Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06511, USA1
The Institute for Genomic Research, 9712 Medical Center Dr., Manassas, VA 20850, USA2
Dept of Human Genetics3 and Dept of Microbiology and Immunology4, University of Michigan, Ann Arbor, MI 48109, USA
Author for correspondence: David Friedman. Tel: +1 734 763 3142. Fax: +1 734 764 3562. e-mail: davidfri{at}umich.edu
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ABSTRACT |
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Keywords: colicins, positive selection, evolution, plasmid, Yanomama Indians
During the time this manuscript was being prepared for publication, our colleague James V. Neel passed away. The other authors wish to dedicate this work to his memory.
The GenBank accession number for the sequence reported in this paper is AF197335.
b Present address: Viropharma Incorporated, 405 Eagleview Blvd, Exton, PA 19341, USA.
c Present address: Washington University Medical School, Molecular Biology, Campus Box 8230, St Louis, MO 62110, USA.
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INTRODUCTION |
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Colicins have served as a model system for investigating the mechanisms of bacteriocin evolution and diversification (Lau et al., 1992 ; Riley & Gordon, 1992
, 1995
; Riley, 1993a
, b
, 1998
; Riley et al., 1994
; James et al., 1996
). Much of this work has involved comparisons of DNA and protein sequences among colicins and their associated immunity and lysis genes, and encoded proteins, to infer evolutionary relationships and molecular mechanisms of diversification.
Two primary mechanisms of diversification have been implicated in colicin evolution: diversifying selection and recombination (Tan & Riley, 1997 ; Riley, 1998
). The role of positive selection in generating colicin diversity was first proposed to explain an unusual pattern of divergence between two pairs of closely related nuclease colicin gene clusters (colicin pairs E3/E6 and E2/E9) (Riley, 1993a
, b
). DNA sequence comparisons reveal an apparent excess of substitutions in the immunity regions (i.e. the immunity gene and the immunity binding domain of the colicin gene) of these two pairs of colicin gene clusters. To account for such an unusual clustering of substitutions, Riley (1993a
, b
) proposed that colicin gene clusters diverge rapidly in the immunity region through a mutation-selection process. Repeated waves of this mutation-selection process result in high levels of substitution in the immunity region (for details see Riley, 1998
). DNA sequence comparisons of the pore-former colicins reveal that recombination generates novel combinations of pore-former colicin functional domains and novel combinations of colicin, immunity and lysis genes (Riley, 1998
). However, the pore-former gene cluster sequences are not similar enough to allow alignments in the immunity region, so that the impact of selection on this region of pore-former colicins cannot be assessed. The high frequencies with which pore-former colicins are recovered from nature argue that some sort of selection is operating but this hypothesis has not been formally tested.
We report here the complete DNA sequence and analysis of a colicin-producing plasmid (pCol-Let). Based on this analysis, we conclude that pCol-Let is related to the recently identified plasmid pColU (Smajs et al., 1997 ). The pore-former colicins encoded by these plasmids have precisely the same pattern of substitution described for nuclease colicins. This observation suggests that diversifying selection may play a similarly important role in the diversification of both nuclease and pore-former colicins.
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METHODS |
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Media.
See Miller & Friedman (1980) for details on the media used in this study.
DNA sequence determination of pCol-Let.
pCol-Let plasmid DNA, isolated using the Qiagen Midi Prep (Qiagen), was digested with HindIII and subcloned into the pUC19 cloning vector (Yanisch-Perron et al., 1985 ). DNA sequencing was initiated with the universal primer designed for pUC19 and successive rounds of primer walking were used to sequence across both strands of the insert DNA. Sequence assembly and manual inspection were done using the ABI Autoassembler.
DNA and protein sequence analysis.
The entire pCol-Let DNA sequence was subjected to a DNA BLAST search employing standard methods (Altschul et al., 1990 ). Translations from all three reading frames and both orientations of the entire pCol-Let DNA sequence were subjected to a protein BLAST search using standard methods (Altschul et al., 1990
).
