1749f12 hprk-f [1,2] in pjrg8 This work plms3289f12 cror-f [1,2] in pjrg8 This work pci3340 E. coli/e. faecalis shuttle vector (Cm R ) (7) pjrg9 constitutive P23s promoter in pci3340 (Cm R ) This work plms148 cror-f [3] in pjrg9 This work plms152 ptsh-f [3] in pjrg9 This work plms ptsh E2G-F [3] in pjrg9 This work plms ptsh E70G-F [3] in pjrg9 This work 4

5 plms168 ptsh H15A-F [3] in pjrg9 This work plms170 ptsh S46A-F [3] in pjrg9 This work pat18 E. coli/e. faecalis shuttle vector (Em R ) (8) pbk200 E. faecalis expression vector, constitutive P23 promoter (Em R ) This work phs11 cror-strep in pbk200 This work pdonr221 Gateway entry clone vector Invitrogen pdonr221- EF1039 pdonr221- EF1714 Gateway entry clone containing EF1039 ORF H. He and C. Kristich, Gateway entry clone containing EF1714 ORF H. He and C. Kristich, phh3 Gateway destination vector based on pjrg8 for fusion to F [1,2] H. He and C. Kristich, phh6 Gateway destination vector based on pbk200 for fusion to Strep H. He and C. Kristich, phh8 Gateway destination vector based on pjrg9 for fusion to F [3] H. He and C. Kristich, phs12 EF1714-Strep in phh6 This work phs13 EF1714-F [1,2] in phh3 This work phs14 EF1039-F [3] in phh8 This work phs22 ptsh H15A S46A-F [3] in pjrg9 This work puab100 source of mdhfr F [1,2] fragment (9) puab200 source of mdhfr F [3] fragment (9) Supplemental References 1. Gold, O. G., H. V. Jordan, and J. van Houte The prevalence of enterococci in the human mouth and their pathogenicity in animal models. Arch. Oral Biol. 20: Dunny, G. M., B. L. Brown, and D. B. Clewell Induced cell aggregation and mating in Streptococcus faecalis: evidence for a bacterial sex pheromone. Proc. Natl. Acad. Sci. U.S.A. 75: Maekawa, S., M. Yoshioka, and Y. Kumamoto Proposal of a new scheme for the serological typing of Enterococcus faecalis strains. Microbiol Immunol 36: Palmer, K. L., P. Godfrey, A. Griggs, V. N. Kos, J. Zucker, C. Desjardins, G. Cerqueira, D. Gevers, S. Walker, J. Wortman, M. Feldgarden, B. Haas, B. Birren, and M. S. Gilmore Comparative genomics of enterococci: variation in Enterococcus faecalis, clade structure in E. faecium, and defining characteristics of E. gallinarum and E. casseliflavus. MBio 3:e Vesic, D., and C. J. Kristich A Rex family transcriptional repressor influences H 2 O 2 accumulation by Enterococcus faecalis. J. Bacteriol. 195: Kristich, C. J., J. L. Little, C. L. Hall, and J. S. Hoff Reciprocal regulation of cephalosporin resistance in Enterococcus faecalis. MBio 2:e Hayes, F., C. Daly, and G. F. Fitzgerald Identification of the Minimal Replicon of Lactococcus lactis subsp. lactis UC317 Plasmid pci305. Appl. Environ. Microbiol. 56:

6 8. Trieu-Cuot, P., C. Carlier, C. Poyart-Salmeron, and P. Courvalin Shuttle vectors containing a multiple cloning site and a lacz alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria. Gene 102: Singh, A., D. Mai, A. Kumar, and A. J. Steyn Dissecting virulence pathways of Mycobacterium tuberculosis through protein-protein association. Proc. Natl. Acad. Sci. U.S.A. 103:

