Grantee Research Project Results
1999 Progress Report: Phage-Mediated Transfer of Genes Between Bacterial Species
EPA Grant Number: R825348Title: Phage-Mediated Transfer of Genes Between Bacterial Species
Investigators: Cohan, Frederick M. , Berger, Evelyn , Mitrica, Ionel , Majewski, Jacek , Palmisano, Margaret , Feldgarden, Michael , Palys, Thomas
Current Investigators: Cohan, Frederick M. , Berger, Evelyn , Mitrica, Ionel , Majewski, Jacek , Libsch, Jason , Palmisano, Margaret , Feldgarden, Michael , Palys, Thomas
Institution: Wesleyan University
EPA Project Officer: Hahn, Intaek
Project Period: January 5, 1997 through January 4, 2000
Project Period Covered by this Report: January 5, 1998 through January 4, 1999
Project Amount: $443,966
RFA: Exploratory Research - Environmental Biology (1996) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Aquatic Ecosystems
Objective:
The objectives of this project are to: (1) determine how the potential for genetic exchange between ecologically distinct groups of bacteria is influenced by sequence divergence, and (2) develop improved methods of identifying ecologically distinct groups of bacteria from sequence data.Progress Summary:
Sexual Isolation and Sequence Divergence. Our earlier work demonstrated that resistance to genetic transformation across Bacillus species increases exponentially with the sequence divergence between donor and recipient. We further found that the exponential function is robust with respect to the genotype and species of the recipient as well as the conditions of transformation. In our current grant, we are investigating the molecular mechanisms underlying the exponential relationship between sexual isolation and sequence divergence. The motivation is that with a better understanding of the mechanisms underlying sexual isolation, we should be able to predict how sexual isolation might be affected by the mode of recombination (i.e., transformation, transduction, and conjugation) and the size of the DNA segment that is recombined. We tested the effect of mismatch repair by comparing a wild-type strain and an isogenic mismatch-repair mutant for the relationship between sexual isolation and sequence divergence in Bacillus transformation. Mismatch repair was shown to contribute to sexual isolation, but was responsible for only a small fraction (16 percent) of the sexual isolation observed. Another possible mechanism of sexual isolation was that more divergent recipient and donor DNA strands have greater difficulty forming a heteroduplex, because a region of perfect identity between donor and recipient is required for initiation of the heteroduplex. A mathematical model showed that this heteroduplex-resistance mechanism yields an exponential relationship between sexual isolation and sequence divergence. Moreover, this model yields an estimate of the size of the region of perfect identity that is comparable to independent estimates for Escherichia coli (E. coli). For these reasons, and because all other mechanisms of sexual isolation may be ruled out, we concluded that resistance to heteroduplex formation is predominantly responsible for the exponential relationship between sexual isolation and sequence divergence in Bacillus transformation.This conclusion has been further supported by transformation experiments using artificially constructed donor DNA segments. In these constructs, a very divergent segment of the rpoB gene from a donor strain was sandwiched between rpoB segments from the recipient strain. By providing flanking sequences that were identical to the recipient, these constructs facilitated recombination of the intervening divergent segment. The constructs completely eliminated sexual isolation between extremely divergent species (provided mismatch repair was eliminated by using mismatch repair mutants as recipient). These experiments demonstrated that it is not the overall divergence between donor and recipient strands that hinders recombination; instead, divergent strains fail to recombine because they lack small regions of identity required as anchors for strand initiation.
We investigated whether regions of identity are required at one or both ends of each recombining segment. We analyzed the junctions of recombination events in 80 recombinants, and found that every junction, at both ends of each integrated segment, corresponded to a region of near identity between recipient and donor. This demonstrates that near identity between recipient and donor is required at both ends. This finding is in contrast to studies with E. coli, where only one junction of recombination needs to be nearly identical.
By analyzing the sequences of recombination junctions, we explored the criteria of sequence similarity that allow a region to act as a junction in recombination. We found that the region must be at least 20 bp in length, with complete identity or at most one mismatch, and high GC content is required when a mismatch is present. All these criteria are related to the stability of the donor-recipient heteroduplex, and one single stability criterion takes into account all three criteria: that the junction region at each end of the recombining segment must be stable enough to have a predicted melting temperature of 51.20 C or more.
