Draft genome sequences of four lactic acid bacteria from fermented chicken meat unveil biosynthetic gene clusters for antimicrobial compounds

13 Lactic acid bacteria play a crucial role in fermented food production and serve as important sources of 14 antimicrobial peptides. This study reports four lactic acid bacteria strains, isolated from fermented 15 chicken meat, which harbor biosynthetic gene clusters encoding antimicrobial compounds. These strains 16 are classified within the genera Pediococcus and Lactiplantibacillus. 17 Announcement 18 Fermented foods have been consumed for centuries, serving as carriers for probiotics, prebiotics, and 19 postbiotics. Lactic acid bacteria (LAB) play a crucial role in fermentation by enhancing food safety, 20 inhibiting spoilage, and offering probiotic benefits like improved immunity and gut health (1, 2). With 21 rising antibiotic resistance, bacteriocins are gaining attention as potential alternatives (3). Genome mining 22 and high-throughput screening of biosynthetic gene clusters present a promising pathway for discovering 23 novel bacteriocins, addressing resistance challenges, and advancing sustainable food preservation (4, 5). 24 Three broiler chickens from a live bird market in Chattogram, Bangladesh, were slaughtered under 25 standard procedures. Breast meat (100 g) was stored anaerobically at 4 °C for three days to propagate 26 probiotic bacteria. One gram of meat was mixed with 5 mL MRS broth (Oxoid, Hampshire, England) and 27 incubated anaerobically at 37 °C for 48 hours, promoting LAB growth. Single colonies were isolated on 28 MRS agar, purified, and confirmed by PCR. Genomic DNA was extracted from 1.5 mL of 24-hour cultured 29 broth using a AllPrep Bacterial DNA/RNA/Protein Kit (Qiagen, Hilden, Germany), and purity was assessed 30 with a Nanodrop One (Thermo Fisher Scientific, MA, USA). DNA libraries were prepared using the Nextera 31 XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA). Genome sequencing was conducted on an 32 Illumina NextSeq2000 platform at the Poultry Research and Training Center, Chattogram Veterinary and 33 Animal Sciences University, Bangladesh, generating 2 × 150 bp paired -end reads. Raw data quality, 34 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.18.633471doi: bioRxiv preprint 3 including read quality, GC content, and adapter contamination, was assessed using FastQC v0.12.1 (6). 35 Trim Galore v0.6.5dev (7) was employed to trim low -quality bases and adapter sequences, followed by 36 normalization of read coverage with bbnorm (8) to reduce redundancy. De novo genome assembly was 37 performed using Unicycler v0.4.8 (9), with subsequent polishing using Pilon v1.24 (10) to enhance 38 accuracy. Genome assembly quality metrics were evaluated using QUAST v5.2.0 (11). Samtools v1.13 (12) 39 was used for alignment file manipulation and indexing to aid downstream analysis. The de novo genome 40 assembly, conducted through the Bacterial and Viral Bioinformatics Resource Center (13) comprising 41 above tools , yielded coverage ranging from 65.4× to 145.6×, with raw reads between 1,452,975 and 42 1,662,521. The assembled genomes were annotated using NCBI PGAP v6. 9 (14) and PATRIC (15), with 43 transport proteins identified via TCDB (16), drug targets predicted using DrugBank v6.0 (17), and 44 additional analyses performed for sequence types using MLST (18, 19), CRISPR arrays and prophages using 45 CRISPRCasFinder (20), and Integrative and Conjugative Elements using ICEFinder (21). Detailed genomic 46 characteristics are provided in Table 1. 47 The genomes of four LAB isolates were mined for biosynthetic gene clusters (BGCs) associated with 48 antimicrobial production using the software antiSMASH 7.0 (22) and BAGEL4 (23). The genomes were 49 found to contain multiple BGCs encoding ribosomally synthesized and posttranslationally modified 50 peptides (RiPP like compounds), Cyclic Lactone Autoinducer, Type III Polyketide Synthase (T3PKS), 51 Terpene Precursors, Terpene, and Class IIa (Pediocin) and IIb (Plantaricin_J) bacteriocins. The isolates or 52 their purified bacteriocins may be used in the future for food preservation and commercial probiotics to 53 manage gut health and related diseases. 