Supplementary materials Whole- Sequencing Analysis of Quorum Quenching Bacterial Strain Acinetobacter lactucae QL-1 Identifies the FadY for Degradation of the Diffusible Signal Factor

Tian Ye1,2# , Tian Zhou1,2# , Xudan Xu1,2#, Wenping Zhang1,2, Xinghui Fan1,2, Sandhya Mishra1,2, Lianhui Zhang1,2, Xiaofan Zhou1,2*, Shaohua Chen1,2* 1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangdong Province Key Laboratory of Microbial Signals and Control, Integrative Microbiology Research Centre, South China Agricultural University, Guangzhou 510642, China 2 Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, China * Correspondence: [email protected]; [email protected]; Tel+86-20-8528 8229 # These authors contributed equally to this work.

3.1.1 Repeat sequences The length distribution of strain QL-1 coding is shown in Figure S4. The repeat sequence statistics of A. lactucae QL-1 are shown in Table S1. The total length of repeat sequences was 14,887 bp, covering 0.3746% of the genomic length. Among the repeat elements, long interspersed nuclear elements (LINEs) accounted for 0.0546% and short interspersed nuclear elements (SINEs) for 0.0332% of the assembled genome. The total length of 87 long terminal repeated sequences (LTR) was 10,433 bp, covering 0.2626% of the genomic length.

3.1.2. Non-coding RNAs The results of non-coding RNAs in the A. lactucae QL-1 genome are shown in Table S2. With regard to RNA, 73 tRNAs, 18 rRNAs, and 1 sRNA were predicted. Among the rRNAs, 6 5S_rRNAs, 6 16S_rRNAs, and 6 23S_rRNAs were obtained.

3.1.3. Functional annotation A total of 3707 -encoding genes were verified using publicly available databases, including the GO, KEGG, COG, NR, Pfam, TCDB, Swiss-Prot, and CAZy protein databases. According to the GO analysis of A. lactucae QL-1, 2544 predicted accounting for 33.07% of the entire genome were identified; they were divided into three major subclasses: Molecular function (10 branches), cellular component (11 branches), and biological process (24 branches). These were mainly distributed across four functional entries, including “Cellular process”, “Metabolic process”, “Binding”, and “Catalytic activity”, for which the numbers of annotated genes were 1370, 1453, 1113, and 1315, respectively (Figure S5). The KEGG function classification is shown in Figure S6. To further understand the functions in A. lactucae QL-1, 3586 putative proteins (accounting for 96.74% of the total number of genes) were successfully assigned to their orthologues in the KEGG database enriched in 217 metabolic pathways (KEGG pathways). “Metabolic pathways” (ko01100) had the highest number of genes (593). This was followed by “ of secondary ” (ko01110) (249), “Microbial in diverse environments” (ko01120) (239), “Biosynthesis of antibiotics” (ko01130) (192), and “Biosynthesis of amino acids” (ko01230) (112), which were the most gene-rich classes in the KEGG pathway groupings. Notably, it was found that 26 genes in A. lactucae QL-1 were involved in fatty acid degradation (ko00071), which further confirmed that QL-1 is capable of the enzymatic inactivation of DSF. NCBI COG mapping revealed that 3299 proteins were assigned to COG categories, accounting for 88.99% of the total number of coding genes. “General function prediction only” had the highest number of genes (334), which were not unambiguously assigned to a particular group. This was followed by “Amino acid transport and metabolism” (299), “” (292), “Lipid transport and metabolism” (259), “, ribosomal structure and biogenesis” (225), and “Energy production and conversion” (215), which were the most gene-rich classes in the COG groupings (Figure S7). Among the protein-coding genes, 3637 genes were annotated in the NR database, accounting for 98.11% of the total number of genes, and 1642 genes were annotated in the SwissProt database, accounting for 44.29% of the total number of genes. The Venn map of A. lactucae QL-1 was obtained according to the annotation results of the COG, NR, Swissprot, and KEGG protein predicted genes, and is shown in Figure S8. It showed 1563 mutual genes, accounting for 42.16% of the total protein-predicted genes. In addition, 55, 2, 0, and 0 specific genes were found in the NR, KEGG, KOG, and Swissport databases, respectively. The gene annotation information indicated that there were 3640, 3586, 2885, 2544, 1642, 394, 261, and 86 annotated genes in the NR, KEGG, COG, GO, Swiss-Prot, TCDB, PHI, and CAZy protein data.

