Vertical and horizontal transmission of ESBL plasmid from Escherichia coli O104:H4


Figure S1. Crude cellular extracts and EMSA in the absence of ParB. (a) SDS-PAGE followed by Coomassie blue staining of an increasing range (1, 3, 10, 30 and 100; black triangle) of total extracts from cells expressing ParBpESBL. The cell extract from the vector control (V) is loaded at the corresponding range 100. The position of ParB is indicated by the arrow. (b) Cy3-labeled dsDNA probes were incubated without (-) or with increasing amounts of cell extract from the vector control (range 10, 30, 100; black triangle). Free DNA fragments of the NS, 7-DR and 8-BS probes were indicated on the left. A faint non-specific complex, labeled with an asterisk (*) is visible at high cell extracts with the 7-DR and 8-BS but not with the NS probes. 

Figure S2.

Transfer efficiency of different pESBL mutants. The presence (+) or absence (-) of par, oriT and pnd genes are indicated on the left, and additional characteristics in pESBL and donor E. coli are indicated with colored bars. Exconjugants were never obtained in all oriT mutants (*), thus the limit of detection was presented with vertical lines. Average and standard deviations for more than three independent experiments are displayed. 

Figure S3

. Representative time-lapse images of the fate of cells that have lost ∆par ∆pnd parSpMT1 + pESBL. The cell culture was loaded in a microfluidic chamber, and Amp and Sytox green nucleic acid stains were added in the flow. Arrowheads represent a cell that loses the plasmid, resulting in the aberrant accumulation of mCherry and sensitivity to Amp, which is reflected by the lysis of the cell and release of the chromosome DNA in the media stained by sytox. Bars = 2 µm. 14,389,584 14,691,470 14,516,201 14,480,809 35,392 #2 5 CGTA 18,531,553 20,124,007 19,864,167 19,765,407 98,760 #3 9 TACTC 14,609,016 15,189,471 14,970,843 14,902,612 68,231 Total 49,351,211 49,148,828 202,383 



  




  




  


Table S1 : Strains used in this study

 S1 
	Strain	Description	Reference
	Host E. coli strains	
	BL21 (DE3)	Host cell for T7 promoter-based protein expression	Laboratory stock
	BW25113	F -DE(araD-araB)567 lacZ4787(del)::rrnB-3 LAM -rph-1 DE(rhaD-rhaB)568 hsdR514	[20]
	MC1061	hsdR2 hsdM+ hsdS+ araD139 Δ(ara-leu)7697 Δ(lac)X74 galE15 galK16 rpsL mcrA mcrB1	Laboratory stock
	SM10 λpir	thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu_kan λpir	Laboratory stock
	β2163	F -RP4-2-Tc::Mu_kan ∆dapA::(erm-pir)	[62]

Table S2 : Plasmids used in this study

 S2 
	Plasmid	Description	Construction/reference
	Vectors		
	pBAD33	p15Aori cat Para_X	[64]
	pDM4	R6Kori cat sacB mobRP4	[65]
	pKD46	oriR101 repA101ts bla Para_exo_bet_gam	[20]
	pXX705	Fori amp-R ∆sopABC	[29]
			m.sf.gfp.mut3 amplified with oYo578 x oYo579
	pEYY240	pBAD33 X-m.sf.gfp-mut3	followed by SacI + XbaI digestion cloned into
			pBAD33 / SacI + XbaI
	Allelic exchange plasmids	
			3 pieces Gibson assembly: left arm amplified with
	pEYY10	pDM4 for hp4::X	oYo17 x oYo24, right arm amplified with oYo25 x
			oYo20 and pDM4 / XbaI + XhoI
			3 pieces Gibson assembly: left arm amplified with
	pEYY40	pDM4 for ∆oriT	oYo107 x oYo108, right arm amplified with oYo110 x
			oYo111 and pDM4 / XbaI + XhoI
			3 pieces Gibson assembly: spc from pVI36 amplified
	pEYY84	pDM4 for hp4::lacZ_spc	with oYo226 x oYo227, lacZ amplified with oYo228 x
			oYo229 and pEYY10 / XbaI
			3 pieces Gibson assembly: left arm amplified with
	pEYY352	pDM4 for ∆par	oYo881 x oYo873, right arm amplified with oYo880 x
			oYo879, and pDM4 / XbaI + XhoI
			4 pieces Gibson assembly: parSpMT1 amplified with
	pEYY371	pDM4 for IG_hp23::parSpMT1	oYo946 x oYo947, left arm amplified with oYo944 x oYo945, right arm amplified with oYo948 x oYo949
			and pDM4 / XbaI + XhoI
			3 pieces Gibson assembly: left arm amplified with
	pEYY377	pDM4 for ∆pnd	oYo1026 x oYo1027, right arm amplified with
			oYo1028 x oYo1029, and pDM4 / XbaI + XhoI
	Other plasmids		
	pEE18	R6Kori mobRP4 cat magellan5-MmeI (kan)	pEE22 [22] but without SC2 reporter
	pSN70	pUCori bla lacI q Plac_mcherry-ParBpMT1 1	[39]
	pR6K biofab-sf.gfp	R6Kori FRT_kan_FRT PBiofab_sf.gfp	Gibson Assembly of annealed oligo nucleotides ol307 + ol308 and PCR fragment from pR6KplaciQ1-sfGFP [39] with ol305 x ol306
	pVI36	R6Kori bla FRT_spc_FRT	[63]
	pEYY367	pBAD33 m.sf.gfp-mut3-parBpESBL	Gibson assembly of parBpESBL (hp7) amplified with oYo960 x oYo961 and pEYY240 /NotI + XbaI
			Gibson assembly of vector from pR6K biofab-sf.gfp
	pEYY373	R6Kori FRT_kan_FRT PBiofab_ mcherry-ParBpMT1 1	amplified with oYo998 x oYo999 and mCherry-parBpMT1 amplified from pSN70 with oYo1000 x
			oYo1001
	pEYY378	pXX705 parpESBL	Gibson assembly of par locus amplified with oYo1032 x oYo1033 and pXX705 / BamHI
	pEYY395	pET28b parBpESBL	Gibson assembly of parBpESBL (hp7) amplified with oYo1092 x oYo1094 and pET28b / HindIII + NcoI