Regions of significant protein or DNA sequence similarity were subjected to more detailed alignments using the CLUSTAL W algorithm of the LASERGENE program (DNASTAR). Distance and parsimony methods were employed to infer evolutionary relationships (Saitou & Nei, 1987 ; Swofford, 1997
). In all cases, both methods yielded similar or identical tree topologies. Only parsimony-based phylogenetic inferences are reported here. The robustness of inferred tree topologies was assessed with bootstrapping (Felsenstein, 1988
). Bootstrap values greater than 80% (of 500 replicates) are reported here.
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RESULTS |
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The pCol-Let colicin gene is predicted to be 1890 bp, which encodes a protein of 629 amino acids, based upon sequence similarity with other characterized colicin genes. The most similar colicin proteins are colicins U, A and B. Levels of protein sequence similarity within this group of colicin proteins range from 87% (between pCol-Let and colicin U) to 41% (between pCol-Let and colicin A). The reduced levels of protein sequence similarity among most characterized pore-former proteins preclude inference of a pore-former colicin protein phylogeny. However, if the protein sequence comparison is restricted to just the C-terminal domain of the protein, levels of protein sequence similarity exceed 20%. Fig. 3 provides the phylogeny inferred from the C-terminal domain of pore-former colicin proteins. Again, the closest relative to the pCol-Let colicin protein is colicin U. In this analysis, the colicin gene was divided into two blocks of sequence of equal length. This was done because previous investigations of nuclease colicin substitution patterns suggest that the killing and immunity domains of the colicin proteins (encoded in the 3' end of the gene) and the immunity protein accumulate substitutions more rapidly than the 5' end of the colicin protein (Riley, 1998
).
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DISCUSSION |
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In addition to these plasmid maintenance functions, pCol-Let also encodes a colicin killing phenotype (Riley, 1993a ). Comparisons of inferred protein sequences of the pCol-Let encoded colicin, immunity and lysis proteins with previously characterized colicin-related proteins reveal that pCol-Let encodes a newly identified member of the pore-former colicin family. Following bacteriocin nomenclature protocols, we have named this colicin gene cluster colicin Y. The recently characterized colicin U gene cluster represents the closest known relative of the colicin Y gene cluster (Smajs et al., 1997
). This close evolutionary relationship is revealed most clearly with a comparison of the lysis and colicin proteins, which retain high levels of protein and DNA sequence similarity (Figs 2
and 3
). The immunity protein encoded in the colicin Y gene cluster, although highly divergent from other characterized immunity proteins, retains unambiguous protein sequence similarities with the colicin U immunity protein (Table 2
).
Extensive DNA and protein sequence comparisons have revealed that the two families of colicin gene clusters (pore-former and nuclease) experience both kinds of mutational events. But the evolutionary significance varies (Riley, 1998 ). It has been suggested that pore-former diversity results from frequent recombination between existing pore-former types, generating additional killing phenotypes. With few exceptions, such as colicins Ia and Ib (Riley, 1993a
), pore-former proteins are highly divergent in protein sequence. However, detailed protein sequence comparisons often reveal relatively short stretches of highly similar protein sequence interspersed within longer stretches of highly divergent sequence (Tan & Riley, 1997
). These restricted regions of high sequence similarity have been interpreted as resulting from recombination events.
By contrast, nuclease colicins are similar in protein sequence. With one exception (colicin E2; Tan & Riley, 1997 ), amino acid sequence comparisons among nuclease colicins have revealed no evidence of the process of recombination-mediated diversification proposed for the pore-former colicins. When pairs of closely related nuclease colicin gene clusters are aligned, there is an elevated level of sequence divergence clustered in the C-terminal region of the colicin protein and in the immunity protein (Tan & Riley, 1997
). However, this clustered divergence has been interpreted as resulting from the action of positive selection for novel immunity phenotypes, rather than resulting from recombination (Tan & Riley, 1997
).