3.1.4. Carbohydrate-active enzyme (CAZyme) The were mapped with the CAZy database to study the presence of CAZymes. A total of 91 genes could be assigned to CAZymes families, as defined in the CAZy database (Figure S9). Glycosyl Transferases (GTs) had the highest number of genes (34), followed by Glycoside Hydrolases (GHs) (30), Carbohydrate-Binding Modules (CBMs) (18), Carbohydrate Esterases (CEs) (6), Auxiliary Activities (AA) (2), and Polysaccharide Lyases (PL) (1).

3.1.5. Gene clusters in secondary prediction The gene clusters involved in the secondary metabolism of A. lactucae QL-1 are shown in Table S3. Six gene clusters, including 2 arylpolyene, 1 bacteriocin, 1 nrps, 1 siderophorerps, and 1 hserlactone-nrps, were predicted in the A. lactucae QL-1 genome.

3.1.6. The pathogen–host interaction (PHI) annotations The PHI is a database of pathogen–host interactions, mainly derived from fungi, oocytes, and bacterial pathogens which are capable of infecting hosts, including animals, plants, fungi, and insects. This database also includes antifungal compounds and their corresponding target genes, which is important in finding target genes for interventions. Each gene in the database contains sequences of nucleic acids and amino acids, as well as a detailed description of the function of the proteins predicted during infection with the host. The number of pathogenicity-related genes (261) in the PHI database of QL-1 is shown in Figure S10. Among these, there were 3 chemistry target genes (resistance to chemicals), 2 effector-related genes (plant avirulence determinant), 21 increased virulence (hypervirulence)-related genes, 9 lethal genes, 12 genes related to a loss of pathogenicity, 157 genes related to reduced virulence, and 57 genes related to unaffected pathogenicity. wt NNGNNCTNCNNATGGAAAAGATTTGGTTTGCAGAATACCA 40 co .....GGATCCATGGAGAAGATCTGGTTCGCCGAATACCA 35 Consensus atgga aagat tggtt gc gaatacca

wt AAAAACAGGGATTCCAGAAACAGTAGCATTGCCTGCGGAA 80 co GAAAACCGGCATCCCAGAAACCGTTGCCCTCCCAGCGGAA 75 Consensusaaaac gg at ccagaaac gt gc t cc gcggaa

wt AATACCTCTCTTGTTGATATTTTTGAGAGTAATTTCCAAA 120 co AACACGAGTCTGGTTGATATCTTCGAAAGCAACTTCCAAA 115 Consensusaa ac tct gttgatat tt ga ag aa ttccaaa

wt AATTTGGTTCTCGTGATGCCTTTATCTTTATGGATAAAGC 160 co AGTTCGGCAGCCGCGACGCCTTCATCTTCATGGACAAGGC 155 Consensusa tt gg cg ga gcctt atctt atgga aa gc

wt GATGTCATTTAACGAGTTAGAACTTGCAAGTCGTAAGTTC 200 co CATGAGCTTTAACGAACTGGAGCTCGCCAGCCGCAAGTTC 195 Consensusatg tttaacga t ga ct gc ag cg aagttc

wt GCGACCTATTTGCAAAATTTGGGGTTAGCAAAAGGAACTC 240 co GCGACCTATCTGCAGAATCTGGGTCTGGCGAAAGGCACGC 235 Consensusgcgacctat tgca aat tggg t gc aaagg ac c

wt GTGTGGCAGTGATGATGCCGAATGTGTTGCAGTATCCTGT 280 co GCGTTGCCGTGATGATGCCAAACGTGCTGCAGTATCCAGT 275 Consensusg gt gc gtgatgatgcc aa gtg tgcagtatcc gt

wt AGTTGCATTAGCAGTGTTACGTGCAGGCTTAGTTTTAGTG 320 co TGTTGCCCTCGCCGTTCTGCGTGCCGGTCTGGTTCTCGTG 315 Consensusgttgc t gc gt t cgtgc gg t gtt t gtg

