CozE is a member of the MreCD complex that directs cell elongation in Streptococcus pneumoniae

1 1CozE is a member of the MreCD complex that directs cell elongation in 2Streptococcus pneumoniae 4Andrew K Fenton1, Lamya El Mortaji1, Derek T C Lau1, David Z Rudner1*, Thomas G 5Bernhardt1* 71Department of Microbiology and Immunobiology 8Harvard Medical School 9Boston, MA 02115 10 11 12*To whom correspondence should be addressed 13 14David Z Rudner, Ph.D 15Harvard Medical School 16Department of Microbiology and Immunobiology 17Boston, Massachusetts 02115 18e-mail: david_rudner@hms.harvard.edu 19 20Thomas G Bernhardt, Ph.D 21Harvard Medical School 22Department of Microbiology and Immunobiology 23Boston, Massachusetts 02115 24e-mail: thomas_bernhardt@hms.harvard.edu 25 Most bacterial cells are surrounded by a peptidoglycan (PG) cell wall that is 26essential for their integrity Major synthases of this exoskeleton are called 27penicillin-binding-proteins (PBPs) 1,2 Surprisingly little is known about how cells 28control these enzymes given their importance as drug targets In the model gram29negative bacterium Escherichia coli, outer membrane lipoproteins are critical 30activators of the class A PBPs (aPBPs) 3,4 , bifunctional synthases capable of 31polymerizing and crosslinking PG to build the exoskeletal matrix Regulators of 32PBP activity in gram-positive bacteria have yet to be discovered but are likely to 33be distinct due to the absence of an outer membrane To uncover gram-positive 34PBP regulatory factors, we used transposon-sequencing (Tn-Seq) to screen for 35mutations affecting the growth of Streptococcus pneumoniae cells when the 36aPBP synthase PBP1a was inactivated Our analysis revealed a set of genes that 37were essential for growth in wild-type cells yet dispensable when pbp1a was 38deleted The proteins encoded by these genes included the conserved cell wall 39elongation factors MreC and MreD 2,6,7 as well as a membrane protein of unknown 40function (SPD_0768) that we have named CozE (coordinator of zonal elongation) 41Our results indicate that CozE is a member of the MreCD complex of S 42pneumoniae that directs the activity of PBP1a to the midcell plane where it 43promotes zonal cell elongation and normal morphology CozE homologues are 44broadly distributed among bacteria, suggesting they represent a widespread 45family of morphogenic proteins controlling cell wall biogenesis by the PBPs 46 To investigate PBP regulation in gram-positive organisms we used the ellipsoid- 47shaped bacterium S pneumoniae as a model system In addition to its interesting 48morphology, this bacterium is an important human pathogen and the causative agent of 49many invasive diseases Antibiotic resistance in S pneumoniae is on the rise 50worldwide New drugs to combat resistance in this and other bacterial pathogens are 51therefore needed A better understanding of the regulation and cellular function of 52proven target enzymes like the PBPs will aid the development of such therapies 53 S pneumoniae encodes three aPBPs (pbp1a, pbp1b and pbp2a), with 54pbp1a and pbp2a forming an essential pair Either gene can be deleted individually, 55but attempts to inactivate both genes have been unsuccessful We reasoned that the 56lethal phenotype of pbp1a pbp2a double mutants could form the basis of a screen for 57gram-positive PBP regulators analogous to previous work that identified the Lpo 58regulators of the E coli aPBPs The set of mutants synthetically lethal with a deletion 59of pbp1a is predicted to include factors required for the in vivo function of PBP2a 60Similarly, a screen for mutants synthetically lethal with ∆pbp2a should identify factors 61required for PBP1a activity To identify synthetic interactions, we performed Tn-Seq (IN62Seq, HITS, TraDIS) 5,10,11 using transposon libraries generated in strain D39 lacking its 63capsule (Δcps) and derivatives inactivated for PBP1a and