These findings have important implications for the spread of new adaptations, such as antibiotic resistance or artificially introduced foreign genes, in the bacterial world. Once a gene for a novel adaptation transfers into a species from a divergent donor, the gene becomes flanked by recipient DNA, and in the absence of barriers other than resistance to strand invasion, this allows efficient homologous recombination of the new adaptation into related species. Our results suggest that even very divergent and heterologous genes may transfer between species at very high rates via homologous recombination.
The heteroduplex-resistance model of sexual isolation predicts that the sensitivity of recombination to sequence divergence should decrease with the size of the DNA segment being recombined, because when a small donor segment is introduced into the cell, the region of identity between donor and recipient needs to be present within, or very close to, the locus of interest. In contrast, when much larger segments are recombined, the initiation of strand exchange may take place at highly conserved regions of the chromosome that are far from the selected locus. Therefore, the exponential function relating sexual isolation and sequence divergence is expected to be steepest for transformation, where very small segments are transferred, and lower for the larger segments transferred by transduction, and lower still for the very large segments recombined in conjugation. Currently, we are testing this prediction by comparing the relationship between sexual isolation and sequence divergence for transduction and transformation in the model system of Bacillus subtilis (B. subtilis).
It is striking how much more mismatch repair contributes to recombination barriers in Escherichia than in Bacillus; in E. coli, mismatch repair is almost entirely responsible for the resistance to interspecies recombination. This raised the question of which role of mismatch repair, that of Escherichia or Bacillus, is the more typical one. This issue was approached by investigating another system of microbial recombination: natural transformation in Streptococcus pneumoniae (S. pneumoniae). We transformed three recipient strains?two wild type and one deficient for the mismatch repair system (Hex-)?with DNA from a diversity of related species with varying degrees of DNA sequence divergence.
Interspecies transformation of the two wild type S. pneumoniae strains followed an exponential decay function of sequence divergence that was very similar to that for Bacillus. The slope of the exponential decay function was much less steep for the mismatch repair mutant. Averaged over all donors, mismatch repair was responsible for 34 percent of the observed sexual isolation in Streptococcus (compared to 16 percent in Bacillus). Because the DNA uptake step in Streptococcus is not sequence specific and all of the recipient strains are nonrestricting, we have proposed that the remaining 66 percent of the barrier to recombination is due to difficulty in forming the donor-recipient DNA heteroduplex molecule, as in Bacillus.
Rates of Recombination, Migration, and Horizontal Transfer in Bacillus Group III Temperate Bacteriophages. Bacillus Group III temperate bacteriophages are harbored as prophage by Bacillus subtilis and closely related species. We investigated the sequence diversity of Bacillus Group III prophages within and among several Bacillus taxa, and from several deserts, to infer rates of horizontal transfer, migration, and intragenic recombination in nature. The phage-borne immunity protein d was amplified from host strains of B. vallismortis, B. mojavensis, B. licheniformis, B. subtilis subsp. subtilis, and B. subtilis subsp. spizizenii. Group III prophages were found to be present in moderately high frequencies within most Bacillus taxa and in all desert environments surveyed. Recombination among Bacillus prophages has occurred at the same low rate as previously found for their host bacteria (? 10-8). Bacillus Group III prophages formed one large sequence cluster, showing no evidence of clustering by host taxon or desert source; therefore, rates of horizontal transfer of prophages between host taxa must be high, indicating no significant barriers to transmission among the taxa surveyed. Rates of intracontinental and intercontinental migration were high, suggesting that Group III bacteriophages are not isolated by distance or differences in local adaptations.
These results suggest a great potential for interspecies and intercontinental transfer of host genes by phage-mediated transduction.
Discovery, Classification, and Quantification of Ecological Diversity in the Bacterial World. We have developed an improved means to identify ecologically distinct bacterial populations that are very closely related.