54 Data availability statement 55 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.18.633471doi: bioRxiv preprint 4 The accession numbers for the genomes of the four LAB strains are shown in Table 1. All genomes are 56 available under BioProject accession number PRJNA1189463. 57 Acknowledgments 58 The genome sequencing was supported by Chattogram Veterinary and Animal Sciences University WGS 59 establishment project. 60 Funding 61 This study was supported by a grant from the University Grants Commission of Bangladesh through 62 Chattogram Veterinary and Animal Sciences University, Bangladesh (Grant number: 32, Session: 2023-63 2024). 64 Author ORCIDs 65 Mahabub Alam https://orcid.org/0000-0001-9837-2598 66 Tamanna Hassan https://orcid.org/0009-0009-4875-9386 67 Tanvir Ahmad Nizami https://orcid.org/0000-0002-7024-4970 68 Lipi Akter https://orcid.org/0000-0002-6317-9410 69 Tofazzal Md Rakib https://orcid.org/0000-0003-2642-9908 70

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It is made The copyright holder for this preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.18.633471doi: bioRxiv preprint 8 Table 1: Genomic features of the sequenced four LAB strains isolated from fermented chicken meat in Chattogram, Bangladesh Strain Name Information Title CVASU1 CVASU3 CVASU2 CVASU4 LAB Species Pediococcus pentosaceus Pediococcus pentosaceus Lactiplantibacillus plantarum Lactiplantibacillus argentoratensis Genome Submission ID SUB14898186 SUB14898186 SUB14898218 SUB14898234 Bioproject ID PRJNA1189463 PRJNA1189463 PRJNA1189463 PRJNA1189463 BioSample accession number SAMN44971012 SAMN44971014 SAMN44971013 SAMN44971015 SRA accession number SRR31442436 SRR31442434 SRR31442435 SRR31442433 GenBank accession number JBJPFK000000000 JBJPFJ000000000 JBJPFL000000000 JBJPFM000000000 FastQC Phred score 36 35 37 35 Raw Reads 1452975 1925165 1600310 1662521 Coverage 113.9× 145.6× 65.4× 70.9× Contig count 24 26 62 99 Coarse consistency (%) 99.1 99.1 97.9 97.9 Fine consistency (%) 98.2 98.2 96 96.3 Completeness (%) 100 100 100 100 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.18.633471doi: bioRxiv preprint 9 Contamination (%) 1.5 1.5 0 0.6 Genome size (bp) 1775093 1775155 3346512 3162126 Contigs N50 (bp) 190398 174289 197047 118879 Contigs L50 4 4 6 9 Largest contig (bp) 297937 297937 471909 305211 GC Content (%) 37.153152 37.153095 44.325077 45.074894 Genes (Total), PGAP 1800 1,799 3247 3008 CDS (PGAP; PATRIC) 1749; 1783 1748; 1783 3175; 3353 2947; 3108 tRNA 45 45 66 56 rRNA 3 3 3 2 CDS Ratio 1.0044544 1.0044193 1.0019387 0.98288304 Hypothetical CDS 350 350 1507 1333 Hypothetical CDS Ratio 0.29220414 0.29220414 0.50611395 0.49131274 PLFAM CDS 1700 1701 0 0 PLFAM CDS Ratio 0.95344925 0.95401007 - - Hypothetical proteins 351 351 1508 1334 Proteins with functional assignments 1432 1432 1845 1774 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.18.633471doi: bioRxiv preprint 10 Proteins with EC number assignments 491 491 687 669 Proteins with GO assignments 407 407 584 561 Proteins with Pathway assignments 336 336 488 470 Proteins with Subsystem assignments 570 570 716 716 Proteins with PATRIC genus-specific family (PLfam) assignments 1700 1701 0 0 Proteins with PATRIC cross-genus family (PGfam) assignments 1727 1728 3233 2990 Proteins with FIGfam assignments 0 0 0 0 Transporter (TCDB) 2 2 15 8 Drug Target (DrugBank) 1 1 1 0 Antibiotic Resistance Genes (PATRIC) 21 21 25 27 Integrative and Conjugative Elements (ICEs) 2 (819699..838639, 18941 bp; 1645942..1683899, 37958 bp) 3 (819699..838639, 18941 bp; 1174724..1193031, 18308 bp; 1662631..1699235, 36605 bp) 2 (2983152..3034384, 51233 bp; 3313868..3325625, 11758 bp) 1 (3096716..3139121, 42406 bp) No. of Clustered Interspaced Short Repeats (CRISPR) (number of Cas clusters) (name of cas genes) 6 (4) (cas9_TypeII, cas1_TypeII, cas2_TypeI-II-III, csn2_TypeIIA) 6 (4) (cas9_TypeII, cas1_TypeII, cas2_TypeI-II-III, csn2_TypeIIA) 2 (1) (cas3_TypeI) 22 (8) (cas6_TypeIE, cas5_TypeIE, cas7_TypeIE, cse2_TypeIE, cse1_TypeIE, .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.18.633471doi: bioRxiv preprint 11 cas3_TypeI, cas2_TypeIE, cas1_TypeIE) Bacteriocins and biosynthetic gene clusters (BGCs) Pediocin, terpene-precursor, T3PKS Pediocin, T3PKS Plantaricin_J, cyclic-lactone- autoinducer, RiPP like, terpene, T3PKS terpene-precursor (3 loci), T3PKS, terpene .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.18.633471doi: bioRxiv preprint