PBP2a This approach 64revealed several factors, which will be investigated in a separate report Here, we focus 65on the characterization of an unexpected class of factors with a distinct and intriguing 66phenotype related to PBP1a, an aPBP that is associated with high-level antibiotic 67resistance 12 and is indispensable for host colonization 13 The genes encoding these 68proteins were found to be virtually devoid of insertions in the wild-type transposon 69library, indicating they are likely essential for growth (Fig 1A, Supplementary Fig 1) 70Strikingly, however, the same genes appeared to be readily inactivated in the Δpbp1a 71library but not the Δpbp2a library (Fig 1A, Supplementary Fig 1), suggesting that 72pbp1a disruption suppresses their essentiality Two of the genes encode MreC and 73MreD, conserved members of the PG biogenesis machinery that promotes cell 74elongation in rod- and ellipsoid-shaped bacteria 2,6,7 The third gene, spd_0768, 75encodes CozE, a conserved polytopic membrane protein of unknown function that 76belongs to the widely-distributed UPF0118 protein family 14 (Fig 1A-C and 77Supplementary Fig 2-3) Like MreC, CozE homologs are absent from the Mollicutes, 78which lack a cell wall, suggesting a role for CozE in PG biosynthesis (Supplementary 79Fig 3) 80 The essentiality of mreC and mreD and its suppression by PBP1a inactivation 81were expected from prior work of Winkler and colleagues 7,15 We confirmed these 82results using a Δpbp1a strain with an ectopic copy of pbp1a under control of a zinc83regulated promoter 16 (Pzn::pbp1a) In this strain background, deletion mutants of mreC, 84mreD, cozE, or both mreC and cozE were viable in the absence of zinc (Fig 1D, 85Supplementary Fig 4) However, the viability of these strains was severely 86compromised on solid medium supplemented with zinc (Fig 1D, Supplementary Fig 874) As an additional confirmation, we deleted mreC or cozE in strain R6, which harbors 88a hypomorphic pbp1a allele 17 and found that both mutants were viable displaying only 89mild morphological defects (Supplementary Fig 5A) Furthermore, expression of the 90pbp1a gene from strain D39 was lethal in mreC or cozE R6 deletion mutants 91(Supplementary Fig 5B) In liquid culture, pbp1a induction was tolerated in wild-type 92or Δpbp1a cells of strain D39, but caused cell lysis in Δpbp1a ΔmreC and Δpbp1a 93ΔcozE double mutants as well as the Δpbp1a ΔmreC ΔcozE triple mutant (Fig 2A-B 94and Supplementary Fig 6B) Upon pbp1a induction, these mutants first displayed a 95cell chaining phenotype followed by significant rounding and swelling of cells in the 96chains before most cells in the culture lysed (Fig 2B and Supplementary Fig 6D) 97Similar phenotypes were observed upon CozE or MreCD depletion in an otherwise wild98type background (Supplementary Fig 7) The PBP1a-induced lysis phenotype 99appeared to be more pronounced in cells lacking both CozE and MreC (Fig 2A) 100However, the drop in viability for single mreC or cozE mutants was similar to that of the 101double mutant (Supplementary Fig 6C), suggesting these factors function in the same 102pathway Deletion of lytA or cbpD encoding the major S pneumoniae autolysins did not 103dramatically alter the lytic effect of PBP1a production in the absence of CozE or MreC, 104indicating that the growth and lysis phenotypes did not result from misactivation of these 105autolysins 18,19 (Supplementary Fig 8) 106 The genetic results suggest a model in which CozE works with the MreCD 107complex to control PG synthesis by PBP1a and that in their absence, deranged PBP1a 108activity causes cell lysis To test this possibility, we monitored PG biogenesis activity 109using the fluorescent D-amino acid TADA (tetramethylrhodamine 3-amino-D-alanine) 11020,21 and the localization of a functional GFP-PBP1a fusion (Supplementary Fig 9) in 111cells inactivated for CozE or MreC As with the untagged version, production