Rosetta(DE3) pLysS Strain used for production of CusD proteins EMD Millipore (Darmstadt, Germany) F. johnsoniae strains UW101 (ATCC 17061 T ) Wild type CJ1808 chiA disruption mutant; (Em r ) [2] CJ1827 rpsL2; (Sm r ) "wild type" strains for construction of all deletion mutants [3] CJ2018 rpsL2 ∆cusD II (Fjoh_4561); (Sm r ) This study CJ2086 rpsL2 ∆cusCI (Fjoh_4559); (Sm r ) This study CJ2121 rpsL2 ∆cusD I (Fjoh_4558); (Sm r ) This study CJ2156 rpsL2 ∆cusD I ∆cusD II ; (Sm r ) This study CJ2340 rpsL2 ∆cusCII (Fjoh_4562); (Sm r ) This study CJ2349 rpsL2 ∆(cusCI -cusDII); (Sm r ) This study CJ2350 rpsL2 ∆chiB (Fjoh_4560); (Sm r ) This study CJ2453 rpsL2 ∆(cusS-cusR); (Sm r ) This study CJ2456 rpsL2 ∆Fjoh_4556; (Sm r ) This study CJ2457 rpsL2 ∆Fjoh_4565; (Sm r ) This study CJ2463 rpsL2 ∆nagB (Fjoh_4557); (Sm r ) This study CJ2590 rpsL2 ∆cusC I ∆cusC II ; (Sm r ) This study CJ2676 rpsL2 ∆(cusCII -cusDII); (Sm r ) This study CJ2677 rpsL2 ∆(cusC I -cusD I ); (Sm r ) This study CJ2679 rpsL2 ∆(cusC I -cusD I ) ∆(cusC II -cusD II ); (Sm r ) This study * Antibiotic resistance phenotypes: ampicillin, Ap r ; erythromycin, Em r ; kanamycin, Km r ; streptomycin, Sm r ; tetracycline, Tc r . Unless indicated otherwise, the antibiotic resistance phenotypes are those expressed in E. coli. The antibiotic resistance phenotypes given in parentheses are those expressed in F. johnsoniae but not in E. coli Table S3: Primers used in this study Primers Sequence and Description ChiA_F_F 
5' TTAAGAAGGAGATATACTATGAAAGTTGTTGGTTATTATGCCCAGTGG 3'; forward primer used to clone ChiA_F (Fjoh_4555); LIC overhang underlined ChiA_F_R GTAATAGCAGTGAACTGAGCAAAGCAGCGCACCATCATCACCACCATT 3'; reverse primer used to clone ChiA_F (Fjoh_4555); LIC overhang underlined ChiA_N_F 5' TTAAGAAGGAGATATACTATGAAAGTTGTTGGTTATTATGCCCAGTGG 3'; forward primer used to clone ChiA_N (Fjoh_4555); LIC overhang underlined ChiA_N_R GATACCAGCTTTGGTAGCGTGGTTGCGCACCATCATCACCACCATT 3'; reverse primer used to clone ChiA_N (Fjoh_4555); LIC overhang underlined ChiA_M_F 5' TTAAGAAGGAGATATACTATGGGTAGCGTGGTTCCGGGTAC 3'; forward primer used to clone ChiA_M (Fjoh_4555); LIC overhang underlined ChiA_M_R CAACCGTGAGCACCAATCGTTTTATTGCGCACCATCATCACCACCATT 3'; reverse primer used to clone ChiA_M (Fjoh_4555); LIC overhang underlined ChiA_C_F 5' TTAAGAAGGAGATATACTATGGTTGTGATTAAAGCAACCGACAATAAAAG 3'; forward primer used to clone ChiA_C (Fjoh_4555); LIC overhang underlined ChiA_C_R GTAATAGCAGTGAACTGAGCAAAGCAGCGCACCATCATCACCACCATT 3'; reverse primer used to clone ChiA_C (Fjoh_4555); LIC overhang underlined GH20_F 5' TTAAGAAGGAGATATACTATGCAGATGCAGAAAGAACAGCTGAATC 3'; forward primer used to clone GH20 (Fjoh_4556); LIC overhang underlined GH20_R CGTTTATGAAAGCCTGAAAAAACTGATTGCGCACCATCATCACCACCATT 3'; reverse primer used to clone GH20 (Fjoh_4556); LIC overhang underlined ChiB_F 5' TTAAGAAGGAGATATACTATGACCAGCGAAAAAGAAAATAACCCGGAAG 3'; forward primer used to clone ChiB (Fjoh_4560); LIC overhang underlined ChiB_R CAAAACCAGCGGTATGTGTGGCAACGCGCACCATCATCACCACCATT 3'; reverse primer used to clone ChiB (Fjoh_4560); LIC overhang underlined CusDI_F 5' CATCATCACCACCATCACGAGAACCTGTACTTCCAGGGCAATGGTATTACACTTCCTGATTTTG 3'; forward primer used to clone CusDI (Fjoh_4558); LIC overhang and TEV site underlined CusD I _R 5' GTGGCGGCCGCTCTATTAGAAATTCGGAGCATTTACATCC 3'; reverse primer used to clone CusDI (Fjoh_4558); LIC overhang underlined CusD II _F1 5' CATCATCACCACCATCACGAGAACCTGTACTTCCAGGGCCTGATAAAGGATCCGGTTGCG 3'; forward primer used to clone CusDII (Fjoh_4561) residues 26-505; LIC overhang and TEV site underlined 

