The Bacillus cereus group: novel aspects of population structure and genome dynamics.

AIMS: To provide new insights into the population and genomic structure of the Bacillus cereus group of bacteria. METHODS AND RESULTS: The genetic relatedness among B. cereus group strains was assessed by multilocus sequence typing (MLST) using an optimized scheme based on seven chromosomal housekeeping genes. A set of 48 strains from different clinical sources was included, and six clonal complexes containing several genetically similar isolates from unrelated patients were identified. Interestingly, several clonal groups contained strains that were isolated from similar human sources. Furthermore, comparative whole genome sequence analysis of 16 strains led to the discovery of novel ubiquitous genome features of the B. cereus group, such as atypical group II introns, IStrons, and hitherto uncharacterized repeated elements. CONCLUSIONS: The B. cereus group constitutes a coherent population unified by the presence of ubiquitous and specific genetic elements which do not show any pattern, either in their sequences or genomic locations, which allows to differentiate between the member species of the group. Nevertheless, the population is very dynamic, as particular lineages of clinical origin can evolve to form clonal complexes. At the genome level, the dynamic behaviour is indicated by the presence of numerous mobile and repeated elements. SIGNIFICANCE AND IMPACT OF THE STUDY: The B. cereus group of bacteria comprises species that are of medical and economic importance. The MLST data, along with the primers and protocols used, will be available in a public, web-accessible database (http://mlstoslo.uio.no).

Selective constraints, amino acid composition, and the rate of protein evolution.

What are the major forces governing protein evolution? A common view is that proteins with strong structural and functional requirements evolve more slowly than proteins with weak constraints, because a stringent negative selection pressure limits the number of substitutions. In contrast, Graur claimed that the substitution rate of a protein is mainly determined by its amino acid composition and the changeabilities of amino acids. In this paper, however, we found that the relative changeabilities of amino acids in mammalian proteins are different for transmembranal and nontransmembranal segments, which have very distinct structural requirements. This indicates that the changeability of a given residue is influenced by the structural and functional context. We also reexamined the relationship between substitution rate and amino acid composition. Indeed, the two kinds of segments exhibit contrasting amino acid compositions: transmembranal regions are made up mainly of hydrophobic residues (a total frequency of approximately 60%) and are very poor in polar amino acids (<5%), whereas nontransmembranal segments have frequencies of 30% and 22%, respectively. Interestingly, we found that within a given integral membrane protein, nontransmembranal segments accumulate, on average, twice as many substitutions as transmembranal regions. However, regression analyses showed that the variability in amino acid frequencies among proteins cannot explain more than 30% of the variability in substitution rate for the transmembranal and nontransmembranal data sets. Furthermore, transmembranal and nontransmembranal segments evolving at the same rate in different proteins have different compositions, and the compositions of slowly evolving and rapidly evolving segments of the same type are similar. From these observations, we conclude that the rate of protein evolution is only weakly affected by amino acid composition but is mostly determined by the strength of functional requirements or selective constraints.

Accounting for evolutionary rate variation among sequence sites consistently changes universal phylogenies deduced from rRNA and protein-coding genes.

Phylogenetic analyses of gene and protein sequences have led to two major competing views of the universal phylogeny, the evolutionary tree relating the three kinds of living organisms, Bacteria, Archaea, and Eukarya. In the first scheme, called "the archaebacterial tree, " organisms of the same type are clustered together. In the second scenario, called "the eocyte tree," the archaeal phylum of Crenarchaeota is more closely related to eukaryotes than are other Archaea. A major property of the evolution of functional ribosomal and protein-encoding genes is that the rate of nucleotide and amino acid substitution varies across sequence sites. Here, using distance-based and maximum-likelihood methods, we show that universal phylogenies of ribosomal RNAs and RNA polymerases built by ignoring this variation are biased toward the archaebacterial tree because of attraction between long branches. In contrast, taking among-site rate variability into account gives support for the eocyte tree.