The process of selection-mediated nuclease colicin diversification is envisioned to require two steps. First, one or a few mutations in the immunity gene results in changes in the immunity function that broadens the immunity of the host strain against several additional colicins. In environments with multiple colicins segregating, this function will be strongly selected for and thus will be retained in the population long enough for a second, paired mutation to produce a super-killer phenotype. The super-killer phenotype results in a strain of E. coli that is immune to its own colicin and to its ancestors colicin. However, the ancestral strain will be immune to self, but not immune to the newly evolved colicin phenotype. Thus, the super-killer strain will rapidly invade the ancestral population. Repeated rounds of this sort of diversifying selection will result in the accumulation of differences in the immunity proteins and in the immunity binding regions of the colicin proteins (reviewed by Riley, 1998 ). The result of diversifying selection is a large and homogeneous class of nuclease colicins (save for the rapidly evolving immunity regions).
The high frequency with which pore-former colicins are encountered in nature argues that, in addition to recombination generating new pore-former colicin phenotypes, positive selection acts to increase the frequency of these new phenotypes (Riley, 1998 ). However, due to the high levels of sequence divergence between the pore-former proteins characterized to date, the importance of selection and the molecular details of this process in the generation of pore-former diversity have been impossible to assess. The colicin Y gene cluster encoded in the pCol-Let plasmid provides the first example in which closely related pairs of pore-former colicin gene clusters have been examined for the signature of diversifying selection. As indicated in Table 2
, colicin Y and its closest known relative, colicin U, have an elevated level of substitution in the immunity gene and in the 3' half of the colicin gene, relative to the remainder of the gene cluster. This pattern of substitution corresponds to the pattern of divergence previously revealed only for nuclease colicins (Riley, 1998
). Recently, a second pair of closely related pore-former gene clusters has been characterized, colicins 5 and 10 (Pilsl & Braun, 1995
). Table 2
provides a summary of the level of substitution between these two closely related pore-former colicin gene clusters. There is an elevated level of substitution in the immunity genes and in the 3' region of the colicin genes. In both these pore-former comparisons, there is a significantly elevated level of DNA sequence divergence in the immunity region, a pattern of substitution predicted by the diversifying selection hypothesis.
The correlation between higher substitutions in the immunity gene and the 3' half of the colicin gene found for nuclease colicins appears easily explained, since these proteins are known to interact (Riley, 1998 ). However, the similar correlation observed here between higher substitutions in the immunity region gene and the 3' half of the colicin gene for pore-former colicins is not as easily explained, since it is not clear yet how these proteins interact. However, it is difficult to explain immunity specificity without assuming some type of interaction between the immunity and colicin protein.
The pCol-Let plasmid was originally characterized with the expectation that understanding the evolutionary relationships of the encoded colicin-associated proteins might provide additional insight into the degree of isolation of the microbial flora of Yanomama Indian. pCol-Let encodes a colicin phenotype that has not previously been described, as might be expected for an isolated E. coli population. However, the colicin Y gene cluster is quite closely related to the colicin U gene cluster. Colicin U was isolated in 1989 from a strain of Shigella boydii isolated in Prague (Horak, cited in Smajs et al., 1997 ). Thus, both significant geographical space and time separate these two colicin gene cluster isolations. Previous DNA sequence studies of colicin E2 gene clusters have revealed that a ColE2 plasmid isolated from Australia was more closely related to a ColE2 plasmid isolated over 15 years earlier from France, than to 12 other ColE2 plasmids isolated at the same time from Australia. These observations argue that E. coli migration rates (or at least plasmid transfer and subsequent migration rates) are substantial and may not reflect the same patterns of isolation as do the host mammals from which they are isolated.
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ACKNOWLEDGEMENTS |
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Received 9 November 1999;
revised 6 April 2000;
accepted 17 April 2000.