of GFP112PBP1a in cells lacking MreC or CozE resulted in a severe growth defect (Fig 3A and 113Supplementary Fig 10A) This phenotype was accompanied by a change in GFP114PBP1a localization and TADA labelling from their normally tightly restricted zone at 115midcell to a widely distributed pattern throughout the cell periphery The ΔmreC cells 116displayed a more severe labelling defect versus ΔcozE cells as expected from the 10 11 117above morphological analysis (Fig 3B-C and Supplementary Fig 11) Similar 118alterations in TADA labelling were observed following the production of untagged PBP1a 119in the mutant strains (Supplementary Fig 12) Importantly, variants of GFP-PBP1a 120inactivated for either PG polymerase/glycosyltransferase activity [GFP-PBP1a(GT -)] or 121PG crosslinking/transpeptidase activity [GFP-PBP1a(TP -)] similarly lost their midcell 122localization in cells lacking CozE or MreC, but this delocalization was not associated 123with a change in TADA labelling nor did it cause a significant growth defect (Fig 3B-C 124and Supplementary Fig 10A) Cells lacking CozE or MreC did not affect midcell 125localization of GFP-PBP2a, suggesting a specific role in PBP1a recruitment 126(Supplementary Fig 13) 127 A functional GFP-CozE fusion (Supplementary Fig 7A) displayed a septal 128localization pattern that was dependent upon MreC (Fig 4A and Supplementary Fig 12914) Reciprocally, the midcell localization of GFP-MreC required CozE (Supplementary 130Fig 14) Moreover, bacterial two-hybrid analysis 22 in E coli indicates that CozE forms a 131complex with MreCD and PBP1a (Fig 4B and Supplementary Fig 15) Finally, a 132functional FLAG-CozE fusion was coimmunoprecipitated with GFP-PBP1a but not GFP133PBP2a (Fig 4C and Supplementary Fig 16) Altogether, these data indicate that 134CozE is a member of the MreCD morphogenic complex in S pneumoniae and that this 135complex coordinates cell elongation in part by interacting with and restricting PBP1a to 136midcell (Fig 4D) 137 In rod-shaped bacteria, MreC and MreD are part of the Rod system that 138elongates the cylindrical portion of the cell wall 23 The system is organized by dynamic 139filaments of MreB that facilitate the incorporation of PG at dispersed locations 12 13 140throughout the cylinder 24 In contrast, S pneumoniae and other ovococci elongate in a 141restricted zone by incorporating PG at the periphery of the cytokinetic ring 25 These 142bacteria lack MreB, but retain the other components of the Rod system, including MreC 143and MreD It was recently shown that the SEDS-family protein RodA is the core PG 144polymerase within the Rod system of Bacillus subtilis and E coli 26,27 Additionally, it was 145found that although aPBP polymerases principally work outside of the MreB-directed 146machinery, the two systems display some interdependence through an as yet ill-defined 147coordination mechanism 27 The results presented in this report suggest the possibility 148that CozE and related proteins might serve as part of this coordination mechanism by 149connecting PBP1a with RodA and other components of the elongation machinery via its 150interactions with PBP1a and the MreCD complex In this case, CozE may be essential 151in S pneumoniae because the spatial localization of aPBPs and their potential 152coordination with SEDS-family PG polymerases is especially critical for proper PG 153biogenesis in organisms where zonal cell wall expansion is the principal mode of 154growth Such a localized mode of cell elongation is not unique to the ovococci In 155addition to the dispersed mode of growth promoted by the Rod system, rod-shaped 156bacteria like E coli and Caulobacter cresentus have also been found to undergo zonal 157elongation for a portion of the cell cycle preceding division 28–30 The broad conservation 158of CozE suggests that it could more generally recruit and coordinate PG synthetic 159functions