wt AATGTTAATCCACTTTATACAGCACGTGAACTCGAGCATC 360 co AACGTGAATCCACTGTACACCGCCCGCGAGCTGGAACATC 355 Consensusaa gt aatccact ta ac gc cg ga ct ga catc

wt AGTTAAATGACTCAGGTGCAGAAGTACTCGTGATTATCGA 400 co AGCTGAACGATAGCGGCGCGGAGGTGCTGGTGATCATCGA 395 Consensusag t aa ga gg gc ga gt ct gtgat atcga

wt AAACTTCGCTAGTGTGTATCAAAGCATTTTAGGTAAAACT 440 co GAACTTCGCCAGCGTGTACCAGAGCATTCTGGGCAAAACG 435 Consensusaacttcgc ag gtgta ca agcatt t gg aaaac

wt CCTGTGAAGCATGTTGTGGTTGCCACAGTAGGAGATATGC 480 co CCAGTTAAACACGTGGTGGTGGCGACCGTGGGTGATATGC 475 Consensuscc gt aa ca gt gtggt gc ac gt gg gatatgc

wt TGGGTACACTTAAAGGTACGTTGGTAAATTTTGTATTGCG 520 co TGGGTACGCTGAAAGGCACCCTCGTGAACTTTGTGCTGCG 515 Consensustgggtac ct aaagg ac t gt aa tttgt tgcg

wt TAAAGTACGTAAGCAAATTCCCGCTTGGAATATTCCAGGG 560 co CAAAGTGCGCAAACAGATCCCGGCGTGGAACATTCCGGGC 555 Consensusaaagt cg aa ca at cc gc tggaa attcc gg

wt TACGTTAAATTTAATACAGCATTAAATAAAGAAAGTCCGA 600 co TACGTGAAATTCAACACGGCGCTGAACAAAGAGAGCCCAA 595 Consensustacgt aaatt aa ac gc t aa aaaga ag cc a

wt GTAATTATAAGCGCCCGAGTTTAACTTTAAGTGATACTGC 640 co GCAATTATAAGCGCCCGAGTCTGACCCTCAGCGATACCGC 635 Consensusg aattataagcgcccgagt t ac t ag gatac gc

wt GGTGCTTCAATATACGGGTGGTACTACAGGTGTTTCAAAA 680 co GGTTCTGCAGTACACCGGTGGTACGACCGGCGTTAGCAAG 675 Consensusggt ct ca ta ac ggtggtac ac gg gtt aa

wt GGTGCGGAACTGACTCACCGTAATTTGGTTGCCAACCTTT 720 co GGCGCCGAACTGACCCATCGCAATCTGGTGGCCAATCTGC 715 Consensusgg gc gaactgac ca cg aat tggt gccaa ct

wt TACAATGTGACGGTATCTTCCAAAGTAAATTTGGTGCAAA 760 co TGCAGTGCGATGGTATCTTCCAGAGCAAGTTCGGTGCGAA 755 Consensust ca tg ga ggtatcttcca ag aa tt ggtgc aa

wt TGATGGTGCTAAAGGCGACCGTATTGTTTGTGCATTACCG 800 co CGATGGTGCGAAAGGCGACCGCATCGTTTGTGCGCTGCCG 795 Consensusgatggtgc aaaggcgaccg at gtttgtgc t ccg

wt CTTTATCATATTTTTGCGTTCATGGTTTGCGCAATGTACG 840 co CTGTACCATATCTTCGCGTTCATGGTTTGCGCGATGTACG 835 Consensusct ta catat tt gcgttcatggtttgcgc atgtacg

wt GTATGTATAAAGGTCAGGCAAATATCTTGATTCCGAACCC 880 co GCATGTACAAGGGCCAAGCCAACATTCTGATTCCGAACCC 875 Consensusg atgta aa gg ca gc aa at tgattccgaaccc

wt ACGTGATTTACCAGCTGTGATTAATGAATTACGTAAATAT 920 co GCGTGATCTGCCGGCGGTGATTAACGAGCTGCGCAAGTAC 915 Consensuscgtgat t cc gc gtgattaa ga t cg aa ta

