Complete Genome Sequence of Lactobacillus johnsonii FI9785, a Competitive Exclusion Agent against Pathogens in Poultry

Abstract

Lactobacillus johnsonii is a member of the acidophilus group of lactobacilli. Because of their probiotic properties, including attachment to epithelial cells, immunomodulation, and competitive exclusion of pathogens, representatives of this group are being intensively studied. Here we report the complete annotated genome sequence of Lactobacillus johnsonii FI9785, a strain which prevents the colonization of specific-pathogen-free chicks by Clostridium perfringens.

The competitive exclusion law formulated by Gause (5) states that two species that compete for the exact same resources cannot stably coexist. In recent times, the exploitation of this concept has gained renewed interest in the agricultural sector. Modern animal production methods rely on stringent hygiene measures, vaccination, and until recently the use of antimicrobial agents in order to control pathogens. A new regulation (1831/2003/EC) preventing the regular use of antibiotic growth promoters previously frequently included in feeds has led to an increase in the occurrence of endemic diseases. Clostridium perfringens, which causes necrotic enteritis in poultry, has emerged as one of the beneficiaries of this policy change (16). Lactobacillus johnsonii FI9785 has been shown to reduce the extent to which C. perfringens colonizes and persists in specific-pathogen-free chicks (8). In order to interrogate the genome sequence with regard to the strain's exclusion properties and to enable the use of “omics”-type techniques for further analyses of these properties, the complete genome sequence of this strain was determined using 454 pyrosequencing technology (454 Life Sciences, Branford, CT).

The initial draft assembly provided by 454 Life Sciences was based on 708,584 pyrosequencing reads with an average read length of 108 nucleotides which assembled into 45 contigs. The genome sequence of L. johnsonii NCC533 (12) was used to order these contigs into large scaffolds. Standard PCR followed by primer walk sequencing on the resulting products was used to close the gaps in combination with inverse PCR for gaps for which no linkage information could be obtained. In the case of one large gap (10 kb), an integration/rescue cloning strategy, followed by transposon-assisted sequencing of the rescued fragment, was used to close the respective gap. Sequence assembly was carried out using the Phred/Phrap (4) software program in conjunction with the Staden software package (14). The predicted error rate of the finished sequence is 1 × 10⁻⁶.

The finished L. johnsonii FI9785 sequence was annotated using the GenDB 2.2 annotation tool (11). Open reading frame (ORF) sequences were determined using the REGANOR5 software program (10), based on the combined ORF predictions of CRITICA (2) and Glimmer (13). Putative ribosomal binding sites and tRNA genes were identified using the RBSfinder (15) and tRNAscan-SE (9) software programs, respectively. An automatic functional annotation was computed based on different analyses, followed by a manual annotation of each predicted gene. Eighteen similarity searches (1) were performed against different databases, including the nonredundant database provided by National Center for Biotechnology Information, SWISS-PROT and TrEMBL, KEGG, Pfam, TIGRFAM, and InterPro. Additionally, SignalP (3), helix-turn-helix, and TMHMM (7) were applied. Predicted ORFs were manually reviewed, and alterations were made on the basis of the presence of potential ribosomal binding sites, sequence alignments, and available literature data. The L. johnsonii FI9785 genome is composed of one circular chromosome of 1,755,993 bp with a GC content of 34.49% and two circular plasmids, p9785S (3,471 bp) (6) and p9785L (25,652 bp), with GC contents of 35.81% and 30.38%, respectively. A total of 1,710 ORFs were identified on the chromosome, of which the majority (77.6%) are encoded on the leading strand of chromosome replication. The small and large plasmids carry 3 and 25 ORFs, respectively. The chromosome contains 4 rRNA gene operons and 53 tRNA genes, as well as 1 complete prophage genome.

Nucleotide sequence accession numbers.

Genome information for the chromosome and the two plasmids of L. johnsonii has been deposited in the EMBL/GenBank databases with accession numbers FN298497, AY862141, and FN357112.