during zonal growth in a range of bacteria Further characterization of CozE in 160S pneumoniae and other organisms will provide deeper mechanistic insight into its 161function and reveal new strategies for disrupting PG biogenesis for antibiotic 162development 14 15 163METHODS 164Strains, plasmids and routine growth conditions Unless otherwise indicated, all S 165pneumoniae strains in this study were derived from D39 Δcps 17 or R6 17,31,32, a non166pathogenic derivative of D39 Cells were grown in Todd Hewitt broth containing 0.5% 167Yeast Extract (THY) at 37oC in an atmosphere containing 5% CO2 Strains were grown 168on pre-poured Tryptic Soy Agar 5% sheep blood plates (TSAII 5%SB, Becton Dickinson; 169BD) with a ml overlay of 1% Nutrient Broth (NB) agar containing additives When finer 170control of media components was required, TSA plates containing 5% defibrinated 171sheep blood were used Tables of all strains, plasmids, and oligonucleotides used in this 172study are provided as supplementary material (Supplementary Table 1-4) 173 174Transformation Cells in mid-exponential phase were grown in THY and back diluted to 175an OD600 of 0.03 Competence was induced with 500 pg ml -1 Competence Stimulating 176Peptide (CSP-1), 0.2% BSA and mM CaCl2 Typically ml of culture was transformed 177with 100 ng of gDNA or plasmid DNA Transformants were selected on TSAII overlay 178plates containing: µg ml-1 chloramphenicol, 0.2 µg ml-1 erythromycin, 250 µg ml-1 179kanamycin, 200 µg ml-1 spectinomycin or 0.2 µg ml-1 tetracycline as appropriate 180 181S pneumoniae strain construction 182 183S pneumoniae deletion strains All S pneumoniae deletion strains were generated 184using linear PCR fragments, similar to the method used by Robertson et al 33 Two ≈1 kb 16 17 185flanking regions of each target gene were PCR amplified and an antibiotic resistance 186marker placed between them using isothermal assembly 34 Assembled PCR products 187were transformed directly into S pneumoniae as described above In all cases, deletion 188primers were given the typical name: ‘gene-designation’_5FLANK_F/R for 5’ regions 189and ‘gene-designation’_3FLANK_F/R for 3’ regions Antibiotic markers were amplified 190from ΔbgaA strains using the AntibioticMarker_F/R primers using gDNA isolated from 191strains: AKF_Spn001-005 A full list of primer sequences can be found in 192Supplementary Table Transformants were picked into ml THY, grown to 193exponential phase and frozen without undergoing autolysis Deletion strains were 194confirmed by diagnostic PCR using the AntibioticMarker_R primer in conjunction with a 195primer binding ≈ 100 bp 5’ of the disrupted gene; these primers were given the typical 196name: ‘ORFdesigation’_Seq_F Diagnostic PCRs gave ≈ 2-2.5 kb PCR products, 197depending on the marker, which was not present in wt controls 198 199Confirmed gDNA preparations of single gene deletions were diluted to 20 ng μl -1 and 200used for the construction of multiple knock out strains For strains containing multiple 201deletions and construct integrations, transformants were verified by diagnostic re202streaking on media containing antibiotics In special cases where more confidence was 203desirable, each construct was confirmed by diagnostic PCR 204 205Antibiotic-marked ΔbgaA strains Strains containing a variety of antibiotic resistance 206cassettes inserted at the bgaA locus served as the source of all markers used in this 207study In all cases cassettes were modified to make them compatible with amplification 18 19 208by the AntibioticMarker_F and AntibioticMarker_R primers This has the advantage of all 209antibiotic markers being compatible with a single set of primers, which makes cloning 210and antibiotic marker replacement a simple process 211 212For construction of the bgaA PCR knock out constructs The chloramphenicol resistance 213cassette was amplified from pAC1000 35 using primers: Chlor_isoT_F/R