Table S3 : Oligo DNAs used in this study

 S3 
	Oligo	Sequence (5' -> 3')
	oYo17	GCGGAGTGTATATCAAGCTTATCGAAGTGATTCGGGAGCTTAGC
	oYo20	TTGTGAGCGGATAACAATTTGTGGGAGCCCTTGCTGTGACAATG
	oYo24	GACCTTTCAGACATTCCAGGTCTAGAATGGCATCAAGACACTCACG
	oYo25	CGTGAGTGTCTTGATGCCATTCTAGACCTGGAATGTCTGAAAGGTC
	oYo107	GCGGAGTGTATATCAAGCTTATCGAAAGCCCTGGTATTTATGCC
	oYo108	CACTCACTTCAGGCTCCTTACTAGTCTTATGCAGACGGCAG
	oYo110	CTGCCGTCTGCATAAGACTAGTAAGGAGCCTGAAGTGAGTG
	oYo111	TTGTGAGCGGATAACAATTTGTGGTTGACCGCAACGTGAACATG
	oYo226	CGTGAGTGTCTTGATGCCATTTAGCTTGCAGTGGGCTTAC
	oYo227	GAGGTCGATATTGACCCAATTCTCATTGGCTGGCACCAAGC
	oYo228	GCTTGGTGCCAGCCAATGAGAATTGGGTCAATATCGACCTC
	oYo229	GACCTTTCAGACATTCCAGGTTGGCTTATTGTGGGGATGAC
	oYo578	AATTCGAGCTCAAGGAGGAAAACCATGAGTAAAGGTGAAGAACTGTTC
	oYo579	ACTCTAGATTAGGCGGCCGCCGCTTTGTAGAGTTCATCCATGC
	oYo873	TTGTGAGCGGATAACAATTTGTGGTGATGAAATCCAGCCCCG
	oYo879	GCGGAGTGTATATCAAGCTTATCGCCGGCTGGCTGGTTTATT
	oYo880	CGACGCAGATGACCAGAAAACAGAGGTATCACGATTGATAGCA
	oYo881	TGCTATCAATCGTGATACCTCTGTTTTCTGGTCATCTGCGTCG
	oYo944	GCGGATAACAATTTGTGGCGACACCTGTCCTGAA
	oYo945	ATTTCGCGCTCTGTTTAATCAGACTCGTGGC
	oYo946	TTAAACAGAGCGCGAAATTATGAGTCACG
	oYo947	CAGGGCTTTAGGATGCCGAAGAGC
	oYo948	CGGCATCCTAAAGCCCTGGTATTTATGCC
	oYo949	GGAGTGTATATCAAGCTTATCGTAGTCTTATGCAGACGGC
	oYo960	TCTACAAAGCGGCGGCCGCCAGAAATAGAGCAAACACTTCACC
	oYo961	TGCCTGCAGGTCGACTCTAGATTATCTTTGTTTTACTTCTGCTATCAA
	oYo998	GGTGAGTAACCCGGGTGTAGGCTG
	oYo999	GCTCACCATATTCACCACCCTGAATTGAC
	oYo1000	GTGGTGAATATGGTGAGCAAGGGCG
	oYo1001	TACACCCGGGTTACTCACCTGATTCTGGAAG
	oYo1026	TTGTGAGCGGATAACAATTTGTGGCATCACTGATAATGTCCTCGC
	oYo1027	GTCAGCCTTCGCAACAAAGCCCGCAGCTATTCTAACG
	oYo1028	CGTTAGAATAGCTGCGGGCTTTGTTGCGAAGGCTGAC
	oYo1029	GCGGAGTGTATATCAAGCTTATCGCCCACTCTGTAACGGAAC
	oYo1032	GACATCCAGCCTGCTGTTGGGTTCCACTGAGCGTCAGACCC
	oYo1033	CTCGTTTCTGACACTTGCAGACTGACAGATTTGCCAGTAGCC
	oYo1092	GGGCCATGGTGAGAAATAGAGCAAACACTTCACC
	oYo1094	CCCAAGCTTTTATCTTTGTTTTACTTCTGCTATCAA
	ns-F	Cy3-CCGTTCTAGCTCATTCTGTTCTTGCTTGCTGCATCATCACTGCATCATCCCAATGCCG ATCTAGCTCATTACTGTTCTAT
	ns-R	ATAGAACAGTAATGAGCTAGATCGGCATTGGGATGATGCAGTGATGATGCAGCAAGCA AGAACAGAATGAGCTAGAACGG
	7DR-F	Cy3-CCATAATATTGTTATCTTGTTATCTTGTTATCTTGTTATCTTGTTATCTTGTTGTCTTGT TATCTTAATTAA
	7DR-R	TTAATTAAGATAACAAGACAACAAGATAACAAGATAACAAGATAACAAGATAACAAG ATAACAATATTATGG
	8BS-F	Cy3-TTGAGATAACAAGATAACAAGATAACAAGATAACACCTTGTTATCTTGTTATCTTG TTATCTTGTCATCGAC
	8BS-R	GTCGATGACAAGATAACAAGATAACAAGATAACAAGGTGTTATCTTGTTATCTTGTTAT CTTGTTATCTCAA
	ol305	TATAATAGATTCATGGATGCAAAATAAGAGAGTCAATTCAGGGTGGT
	ol306	GCGAAGTCAATACTCTATCGATGCTTCCAGTCGGGAAACCTGTCGTG
	ol307	GCATCGATAGAGTATTGACTTCGCATCTTTTTGTACCTATAATAGATTCATGGATGCAAA AT
	ol308	ATTTTGCATCCATGAATCTATTATAGGTACAAAAAGATGCGAAGTCAATACTCTATCGA TGC