Table S2: Plasmids used in this study

Helper plasmid for triparental conjugation; IncP Tra + Km r [5] pRR51 rpsL-containing suicide vector for construction of deletions; Ap r (Em r ) [ This study * Antibiotic resistance phenotypes: ampicillin, Ap r ; erythromycin, Em r ; kanamycin, Km r ; streptomycin, Sm r ; tetracycline, Tc r . Unless indicated otherwise, the antibiotic resistance phenotypes are those expressed in E. coli. The antibiotic resistance phenotypes given in parentheses are those expressed in F. johnsoniae but not in E. coli. 
5' GCTAG TCTAGA AGTTTACATCCCACCAAACCTTACCAG 3'; forward primer used in construction of pSSK29; XbaI site underlined 5' GCTAG GCATGCCTGCTGCAAATTCTCTAAAAGC 3'; reverse primer used in construction of pSSK33; SphI site underlined 5' GCTAG GTCGACAGCAGATGCCTGAATCGTATACATACC 3'; forward primer used in construction of pSSK33; SalI site underlined 5' GCTAGGGATCCTTTACTCAATTATGTATGTCTGGAGAC 3'; forward primer used in construction of pSSK38; BamHI site underlined 5' GCTAGGTCGACAAGGATGCCTAATAAGGCTTTATTTTT 3'; reverse primer used in construction of pSSK38; SalI site underlined 5' GCTAGGTCGACTACAAGACTTCAGGCATGTGCGGT 3'; forward primer used in construction of pSSK40; SalI site underlined 5' GCTAGGCATGCTATAATAATTAAAGTCATTTCCTCTTG 3'; reverse primer used in construction of pSSK40; SphI site underlined 5' GCTAGGGTACCGGAACTGGCTCAGGATTCTT 3'; forward primer used in construction of pYT351 and pYT353; KpnI site underlined 5' GCTAGGCATGCCTGCTTGTACCATTTGCTAACC 3'; reverse primer used in construction of pLW01 and pYT351; SphI site underlined 5' GCTAGGCTAGCACAGATAATTTTGAAGACATTAATACT 3'; forward primer used in construction of pSSK49; NheI site underlined 5' GCTAGCTCGAGTTAGAAATTCGGAGCATTTACATCCCA 3'; reverse primer used in construction of pSSK49; XhoI site underlined 5' GCTAGGCTAGCACAGAAAATTTTGACGAACTGATAAAG 3'; forward primer used in construction of pSSK50; NheI site underlined 5' GCTAGCTCGAGTTAGTTTACATCCCACCAAACCTTACC 3'; reverse primer used in construction of pSSK50; XhoI site underlined 5' GCTAGGGATCCGAAGCTTCGGTAATTGTAGCACAGG 3'; forward primer used in construction of pYT248; BamHI site underlined 5' GCTAGGTCGACTTCTTTTTGCATTTGAGCATTTGC 3'; reverse primer used in construction of pYT248; SalI site underlined 5' GCTAGGTCGACAAAGATCATGCTGACGTAGAGTTG 3'; forward primer used in construction of pYT255; SalI site underlined 5' GCTAGGCATGCGCATTTCGTAATTCAACTGTAACAT 3'; reverse primer used in construction of pYT255; SphI site underlined 5' GCTAGTCTAGAGTACTTTGTTACTCGCCGCTGTG 3'; forward primer used in construction of pDE01; XbaI site underlined 5' GCTAGGTCGACGGCGCTCTTATAACTGATATCAGG 3'; reverse primer used in construction of pDE01; SalI site underlined 5' GCTAGGTCGACAGAGAATTCTGGGTTAGAGCCGAAG 3'; forward primer used in construction of pDE02; SalI site underlined 5' GCTAGGCATGCGGCAAGTTCTGTCCACATTGTAGC 3'; reverse primer used in construction of pDE02; SphI site underlined 5' GCTAGGGATCCTCCTGTCTTCTAAAATCTCTGCGG 