The kanamycin 214and erythromycin resistance cassettes were amplified from pDR240 and pDR242 215respectively, using primers: AntibioticMarker_F/R The Janus cassette was used as the 216original source of the kanamycin marker 36 The spectinomycin resistance cassette was 217amplified from pMagellan6 using primers: Spec_isoT_F/R Finally, the tetracycline 218resistance cassette was amplified from pJWV025 37 using primers: Tet_isoT_F/R A 5’ 219flanking region of bgaA and a 3’ bgaA ORF fragment was amplified using primers: 220BgaA_5FLANK_F/R and BgaA_3ORF_F/R Amplified bgaA fragments were combined 221with each resistance marker using isothermally assembly 34, transformed into S 222pneumoniae and selected on media containing the appropriate antibiotic Integration of 223each resistance cassette at the bgaA locus was confirmed by diagnostic PCR using the 224bgaA flanking primer: bgaA_FLANK_F and AntibioticMarker_R Resulting strains were 225given the names AKF_Spn001-005 (see Supplementary Table 1) 226 227Pzn::pbp1a The Pczc promoter 16, henceforth known as Pzn, was amplified from pJW025 22837 using primers: oSP104 and oSp105 The pbp1a ORF was amplified from the D39 229genome using oSp106 and oSp107 and added to the first ‘P zn’ fragment by isothermal 230assembly The resulting product was digested with BamHI and XhoI and ligated into 20 10 43 480for signal heterogeneity Single colonies were picked into 150 μl LB Amp 50 Kan25 481containing 500 µg ml−1 Isopropyl β-D-1-thiogalactopyranoside (IPTG500) in 96 deep-well 482plates and incubated at 30oC μl of resulting cultures were spotted onto LB Amp 50 483Kan25 IPTG500 Xgal40 plates Plates were incubated at 30oC in an environment protected 484from light and imaged Plates were picked in triplicate with selected images 485representative of three biological replicates 486 487Immunoblot analysis S pneumoniae cultures were normalized to an OD600 of 0.3 488and ml harvested Cell extracts were prepared by resuspension of cell pellets in 100 μl 489lysis buffer (20 mM Tris pH 7.5, 10 mM EDTA, mg ml−1 lysozyme, 10 μg ml−1 DNase I, 490100 μg ml−1 RNase A, with protease inhibitors: mM phenylmethane sulfonyl fluoride 491[PMSF], μg ml−1 leupeptin, μg ml−1 pepstatin) and incubation at 37°C for 10 min, 492followed by addition of 10 μl 10% Sarcosyl for 100 μl SDS sample buffer (0.25 M 493Tris pH 6.8, 4% SDS, 20% glycerol, 10 mM EDTA) containing 10% 2-mercaptoethanol 494was added to each prep and samples were heated for 15 at 50°C prior to loading 49510 μl per lane Proteins were separated by SDS-PAGE on 12.5% polyacrylamide gels, 496electroblotted onto a PVDF membrane and blocked in 5% non-fat milk in PBS-0.5% 497Tween-20 The blocked membranes were probed with rabbit anti-FtsE (1:20,000) 46 and 498affinity-purified rabbit anti-GFP (1:10,000) diluted into 3% BSA in 1x PBS-0.05% Tween49920 Primary antibodies were detected using horseradish peroxidase-conjugated goat 500anti-rabbit IgG (1:20,000, BioRad) and the Western Lighning Plus ECL reagent as 501described by the manufacturer (PerkinElmer) Membrane chemiluminescence was 502imaged on a FluorChem R system (ProteinSimple) 44 22 45 503 504Co-immunoprecipitation Assay S pnemoniae strains were grown in 60 ml THY in 505the presence of 400 μM ZnCl2 at 37oC in 5% CO2 50 ml of these cultures was matched 506to a starting OD600 of 0.5 Cells were harvested by centrifugation at 5,000 g for 507and cell pellets re-suspended in 25 ml SMM (1 M Sucrose, 40 mM Maleic acid, 40 mM 508MgCl2, pH 6.5) Cells were washed a second time and finally re-suspended in ml 509SMM Cell protoplasts were generated by enzymatic digestion of the cell wall with mg 510ml-1 lysozyme, cells not lyse due to the osmotic potential of the SMM Protoplasts 511were pelleted at 5,000 g for and re-suspended in ml cold Hypotonic Buffer 512‘Buffer-H’ (20 mM HEPES [Na+], 100 mM NaCl, 1mM dithiothreitol [DTT], mM MgCl2, 513mM CaCl2, mM