7 Fig S1. Fig S1. Simplified overview of the PTS in Gram-positive bacteria. Enzyme I (Enz I) and HPr are general PTS proteins that function in transport of all PTS substrates. During uptake of carbohydrates that are substrates of the PTS, Enz I uses phosphoenol pyruvate (PEP)) to autophosphorylate. This phosphoryl group is transferred to HPr on His15 in an intermediate step. HPr-His15-P subsequently transfers this phosphoryl group to a carbohydrate-specific transporter known as an Enzyme II ( Enz II). Enz IIs are comprised of several domains, minimally including the Enz IIa, Enz IIb, and integral membrane Enz IIc domains. The phosphoryl group is passed from HPr-His15-P (glucose in this example) that passes through the Enz IIc component. HPr can also be reversibly phosphorylated on Ser46 by the bifunctional kinase/phos phorylase HprK, whose activities are modulatedd by metabolites (not shown). HPr-Ser46-PP interacts as a co-repressor with the DNA-binding transcription factor CcpA, enabling the complex to bind DNA and modulate transcriptionn of target genes (catabolite repression or activation). Our results indicate that HPr can also associate with CroR, the response regulator component of the CroR/S two-component signaling system that is required forr cephalosporin resistance in E. faecalis, to modulate CroR-dependent gene expression and cephalosporin resistance. We have not yet established with isoform of HPr is important for association with CroR and thereforee have depicted potential associations with HPr-His15-P or HPr-Ser46-P. Association of CroR with unphosphorylated HPr is also a formal possibility, although our results suggest this is the least likely to Enz IIa and subsequently to Enz IIb before transfer to the incoming carbohydrate alternative. 7

8 Fig S2. Fig S2. HPr mutants with substitutions at the sites of phosphorylation are impaired at association with CroR. Mutants of E. faecalis HPr with substitutions at the sites of phosphorylation exhibit impaired association with CroR in vivo. (A) E. faecalis strains co-expressing the indicated fusions weree subjected to 10-fold serial dilutions and inoculated on MH agar supplemented with Cm and Em (for plasmid selection) in the presence or absence of trimethoprim. The HPr mutants exhibit a moderately reduced ability to promote growth in the presence of trimethoprim compared to wild-type HPr (colony formation reduced by 10-fold compared to wild-type), OG1RF; plasmid combinations were: plms3289f12 + plms152 (top); plms3289f12 + plms168 (upper middle); plms3289f12 + plms170 (lower middle); and plms3289f12 + phs22 (bottom) (B) Co-immunoprecipitation from E. faecalis lysates using anti-f [3] antisera. Lysates and immunoprecipitates were subjected to immunoblot analysis with antiseraa specific for the strep or F [3] fusions, revealing that CroR no longer co-precipitates with non-phosphorylatable HPr. Host strain was E. faecalis OG1RF; plasmid combinations were: phs11 + plms152 (left lane) and phs11 + phs22 (right lane). Lysates corresponds to the whole-cell lysates used as input for co-immunoprecipitation; IP corresponds to the immune complexes recovered after co-immunoprecipitation. indicative of reduced in vivo association of the fusions. Host strain was E. faecalis 8

9 Fig S3. Fig S3. mdhfr analysis of HPr mutants with impaired CroR association. Mutants of E. faecalis HPr that exhibit impaired association with CroR in vivo weree identified using mdhfr PCA, as described in Materials and Methods. E. faecalis strains co-expressing the indicated fusions were subjected to 10-fold serial dilutions and inoculated on MH agar supplemented with Cm and Em (for plasmid selection) and trimethoprim at 1 μg/ml. Whereas thee wild-type HPr-F [3] fusion enables growth when co-expressed with F [1,2] fusionss of both CroR (left) andd HPr (right),, indicative of in vivo association, the HPr mutants only enable growth when co-expressed with HPr. Host strain was E. faecalis OG1RF. 9

10 Fig S4 Fig S4. HPr point mutants that do not interact with CroR, as determined by mdhfr PCA, also do not co-immunoprecipitate with CroR. Association of CroR and HPr in whole-cell lysates of E. faecalis was assessed by co-immunoprecipitation with anti-f [3] antisera. CroR-Strep was co-expressed with wild-type HPr, HPr E2G, or HPr E70G fused to mdhfr F [3] as indicated. Lysates and immunoprecipitates weree subjected to immunoblot analysis with antisera specific for the Strep or F [3] fusions, revealing that CroR specifically co-precipitates with wild-type HPr but not the HPr mutants. Host strain was E. faecalis OG1RF. Lysates corresponds to the whole-cell lysatess used as input for co-immunoprecipitation; IP corresponds to the immune complexes recovered after coimmunoprecipitation. 10