3'; forward primer used in construction of pYT249; BamHI site underlined 5' GCTAGGTCGACGTAGCTTCCCCAAAACCATATATGA 3'; reverse primer used in construction of pYT249; SalI site underlined 5' GCTAGGTCGACCAAATTCAGTCGTACACGATTCCTA 3'; forward primer used in construction of pYT256; SalI site underlined 5' GCTAGGCATGCCGCTAAGTAAAAGGGCCAAAGGT 3'; reverse primer used in construction of pYT256 and pYT260; SphI site underlined 5' GCTAGGGATCCTTTACTCTCAACTTTTTACGATGGG 3'; forward primer used in construction of pYT250; BamHI site underlined 5' GCTAGGTCGACAAACTGTCCCCATTTGGTTTGAAC 3'; reverse primer used in construction of pYT250; SalI site underlined 5' GCTAGGTCGACTTGAAAAAACAAGGAATCAGCCAC 3'; forward primer used in construction of pYT257; SalI site underlined 5' GCTAGGCATGCCGGATAAGGTTTACGAAAAAATGCT 3'; reverse primer used in construction of pYT257; SphI site underlined 5' GCTAGGGATCCTTGAATAATAATACTTACTCCCGGC 3'; forward primer used in construction of pYT259; BamHI site underlined 5' GCTAGGCATGCCCAACTACCTTATTTATTCAACAATGTG 3'; reverse primer used in construction of pYT259; SphI site underlined 5' GCTAGGGATCCCGATACCACAAAAGGCGATTCAC 3'; forward primer used in construction of pYT260; BamHI site underlined For whole cells, 10 µg cell protein was loaded on gels. For secreted proteins, the amount of spent medium that contained 10 µg cell protein before cell removal was loaded. Western blot analyses were performed using antibodies against ChiA at 1: 5,000 dilution. pYT259 expresses the two component signal transduction proteins CusS and CusR, and pSSK05 expresses ChiA. The observation of ChiA as a ~95 kDa band (the expected size is 169 kDa) is consistent with previous studies, which used the same antibodies as in the current study, raised against a part of the N-terminal region of the protein [2]. The ChiUL does not encode proteases, and we assume that ChiA_F is proteolytically cleaved by secreted proteases during growth, similar to how the well-studied Serratia marcescens chitinases are susceptible to cleavage by endogenously produced proteases, whereby chitinolytic activity is decreased [6,7]. ChiA_F does not exhibit detectable auto-lytic activities (no degradation observed by SDS-PAGE after incubation at 22°C for two days in 50 mM sodium phosphate, pH 6.5). Given that ChiA_F is dramatically more active on chitin as fulllength protein compared to the individual GH18 domains (ChiA_N and ChiA_C, singly or combined), it is likely that full-length ChiA is responsible for chitin utilization by F. johnsoniae. Further, the strong affinity of the middle region of ChiA (ChiA_M, Figure 6) to chitin crystals, suggests that ChiA_F is in intimate contact with the substrate during degradation. Note that only soluble fractions were analyzed in the Western blot immunodetection experiments. It is possible that ChiA_F, protected from proteases while bound to its insoluble substrate, was not detected for this reason. 