phenylmethane sulfonyl fluoride [PMSF], µM leupeptin, µM 514pepstatinA) The protoplasts lysed in H-buffer in due to the change in buffer osmolarity 515Lysates were further treated with: DNase µg ml-1, RNaseA 12 µg ml-1 and mg ml-1 516lysozyme to form a crude extract These extracts were pelleted by ultracentrifugation at 517100,000 g for h at 4oC Crude membrane pellets were dispersed in 420 µl Glycerol 518Buffer ‘Buffer-G’ (20 % glycerol, 20 mM HEPES [Na +], 100 mM NaCl, 1mM dithiothreitol 519[DTT], mM MgCl2, mM CaCl2, mM PMSF, µM leupeptin, µM pepstatinA) and 520stored at -80oC 50 µl aliquots of crude membranes were solubilized with 450 µl Buffer521G-DIG (Buffer-G, 5% digitonin) for h at 4oC with gentle agitation followed by 522ultracentrifugation at 100,000 g for h at oC 400 µl of digitonin-solubilized membrane 523proteins were incubated with anti-GFP sepharose resin for h at oC with slow 524agitation The unbound material was removed (Flow Through) and the resin washed 525four times with 400 µl buffer Buffer-G-DIG (decreasing to a final concentration of 0.05% 46 23 47 526digitonin) Proteins were eluted from the resin with 100 µl SDS-PAGE sample buffer 527heated to 50oC for 15 Samples were denatured for 15 at 50°C prior to loading 528Proteins were separated by SDS-PAGE on 15% polyacrylamide gels, electroblotted 529onto a PVDF membrane and blocked in 5% non-fat milk in PBS-0.5% Tween-20 The 530blocked membranes were probed with mouse monocolonal anti-GFP (1:5000, Sigma) or 531mouse monocolonal anti-FLAG M2 (1:1000, Sigma) diluted into 3% BSA in 1x PBS5320.05% Tween-20 Primary antibodies were detected using horseradish peroxidase533conjugated goat anti-mouse IgG (1:10000, Biorad) and the Western Lightning Plus ECL 534reagent as described by the manufacturer (PerkinElmer) Membrane 535chemiluminescence was imaged on a FluorChem R system (ProteinSimple) 48 24 49 536SUPPLEMENTARY INFORMATION 537Supplementary Information is available online and includes Supplementary Figures and 538Supplementary Methods 539 540ACKNOWLEDGEMENTS 541The authors would like to thank all members of the Bernhardt and Rudner laboratories 542for support and helpful comments Andrew Fenton was a jointly mentored postdoctoral 543fellow bridging work in both labs Special thanks to Rachel Yunck, Harvey Kimsey, 544Malcolm Winkler, Tim van Opijnen, Andy Camilli, Nathalie Campo, Jan-Willem Veening, 545Thierry Vernet and Don Morrison for strains, reagents, and technical assistance This w 546ork was supported by the National Institutes of Health (R01AI083365 to TGB, CETR 547U19 AI109764 to TGB and DZR, GM073831 to DZR, and RC2 GM092616 to DZR) 50 25 51 548AUTHOR CONTRIBUTIONS 549AKF performed all experiments, designed part of the experimental program and co550authored the manuscript LEM carried out essential pilot experiments for the project 551DTCL helped adopt the Tn-seq data analysis pipeline and proofread the manuscript 552DZR and TGB co-supervised the project and co-authored the manuscript 553 554ADDITIONAL INFORMATION 555Reprints and permissions information is available at www.nature.com/reprints The 556authors declare that they have no competing financial interests Correspondence and 557requests for materials should be addressed to TGB and DZR 558 559DATA AVAILABILITY 560The data that support the findings of this study are available from the corresponding authors 561upon request 52 26 53 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of the Himar1 mariner transposon Proc Natl 683 Acad Sci U S A 96, 11428–33 (1999) 68443 Langmead, B., Trapnell, C., Pop, M & Salzberg, S L Ultrafast and memory- 685 efficient alignment of short DNA sequences to the human genome Genome Biol 686 10, R25 (2009) 68744 Carver, T., Harris, S R., Berriman, M., Parkhill, J & McQuillan, J A Artemis: An 64 32 65 688 integrated platform for visualization and analysis of high-throughput sequence- 689 based experimental data