Table S4 : Summary of Tnseq

 S4 
	Experiment	Illumina barcode 1	P5 adaptor 2	Raw reads	Bowtie-mapped	# of reads Total	TA-mapped Chr 3	pESBL
	Input	library	#1 #2 #3	41 1 8	ATGCTA 15,444,882 16,905,660 14,040,424 13,956,375 CAGT 14,776,122 15,414,730 15,214,960 15,188,012 ACAGT 12,687,763 12,876,419 12,699,636 12,654,016	84,049 26,948 45,620
			Total					41,955,020 41,798,403 156,617
	Output	library	#1	42	AGCATA			

ParB pMT1 lacks N-terminal region to (pMT1 ∆23ParB, see[33]) to avoid dimerization and interaction to ParA. 

Index number (visit https://support.illumina.com for details). 

See [22]. 

Chromosome. 

YBA268 MC1061 / pXX705 [63] bEYY2082 MC1061 / pEYY367 This study bEYY2092 MC1061 / pEYY378 This study bEYY2118 BW25113 ∆galK::mcherry-parBpMT1_kan This study bEYY2125 MC1061 ∆galK::mcherry-parBpMT1_kan This study Strains for allelic exchange bEYY1101 SM10 λ pir / pEYY40 This study bEYY1204 β2163 / pEYY84 This study bEYY2009 SM10 λ pir / pEYY352 This study bEYY2060 SM10 λ pir / pEYY371 This study bEYY2091 SM10 λ pir / pEYY377 This study Strains harboring pESBL YBB1195 MC1061 / pESBL [14] bEYY1116 MC1061 / pESBL ∆oriT This study bEYY1230 MC1061 / pESBL hp4::lacZ_spc This study bEYY1438 MC1061 / pESBL ∆oriT hp4::lacZ_spc This study bEYY2026 MC1061 / pESBL ∆par This study bEYY2051 MC1061 / pESBL ∆oriT ∆par This study bEYY2072 MC1061 / pESBL IG_hp23::parSpMT1 This study bEYY2093 MC1061 / pESBL ∆pnd This study bEYY2094 MC1061 / pESBL ∆oriT ∆pnd This study bEYY2095 MC1061 / pESBL ∆par ∆pnd This study bEYY2096 MC1061 / pESBL ∆oriT ∆par ∆pnd This study bEYY2115 MC1061 / pESBL ∆par ∆pnd IG_hp23::parSpMT1 This study bEYY2127 bEYY2125 / pESBL IG_hp23::parSpMT1 This study bEYY2166 bEYY2125 / pESBL ∆par ∆pnd IG_hp23::parSpMT1 This study bEYY2167 bEYY2125 / pESBL ∆oriT ∆par ∆pnd IG_hp23::parSpMT1 This study bEYY2202 MC1061 / pESBL ∆par ∆pnd hp4::lacZ_spc This study bEYY2203 MC1061 / pESBL ∆oriT ∆par ∆pnd hp4::lacZ_spc This study bEYY2242 MC1061 / pESBL ∆par hp4::lacZ_spc This study
Vertical and horizontal transmission of ESBL plasmid from Escherichia coli O104:H4