  / F. johnsoniae shuttle vector; Ap r (Tc r )[4] pDE01 1.7 kbp region upstream of nagB amplified with primers 1626 and 1627 and cloned into XbaI and SalI sites of pRR51; Ap r (Em r ) This study pDE02 Construct used to delete nagB; 1.7 kbp region downstream of nagB amplified with primers 1628 and 1629 and cloned into SalI and SphI sites of pDE01; Ap r (Em r ) This study pETite N-His Expression plasmid, including an N-terminal His6-tag and TEV protease cleavage site; (Km r ) Lucigen pLW01 pCP23 carrying cusD I ; Ap r (Tc r ) This study pNIC-CH Expression plasmid, including a C-terminal His6-tag; (Km r ) Addgene pRK2013 


1871 5 '

 5 GCTAGGGTACCCTACACAACTGTTGGAGGAAGA 3'; forward primer used in construction of pLW01; KpnI site underlined 1955 5' GCTAGGGTACCATCATTAAAGTAAGCACCCCATCG 3'; forward primer used in construction of pYT352; KpnI site underlined 1956 5' GCTAGGCATGCCCTTATCAGGCATAACTATATTTTAGG 3'; reverse primer used in construction of pYT352; SphI site underlined 1957 5' GCTAGGCATGCCGGCGAGTAACAAAGTACAAATAG 3'; reverse primer used in construction of pYT353; SphI site underlined 


FiguresFigure S1 :

 S1 Figures 


Figure S2 :

 S2 FigureS2: Western blot immunodetection of ChiA. F. johnsoniae wild type and mutant cells were grown overnight in motility medium at 25°C with shaking. Cells and supernatants (spent media) were separated by centrifugation. For whole cells, 10 µg cell protein was loaded on gels. For secreted proteins, the amount of spent medium that contained 10 µg cell protein before cell removal was loaded. Western blot analyses were performed using antibodies against ChiA at 1: 5,000 dilution. pYT259 expresses the two component signal transduction proteins CusS and CusR, and pSSK05 expresses ChiA. The observation of ChiA as a ~95 kDa band (the expected size is 169 kDa) is consistent with previous studies, which used the same antibodies as in 


Figure S3 :

 S3 Figure S3: Growth curves of cells carrying pYT353 (cusC I ) on chitin (A) and glucose (B). For growth on chitin, cells (0.1 ml, OD600=1.0) were introduced into 50 ml of Stanier medium supplemented with 0.05% chitin in 250-ml flasks, and incubated with shaking at 25°C. Growth presented as log (µg cell protein/ml). For growth on glucose, cells (0.1 ml, OD 600 =1.0) were introduced into 50 ml of Stanier medium supplemented with 0.1% glucose in 250-ml side-arm flasks and incubated with shaking at 25°C. Growth presented as log (Klett Units). Growth curves were performed in triplicate and error bars indicate standard deviations. pYT353 is derived from pCP23, which has a copy number of approximately 10 in F. johnsoniae. 


Figure S4 :

 S4 Figure S4: Growth curves of wild type cells and predicted cytoplasmic inner membrane sugar transporter mutants. pYT260 expresses the predicted cytoplasmic membrane sugar transporter Fjoh_4565. Cells (0.1 ml, OD 600 =1.0) were introduced into 50 ml of medium in 250-ml side-arm flasks and incubated with shaking at 25°C. A: Stanier medium supplemented with 0.1% glucose as sole source of carbon and energy. B: Stanier medium supplemented with 0.1% N-acetylglucosamine as sole source of carbon and energy. C: CYE medium. Growth presented as log (Klett Units). Growth curves were performed in triplicate and error bars indicate standard deviations. 


