Analysis of pan-genome content and its application in microbial identification

Six bacterial genera containing species commonly used as probiotics for human consumption or starter cultures for food fermentationwere compared and contrasted, based on publicly available complete genome sequences. The analysis included 19 Bifidobacterium genomes, 21 Lactobacillus genomes, 4 Lactococcus and 3 Leuconostoc genomes, as well as a selection of Enterococcus (11) and Streptococcus (23) genomes. The latter two genera included genomes from probiotic or commensal as well as pathogenic organisms to investigate if their non-pathogenic members shared more genes with the other probiotic genomes than their pathogenic members. The panand core genome of each genus was defined. Pairwise BLASTP genome comparison was performed within and between genera. It turned out that pathogenic Streptococcus and Enterococcus shared more gene families than did the non-pathogenic genomes. In silico multilocus sequence typing was carried out for all genomes per genus, and the variable gene content of genomes was compared within the genera. Informative BLAST Atlases were constructed to visualize genomic variation within genera. The clusters of orthologous groups (COG) classes of all genes in the panand core genome of each genus were compared. In addition, it was investigated whether pathogenic genomes contain different COG classes compared to the probiotic or fermentative organisms, again comparing their panand core genomes. The obtained results were compared with published data from the literature. This study illustrates how over 80 genomes can be broadly compared using simple bioinformatic tools, leading to both confirmation of known information as well as novel observations. Introduction The first bacterial genome sequences were published in 1995, and within 15 years, over a thousand fully sequenced bacterial genomes have become publicly available [16]. A number of these genome sequences are derived from bacteria used as probiotics or starter cultures in food fermentation, or both. Reid and co-workers [21] defined probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. A number of bacterial species from various genera are in use as probiotics, including members of Lactobacillus, Lactococcus and, less commonly, Leuconostoc. These Firmicutes are sometimes collectively described as lactic acid bacteria (LAB). Other commonly used probiotic species belong to Bifidobacterium, a genus within the phylum Actinobacteria. These genera exclusively contain species that are unlikely to cause disease while colonizing the intestine, and although some species (e.g. Bifidobacterium dentium) have been associated with dental disease, these are more commonly members of a normal oral flora. The distinction between normal gut flora (commensals) and Electronic supplementary material The online version of this article (doi:10.1007/s00248-011-9948-y) contains supplementary material, which is available to authorized users. O. Lukjancenko :D. W. Ussery Center for Biological Sequence Analysis, Department of Systems Biology, The Technical University of Denmark, Building 208, 2800 Kgs, Lyngby, Denmark T. M. Wassenaar (*) Molecular Microbiology and Genomics Consultants, Tannenstrasse 7, 55576 Zotzenheim, Germany e-mail: trudy@mmgc.eu Microb Ecol (2012) 63:651–673 DOI 10.1007/s00248-011-9948-y probiotic bacteria having a beneficial effect on their host’s health cannot always be made, for which reason we collectively describe them here as ‘non-pathogens’. Species belonging to LAB or Bifidobacterium are also frequently used in food fermentation, another application where the bacterial load of food is desirably increased. Besides LAB and Bifidobacterium, fermentation starter cultures can typically comprise of Streptococcus thermophilus, a nonpathogenic member of this genus that mostly contains pathogenic species. Some strains of Enterococcus are also in use as starter cultures or probiotics, whereby the used species also contain pathogenic strains. These two genera are therefore of interest, and their species that are used as starter cultures are included in our general description of ‘non-pathogens’. Other types of bacteria (particular strains of Escherichia coli, Pediococcus species and others) or yeasts used as starter cultures or probiotics are not treated here. For all six genera of interest, multiple genome sequences are publicly available. In many cases, several genomes per species have been sequenced, so that the variation between and even within species can be assessed. One obvious question that could be addressed by comparison of these genomes is: what genes (if any) are common to all genomes of non-pathogens and distinct from genes found in (related) pathogens? Such a comparison requires including multiple species and genera of multiple bacterial phyla (in this case, the phylum of Firmicutes and Actinobacteria). As a general rule, genetic diversity increases with evolutionary