All living organisms fall into discrete clusters of closely related individuals on the basis of gene sequence similarity. We developed an evolutionary genetic theory that predicts that in bacteria, each such sequence similarity cluster should correspond to an ecologically distinct population. Surveys of sequence diversity in protein-coding genes corroborated this theory by showing that sequence clusters correspond to ecological populations. We have predicted that future population surveys of protein-coding gene sequences can be expected to disclose many previously unknown ecological populations of bacteria. Moreover, sequence clusters should provide a useful tool for characterizing the ecological diversity of a habitat. Sequence similarity clustering in protein-coding genes is recommended as a primary criterion for demarcating taxa.
We have distinguished two very different uses for sequence data in the systematics of closely related species. One use is to diagnose and classify new isolates into existing species where sequences already have been characterized. The second use is to discover cryptic populations and species within existing taxa. Strain diagnosis can be accomplished by either protein-coding genes or the more slowly evolving 16S rRNA genes. Others previously have shown that 16S rRNA sequences can distinguish the most closely related species, because only one diagnostic nucleotide site is required for this purpose; however, it has been shown that the extremely low rate of 16S rRNA evolution frequently prevents this gene from allowing discovery of cryptic populations. We have shown that the greater rate of evolution in protein-coding genes makes these genes useful in both the diagnosis of new strains and the discovery of new taxa.
We recently have improved our sequence-based approach for discovering the ecological diversity within a bacterial species. Our "star method" is based on a theory demonstrating that a phylogeny of strains from the same ecotype should appear as a star radiation, with all strains equally related. With this premise, we concluded that when a clade of strains within a taxon is not consistent with a star phylogeny, that clade must contain more than one ecotype. We have developed a method for identifying the most inclusive clades that are each consistent with a star phylogeny. Our interpretation is that each such clade is a distinct ecotype.
Application of this method shows that most bacterial species tested cannot be resolved into a star phylogeny. This implies that each species must contain more than one ecotype. Each analyzed bacterial species has yielded about 10 ecotypes, each consistent with a star phylogeny. For example, E. coli has yielded at least 14 ecotypes. We have concluded that the bacterial world has at least an order of magnitude more ecologically distinct and coexisting populations than was previously expected.
Future Activities:
We have refined our methods for quantifying the rate of transduction between Bacillus species, and we will spend the next year investigating the relationship between sequence divergence and sexual isolation in Bacillus transduction.Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 9 publications | 9 publications in selected types | All 6 journal articles |
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Majewski J, Cohan FM. The effect of mismatch repair and heteroduplex formation on sexual isolation in Bacillus. Genetics 1998;148(1):13-18. |
R825348 (1999) R825348 (Final) |
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Majewski J, Cohan FM. DNA sequence similarity requirements for interspecific recombination in Bacillus. Genetics 1999;153(4):1525-1533. |
R825348 (1999) R825348 (Final) |
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Majewski J, Zawadzki P, Pickerill P, Cohan FM, Dowson CG. Barriers to genetic exchange between bacterial species: Streptococcus pneumoniae transformation. Journal of Bacteriology 2000, Volume: 182, Number: 4 (FEB), Page: 1016-1023. |
R825348 (1999) R825348 (Final) |
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Palys T, Nakamura LK, Cohan FM. Discovery and classification of ecological diversity in the bacterial world: the role of DNA sequence data. International Journal of Systemic Bacteriology 1997;47(4):1145-1156. |
R825348 (1999) R825348 (Final) |
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Palys T, Berger E, Mitrica I, Nakamura LK, Cohan FM. Protein-coding genes as molecular markers for ecologically distinct populations: the case of two Bacillus species. International Journal of Systemic and Evolutionary Microbiology 2000;50(Part 3):1021-1028. |
R825348 (1999) R825348 (Final) |
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Supplemental Keywords:
sexual isolation, interspecies recombination, bacteria, migration., RFA, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Ecology, Ecosystem Protection, exploratory research environmental biology, Chemical Mixtures - Environmental Exposure & Risk, Ecosystem/Assessment/Indicators, Environmental Chemistry, Genetics, Chemistry, Ecological Effects - Environmental Exposure & Risk, Ecological Effects - Human Health, Biology, Ecological Indicators, risk assessment, transduction, ecological effects, bacteria, gene-environment interaction, Bacillus subtilis, phylogenetics, microorganisms, soil, gene transfer, DNA, phage mediatedRelevant Websites:
http://www.wesleyan.edu/bio/cohan/cohan.htmlProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.