Figure S5 :

 S5 Figure S5: Binding analysis of the CusD proteins to insoluble polysaccharides. Lanes are labelled as C for control (protein incubated in buffer), S for soluble proteins after incubation with polysaccharides, and B for bound proteins, where the polysaccharides had been washed once in buffer and subsequently resuspended in SDS-PAGE loading buffer to release bound proteins. Bovine serum albumin (BSA) was used as a nonbinding reference protein. 


Figure S6 :

 S6 Figure S6: Section of a structural alignment of Serratia marcescens chitinases (ChiA, B and C) to which the ChiUL chitinase domains were added. The red box highlights the α+β-insert region. The characteristic catalytic motif, DXXDXDXE, is indicated by a black bar.The alignment was prepared using Clustal Omega[8], manually edited and displayed using BioEdit. 


Figure S7 :

 S7 Figure S7: pH profiles for all enzymes. A: ChiA_N, B: ChiA_C, C: ChiB, D: GH20. The buffers (50 mM) used were: citrate (closed squares), sodium phosphate (open squares), tris (closed circles), MES (open circles), sodium acetate (closed triangles), Bis-Tris (open triangles), and sodium carbonate (crosses). The substrate was chitotetraose and the incubation temperature was 25°C. Data points are the average of duplicate experiments and error bars represent the standard error of the mean. 




  region downstream of cusD II amplified with primers 1055 and 1056 and cloned into BamHI and SalI sites of pRR51; Ap r (Em r ) 


			3]
	pSSK05	pCP23 carrying chiA; Ap r (Tc r )	[2]
	pSSK08	1.8 kbp This study
	pSSK09	Construct used to delete cusDII; 1.3 kbp region upstream of cusDII amplified with	This study
		primers 1057 and 1058 and cloned into SalI and SphI sites of pSSK08; Ap r (Em r )	
	pSSK13	cloned into XbaI and SalI sites of pRR51; Ap r (Em r ) 1.7 kbp region downstream of cusD I amplified with primers 1166 and 1167 and	This study
	pSSK15	1.5 kbp region downstream of cusCI amplified with primers 1170 and 1052 and	This study
		cloned into XbaI and SalI sites of pRR51; Ap r (Em r )	
	pSSK17	primers 1053 and 1054 and cloned into SalI and SphI sites of pSSK15; Ap r (Em r ) Construct used to delete cusC I ; 1.8 kbp region upstream of cusC I amplified with	This study
	pSSK18	Construct used to delete cusDI; 1.7 kbp region upstream of cusDI amplified with	This study
		primers 1168 and 1169 and cloned into SalI and SphI sites of pSSK13; Ap r (Em r )	
	pSSK29	cloned into XbaI and SalI sites of pRR51; Ap r (Em r ) 1.9 kbp region downstream of cusC II amplified with primers 1250 and 1251 and	This study
	pSSK33	Construct used to delete cusCII; 1.8 kbp region upstream of cusCII amplified with	This study
		primers 1252 and 1253 and cloned into SalI and SphI sites of pSSK29; Ap r (Em r )	
	pSSK38	1.5 kbp region upstream of chiB amplified with primers 1423 and 1424 and cloned	This study
		into BamHI and SalI sites of pRR51; Ap r (Em r )	
	pSSK40	Construct used to delete chiB; 1.6 kbp region downstream of chiB amplified with	This study
		primers 1425 and 1426 and cloned into SalI and SphI sites of pSSK38; Ap r (Em r )	
	pSSK49	1,518-bp NheI-XhoI fragment spanning cusDI amplified with primer 1561 and 1562	This study
		and inserted into pET28a	
	pSSK50	1,461-bp NheI-XhoI fragment spanning cusD II amplified with primer 1563 and 1564	This study
		and inserted into pET28a	
	pYT248	2.1 kbp region upstream of Fjoh_4556 amplified with primers 1622 and 1623 and	This study
		cloned into BamHI and SalI sites of pRR51; Ap r (Em r )	
	pYT249	2.0 kbp region upstream of cusS amplified with primers 1630 and 1631 and cloned	This study
		into BamHI and SalI sites of pRR51; Ap r (Em r )	
	pYT250	1.9 kbp region upstream of Fjoh_4565 amplified with primers 1634 and 1635 and	This study
		cloned into BamHI and SalI sites of pRR51; Ap r (Em r )	
	pYT255	Construct used to delete Fjoh_4556; 2.0 kbp region downstream of Fjoh_4556	This study
		amplified with primers 1624 and 1625 and cloned into SalI and SphI sites of	
		pYT248; Ap r (Em r )	
	pYT256	Construct used to delete cusS -cusR; 1.8 kbp region downstream of cusR amplified	This study
		with primers 1632 and 1633 and cloned into SalI and SphI sites of pYT249; Ap r	
		(Em r )	
	pYT257	Construct used to delete Fjoh_4565; 1.8 kbp region downstream of Fjoh_4565	This study
		amplified with primers 1636 and 1637 and cloned into SalI and SphI sites of	
		pYT250; Ap r (Em r )	
	pYT259	pCP23 carrying cusS -cusR; Ap r (Tc r )	This study
	pYT260	pCP23 carrying Fjoh_4565; Ap r (Tc r )	This study
	pYT351 pYT352	pCP23 carrying cusC I -cusD I ; Ap r (Tc r ) pCP23 carrying cusCII -cusDII; Ap r (Tc r )	This study This study
	pYT353	pCP23 carrying cusCI; Ap	