distance, so that the genetic variation in such a collection of genomes will be enormous. One way of extracting information from such complex data is by grouping genes into functional groups or families, so that gene families rather than individual genes are compared. Such grouping is based on protein sequence similarity, as this approximately predicts conservation of gene function, ignoring the exceptions resulting from parallel evolution where function similarity does not coincide with sequence conservation. Slight differences in function, resulting from minor differences in sequences, are usually ignored in these groupings, so that fewer but broader groups can be achieved. In this contribution, 2 approaches were used to compare over 80 genomes from 6 bacterial genera of interest. First, all protein-coding genes from these genomes were grouped into gene families based on sequence identity using a defined similarity cut-off, after which comparisons between and across genera could be performed. Genomes were then compared within their genus for both conserved and variable genes. Second, clusters of orthologous groups (COG) of genes were used to produce functional groups of genes. An attempt was made to identify differences in functional gene distribution between pathogenic and nonpathogenic members of the six genera of interest. Materials and Methods Selection of Genomes Used in This Study Publicly available genomes of the six bacterial genera analyzed here were identified from the NCBI web pages. All completely sequenced genomes (as of July 2010) of 4 Lactococcus lactis strains, 3 Leuconostoc species and 21 Lactobacillus strains from 14 species were included. For Bifidobacterium, 11 completely sequenced and 8 incomplete genomes were selected; the latter were chosen when fewer than 70 contigs resulting in 19 genomes from 9 species. Since only 1 complete Enterococcus genome was available at the time of analysis, this genome was combined with 10 incomplete sequences, provided they were represented in fewer than 80 contigs, whereby animal isolates were excluded. This allowed inclusion of 2 strains obtained from normal gut flora to give 11 genomes from 4 species. For Streptococcus, all S. thermophilus genomes were included. All other species of this genus for which genome sequences were available are pathogens, and a selection of these was made of three genomes per species. These were chosen based on their strain characteristics to cover common but diverse serotypes. Animal isolates were excluded, although Streptococcus suis (a typical pig pathogen) was included as it has been responsible for a large human outbreak in China. This resulted in 23 genomes from 12 species. All genomes are listed in Table 1, which also provides characteristics such as their size, GC content and the strain description. The latter was extracted from the Genome Project pages at NCBI but checked in the corresponding genome publication when available. This resulted in a few small differences from descriptions listed on the Genome Project Description pages at NCBI. The derived proteomes (protein-coding sequences translated from the DNA sequence) were extracted from GenBank for completed sequences or produced with Prodigal [14] for incomplete sequences. Definition of Gene Families and Panand Core Genome The pan-genome of a collection of genomes represents all genes encountered in these genomes [27]. In order to define a pan-genome, the criteria to score a gene as ‘conserved’ or ‘novel’ were used as previously described [12]. Simply put, two genes are considered to belong to the same gene family and thus ‘conserved’ when their amino acid sequence is at least 50% identical over at least 50% of the length of the longest gene. All genes of a genome are thus grouped into gene families. Multiple genes per genome can belong to a single gene family, resulting in a lower number of gene families per genome than the reported number of genes. A gene not finding a match with the given criteria is put in its own gene family as a singleton. 652 O. Lukjancenko et al. An accumulative pan-genome was constructed according to Friis et al. [11], who built on work by Tettelin and coworkers [27]. A resulting pan-genome curve increases in size as more genomes are analyzed, and its shape is order-dependent, though the accumulative pan-genome is not influenced by the order of analysis. Similarly, a core genome is defined as all gene families conserved in all analyzed genomes, and this decreases in size as more genomes are analyzed. Pairwise panand core genomes were calculated for all genome combinations as above, and for each combination, the obtained core genome was expressed as the fraction of the pan-genome. These percentages were visualized in a BLAST Matrix [11]. Core Genome Consensus Tree Phylogenetic trees were constructed of all core genes that were conserved within the analyzed Firmicute genomes. Multiple alignments of all core sequences were performed with MUSCLE so