Bioinformatics 28, 464–469 (2012) 69045 Letunic, I & Bork, P Interactive Tree Of Life (iTOL): An online tool for 691 phylogenetic tree display and annotation Bioinformatics 23, 127–128 (2007) 69246 Meisner, J et al FtsEX is required for CwlO peptidoglycan hydrolase activity 693 during cell wall elongation in Bacillus subtilis Mol Microbiol 89, 1069–83 (2013) 694 66 33 67 695FIGURE LEGENDS 696Figure | The essential genes cozE and mreCD can be deleted in cells lacking 697PBP1a a, Mariner transposon libraries were generated in wt (D39 Δcps) and Δpbp1A 698mutant strains Transposon insertions in each library were identified by deep 699sequencing and mapped onto the D39 genome The height of each line reflects the 700number of sequencing reads at this position Boxes highlight loci significantly enriched 701(p700 cell units were scored per time 762point, n = Demograms of GFP-PBP1a and TADA signal profiles at the h time point 763are shown in Supplementary Fig 11 Similar TADA labelling to those shown in b and c, 764using untagged PBP1a can be found in Supplementary Fig 12 765 766Figure | CozE is a member of the MreCD cell elongation complex 767a, CozE is enriched at mid-cell S pneumoniae cells expressing GFP-CozE under the 768control of a fucose-inducible promoter were grown to mid-exponential phase in THY 769+0.4% fucose, spotted onto 2% agarose pads and imaged In rare cases GFP-CozE 770can be detected at mid-cell before the onset of membrane invagination (arrow) Two 771representative images are shown, n = 3, scale bars = μm 70 35 71 772b, Bacterial two-hybrid interactions of CozE with members of the PG biosynthetic 773complex E coli strain BTH101 (Δcya) expressing protein fusions to domains (T25 and 774T18) of adenylate cyclase Positive interactions reunite T25 and T18 domains resulting 775in lacZ expression and blue colonies on LB agar containing X-gal Strains were grown 776to stationary phase in LB at 30oC and µl spotted onto LB agar plates supplemented 777with X-gal, incubated at 30oC and imaged The ‘zip’ fusions are to a leucine zipper 778domain derived from the yeast protein GCN4 and serve as both a positive and negative 779controls Representative image from biological replicates is shown Additional controls 780are provided in Supplementary Fig 15 781c, A functional FLAG-CozE fusion co-immunoprecipitates with GFP-PBP1a but not 782GFP-PBP2a Digitonin-solubilized membrane preparations from the indicated strains 783were incubated with anti-GFP resin, washed, and eluted in sample buffer Immunoblots 784show matched samples of solubilized membrane (input) and elution (eluate, 20X 785concentrated relative to input), fractions were probed with anti-GFP and anti-FLAG 786antibodies Representative blots are shown, n = Evidence of FLAG-functionality, 787antibody specificity, and full immunoblot analysis are provided in Supplementary Fig 78816 An untagged PBP1a control showing FLAG-CozE does not precipitate with the resin 789is included in Supplementary Fig 16c 790d, Schematic model of CozE and MreCD function In wt cells, the PBP1a (green) is 791held at mid-cell by the CozE/MreCD complex, resulting in nascent cell wall synthesis at 792this location (red shading) In the absence of CozE or MreCD, PBP1a catalyzes 793delocalized PG synthesis causing lethal morphological defects 72 36 ... (SPD_0768) that we have named CozE (coordinator of zonal elongation) 41Our results indicate that CozE is a member of the MreCD complex of S 4 2pneumoniae that directs the activity of PBP 1a to the midcell... D-amino acids (TADA) The 457Tetramethylrhodamine(TAMRA) 3-amino-D-alanine (TADA) used in this study was 40 20 41 458synthesized by Tocris TADA was used to label nascent S pneumoniae cell wall... S pneumoniae CozE and MreC protein sequences were used as queries 428against a database of bacterial genomes with an e-value cut off of 1x10-4 BLAST 429analysis was carried out using the Harvard