wt CAGCCATCATTCTTCCCAGCCGTAAATACATTATTTAATG 960 co CAACCGAGCTTCTTTCCGGCCGTGAACACCCTCTTTAACG 955 Consensusca cc ttctt cc gccgt aa ac t tttaa g

wt CTTTAGTGAATAATGAAGAATTCAAACAACTTGACCATAG 1000 co CGCTCGTGAACAACGAGGAGTTCAAGCAACTGGACCACAG 995 Consensusc t gtgaa aa ga ga ttcaa caact gacca ag

wt CAATTTAAAAATGGCGATGGGTGGTGGTATGGCAGTTTTA 1040 co CAATCTGAAAATGGCGATGGGCGGTGGCATGGCCGTGCTG 1035 Consensuscaat t aaaatggcgatggg ggtgg atggc gt t

wt CCTTCTACAGCAGAAGCTTGGAAGAAAATTACTGGTACAA 1080 co CCGAGTACCGCCGAAGCGTGGAAAAAGATCACCGGCACCA 1075 Consensuscc tac gc gaagc tggaa aa at ac gg ac a

wt CCATTATCGAAGGTTATGGCTTGTCTGAGACTTCTCCAGT 1120 co CCATCATCGAAGGCTACGGTCTGAGTGAAACCAGTCCGGT 1115 Consensusccat atcgaagg ta gg tg tga ac tcc gt

wt AGCGACTGCGAACCCACCAGCTTCTACCGAATTTAGCGGC 1160 co TGCGACGGCCAATCCACCAGCGAGCACCGAGTTTAGCGGC 1155 Consensusgcgac gc aa ccaccagc accga tttagcggc

wt ACAATTGGTATTCCATTACCTTTAACAGAAGTTGCTATTT 1200 co ACGATTGGTATCCCACTGCCACTGACCGAAGTGGCGATCC 1195 Consensusac attggtat cca t cc t ac gaagt gc at

wt TAGATGATGACGGTAATGAAGTGGCTTTGGGAGAACAAGG 1240 co TCGATGATGATGGTAACGAGGTTGCGCTGGGTGAACAAGG 1235 Consensust gatgatga ggtaa ga gt gc tggg gaacaagg

wt TGAAATTTCAATTCGTGGTCCTCAAGTCATGAAAGGCTAT 1280 co CGAGATCAGCATCCGCGGCCCACAAGTTATGAAGGGCTAC 1275 Consensusga at at cg gg cc caagt atgaa ggcta

wt TGGAACCGTCCGGATGAAACAGCTAAAGTCATGACAGCAG 1320 co TGGAATCGCCCGGATGAGACCGCCAAAGTGATGACGGCCG 1315 Consensustggaa cg ccggatga ac gc aaagt atgac gc g

wt ATGGTTTCTTCCGTACAGGTGACATTGGTGTAATGGACAG 1360 co ATGGCTTCTTCCGTACCGGCGATATCGGTGTTATGGATAG 1355 Consensusatgg ttcttccgtac gg ga at ggtgt atgga ag

wt CCGTGGATACGTTAAAATTGTAGACCGTAAAAAAGATATG 1400 co CCGCGGCTACGTGAAGATCGTGGACCGCAAGAAAGACATG 1395 Consensusccg gg tacgt aa at gt gaccg aa aaaga atg

wt ATTTTGGTATCTGGCTTTAACGTTTATCCAAGTGAAATTG 1440 co ATCCTCGTGAGCGGCTTCAATGTGTACCCGAGTGAGATCG 1435 Consensusat t gt ggctt aa gt ta cc agtga at g

wt AAGAAATTATTGCTAAACATCCAAAAGTATTGGAAGTGGC 1480 co AGGAGATCATCGCGAAGCACCCGAAAGTTCTGGAAGTGGC 1475 Consensusa ga at at gc aa ca cc aaagt tggaagtggc

wt TGCAATTGGTGTTCCAGACGAAAAATCAGGTGAAGTGCCT 1520 co GGCCATTGGCGTGCCAGACGAGAAGAGCGGCGAAGTTCCG 1515 Consensusgc attgg gt ccagacga aa gg gaagt cc