Performance of the relative-rate test under nonstationary models of nucleotide substitution.

Relative-rate tests have previously been developed to compare the substitution rates of two sequences or two groups of sequences. These tests usually assume that the process of nucleotide substitution is stationary and the same for all lineages, i.e., uniform. In this study, we conducted simulations to assess the performance of the relative-rate tests when the molecular-clock (MC) hypothesis is true (i.e., there is no rate difference between lineages), but the stationarity and uniformity assumptions are violated. Kimura's and bias-corrected LogDet distances were used. We found that the computation of the variances and covariances of LogDet distances had to be modified, because the constraint that the sum of the frequencies of the 16 nucleotide pair types is equal to 1 must be imposed. Comparison of the rates of two single sequences (Wu and Li's test) or two groups of sequences (Li and Bousquet's test) gave similar results. When the sequences are long (> or = 500 nt), the test based on LogDet distances and their appropriate variances and covariances is appropriate even when the substitution process is not stationary and/or not uniform. That is, at the 5% significance level, the test rejects the MC hypothesis in about 5% of the simulation replicates. In contrast, if the sequences are short (< or = 200 bases) and highly divergent, the LogDet test is very conservative due to overestimation of the variances of the distances. When the uniformity assumption is violated, the relative-rate test based on Kimura's distances can be severely misleading because of differences in base composition between sequences. However, if the uniformity assumption held and so the base frequencies remained similar among sequences, the rate of rejection turned out to be close to 5%, especially with short sequences. Under such conditions, the test using Kimura's distances performs better than the LogDet test. The reason seems to be that these distances are less affected by a reduction in the number of sites than the LogDet distances because they depend on only two parameters.

Evolutionary distances between nucleotide sequences based on the distribution of substitution rates among sites as estimated by parsimony.

The rate of evolution of macromolecules such as ribosomal RNAs and proteins varies along the molecule because structural and functional constraints differ between sites. Many studies have shown that ignoring this variation in computing evolutionary distances leads to severe underestimation of sequence divergences, and thus can lead to misleading evolutionary tree inferences. We propose here a new parsimony-based method for computing evolutionary distances between pairs of sequences that takes into account this variation and estimates it from the data. This method applies to the number of substitutions per site in ribosomal RNA genes as well as to the number of nonsynonymous substitutions per codon for protein-coding genes and is especially suitable when large data sets (> or = 100 sequences) are analyzed. First, starting from a phylogeny constructed with usual distances, the maximum-parsimony method is used to infer the distribution of the number of substitutions that have occurred at each site (or codon) along this tree. This distribution is then fitted to an "invariant + truncated negative binomial" distribution that allows for invariant sites. Maximum-likelihood fitting of this distribution to different data sets showed that it agreed very well with real data. Noticeably, allowing for invariant sites seemed to be very important. Finally, two distance estimates were developed by introducing the distribution of site variability into the substitution models of Jukes and Cantor and of Kimura. The use of different numbers of aligned sequences (up to 1,000 rRNA sequences) showed that the parameters of the model are very sensitive to the number of sequences used to estimate them. However, if at least 100 sequences are considered, the two new distance estimates are quite stable with respect to the number of sequences used to fit the distribution. This stability is true for low as well as for high evolutionary distances. These new distances appeared to be much better estimates of the number of substitutions per site than the classical distances of Jukes and Cantor and of Kimura, which both greatly underestimate this number, so that they can serve as indexes to detect saturation. We conclude that the new distances are particularly suitable for phylogenetic analysis when very distantly related species and relatively large data sets are considered. Trees reconstructed using these distances are generally different from those constructed by means of the classical estimates. Using this new method, we showed that the mean evolutionary distance between Prokaryotes and Eukaryotes is substantially higher for the small-subunit than for the large-subunit rRNAs. This suggests than the former might have experienced a drastic change during the early evolution of Eukaryotes.