r (Tc r ) 
Table S4

 S4 
	: X-ray Data Collection and Refinement Statistics	
		CusD I (Fjoh_4558)	CusD II (Fjoh_4561)
	PDB	5J90	5J5U
	Resolution range (Å)	36.57 -1.393	44.49 -2.3
	Space group	P 21 21 21	P 1
	Unit cell (Å)	79.33, 112.14, 121.57	82.71 82.89 93.91
		===90°	=66.4°, =78.3° =67.6°
	Total reflections	1693675 (129785)	357070 (35307)
	Unique reflections	215491 (21033)	91093 (9016)
	Multiplicity	7.9 (6.2)	3.9 (3.9)
	Completeness (%)	99.81 (98.30)	97.26 (96.68)
	Mean I/sigma(I)	26.56 (4.63)	7.88 (4.14)
	Wilson B-factor	11.53	22.6
	R merge	0.05585 (0.3396)	0.123 (0.3061)
	Rmeas	0.05968	0.1424
	CC1/2	0.998 (0.941)	0.986 (0.909)
	CC*	0.999 (0.985)	0.996 (0.976)
	R work	0.1503 (0.1911)	0.2139 (0.2566)
	R free	0.1673 (0.2213)	0.2626 (0.3454)
	Number of non-hydrogen atoms	9149	14777
	macromolecules	7591	14157
	ligand (ethylene glycol)	44	0
	water	1514	620
	Protein residues	964	1806
	RMS bonds (Å)	0.006	0.009
	RMS angles (°)	1.08	1.14
	Ramachandran favored (%)	97	97
	Ramachandran allowed (%)	3	3
	Ramachandran outliers (%)	0	0
	Clashscore	0.8	3.1
	Average B-factor	15.7	26
	macromolecules	13.5	26
	ligands (ethylene glycol)	24.1	
	solvent	26.4	25.3
	Values in parenthesis are for the highest resolution shell	

Strain

Description * Source E. coli strains BL21(DE3) 
Strain used for production of ChiUL enzymes Invitrogen (Carlsbad, US) DH5MCR 
Strain used for general cloning Life Technologies (Grand Island, NY, USA) HB101 
Strain used for triparental conjugation [1] 


Supplementary information

Tables Table S1: Bacterial strains used in the study