wt AAACTCTTTATCGTGAAAAAAGATCAAAGCTTAACGACTG 1560 co AAACTGTTCATTGTGAAAAAAGACCAGAGTCTGACCACCG 1555 Consensusaaact tt at gtgaaaaaaga ca ag t ac ac g

wt AAGAAGTTTTGAACTTTGCTAAAGAGAACTTAACAGGCTA 1600 co AAGAGGTGCTGAACTTCGCCAAGGAGAATCTGACCGGCTA 1595 Consensusaaga gt tgaactt gc aa gagaa t ac ggcta

wt TAAACGCCCTCGTTATGTTGAGTTTATGGATGAATTACCA 1640 co CAAACGCCCGCGCTATGTGGAGTTTATGGACGAGCTGCCG 1635 Consensusaaacgccc cg tatgt gagtttatgga ga t cc

wt AAATCAAATGTAGGCAAAATTTTACGTAAAGACTTACGTA 1680 co AAAAGCAACGTGGGTAAGATTCTGCGCAAAGATCTCCGCA 1675 Consensusaaa aa gt gg aa att t cg aaaga t cg a

wt AACCAACCTAA..... 1691 co AGCCAACCTAAGAATT 1691 Consensusa ccaacctaa

Figure 1. Sequence comparison of wild-type and codon-optimized fadY genes. wt: wild-type fadY genes; co: codon-optimized fadY genes. 100kDa 87kDa 75kDa

M 1 2 3 4 5 6

Figure 2. Expression and purification of enzyme FadY. M: marker; 1: total protein of BL21 harboring pGEX-6p-1; 2: total protein BL21 harboring pGEX-6p-1 with IPTG; 3: total protein of BL21 harboring recombinant pGEX-6p-1-fadY; 4: total protein of BL21 harboring recombinant pGEX-6p-1-fadY with IPTG; 5: pellet protein of BL21 harboring recombinant pGEX-6p-1-fadY with IPTG; 6: supernatant protein of BL21 harboring recombinant pGEX-6p-1-fadY with IPTG.

Figure 3. From left to right are 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), sample mixed with DTNB, and control mixed with DTNB, respectively.

Figure 4. The gene length distribution of Acinetobacter lactucae QL-1 coding genes.

Figure 5. (GO) functional annotation of Acinetobacter lactucae QL-1.

Figure 6. The Kyoto Encyclopedia of Genes and Genomes (KEGG) function annotation of Acinetobacter lactucae QL-1.

Figure 7. Clusters of orthologous groups of proteins (COG) function classification of proteins in Acinetobacter lactucae QL-1.

Figure 8. Venn diagrams of the COG, Non-Redundant Protein Database (NR), Swissport, and KEGG annotated genes.

Figure 9. The carbohydrate-active enzyme (CAZyme) annotations of Acinetobacter lactucae QL-1.

Figure 10. The pathogen–host interaction (PHI) annotations of Acinetobacter lactucae QL-1. Table 1. Statistical results of repeat sequences in the strain QL-1 genome.

Number of Total length Percentage of genome Average length Type elements (bp) (%) (bp) LTR 87 10,433 0.2626 120 DNA 16 1,167 0.0294 73 LINE 29 2,168 0.0546 75 SINE 20 1,321 0.0332 66 RC 0 0 0 0 Unknown 1 71 0.0018 71 Total 153 14,887 0.3746 99

Table 2. Statistical results of ncRNA in the strain QL-1 genome.

Average Total length Percentage of Type Number length (bp) (bp) genome (%) tRNA 73 77 5,680 0.1429 5s_rRNA 6 114 684 rRNA_ 16s_rRNA 6 1,526 9,156 0.6839 23s_rRNA 6 2,889 17,334 sRNA 1 89 89 0.0022

Table 3. The gene clusters involved in the secondary metabolism of Acinetobacter lactucae QL-1.

Clusters Clusters number Gene number Bacteriocin 1 16 Arylpolyene 2 84 Nrps 1 51 Siderophore 1 14 Hserlactone-nrps 1 48