- Email: [email protected]
Ingenious gene generation
Research Update
of membrane proteins of any of these rickettsiae that are only expressed in ticks. It is possible that, as with the OspA vaccine against the tick-transmitted spirochete that causes Lyme disease17, such proteins could be targets for blocking transmission from ticks. The rickettsiae might not have evolved mechanisms for dealing with immune responses against these molecules. Sequencing projects are in progress for several of these genomes, and indeed the description by Brayton et al. of the msp2/msp3 pseudogene structure is the first result from A. marginale genome sequencing. It will not be long before we have a full catalog of genes and their stage-specific expression. This should identify many new vaccine targets and help our understanding of how each pseudogene family contributes to immune evasion. References 1 Dumler, J.S. et al. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales; unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia, and Ehrlichia with Neorickettsia; descriptions of five new species combinations; and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. Int. J. Syst. Evol. Bacteriol. (in press) 2 Palmer, G.H. et al. (2000) Antigenic variation in the persistence and transmission of the ehrlichia Anaplasma marginale. Microbes. Infect. 2, 167–176
TRENDS in Microbiology Vol.9 No.8 August 2001
3 Alleman, A.R. et al. (1997) Anaplasma marginale major surface protein 3 is encoded by a polymorphic, multigene family. Infect. Immun. 65, 156–163 4 Palmer, G.H. et al. (1994) The immunoprotective Anaplasma marginale major surface protein 2 is encoded by a polymorphic multigene family. Infect. Immun. 62, 3808–3816 5 Brown, W.C. et al. (1998) The repertoire of Anaplasma marginale antigens recognized by CD4(+) T-lymphocyte clones from protectively immunized cattle is diverse and includes major surface protein 2 (MSP-2) and MSP-3. Infect. Immun. 66, 5414–5422 6 Brayton, K.A. et al. (2001) Efficient use of a small genome to generate antigenic diversity in tickborne ehrlichial pathogens. Proc. Natl. Acad. Sci. U. S. A. 98, 4130–4135 7 Eid, G. et al. (1996) Expression of major surface protein 2 antigenic variants during acute Anaplasma marginale rickettsemia. Infect. Immun. 64, 836–841 8 French, D.M. et al. (1998) Expression of Anaplasma marginale major surface protein 2 variants during persistent cyclic rickettsemia. Infect. Immun. 66, 1200–1207 (Erratum: Infect. Immun. (1998) 66, 2400) 9 French, D.M. et al. (1999) Emergence of Anaplasma marginale antigenic variants during persistent rickettsemia. Infect. Immun. 67, 5834–5840 10 Barbet, A.F. et al. (2000) Antigenic variation of Anaplasma marginale by expression of MSP2 mosaics. Infect. Immun. 68, 6133–6138 11 Barbet, A.F. et al. (2001) Antigenic variation of Anaplasma marginale: major surface protein 2 diversity during cyclic transmission between ticks and cattle. Infect. Immun. 69, 3057–3066 12 Ohashi, N. et al. (2001) Analysis of transcriptionally active gene clusters of major
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17
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outer membrane protein multigene family in Ehrlichia canis and E. chaffeensis. Infect. Immun. 69, 2083–2091 Yu, X. et al. (2000) Characterization of the complete transcriptionally active Ehrlichia chaffeensis 28 kDa outer membrane protein multigene family. Gene 248, 59–68 Gilbert, S.C. et al. (1998) Association of malaria parasite population structure, HLA, and immunological antagonism. Science 279, 1173–1177 de la Fuente, J. et al. (2001) Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells. Int. J. Parasitol. 31, 145–153 McGarey, D.J. et al. (1994) Putative adhesins of Anaplasma marginale: major surface polypeptides 1a and 1b. Infect. Immun. 62, 4594–4601 Poland, G.A. and Jacobson, R.M. (2001) The prevention of Lyme disease with vaccine. Vaccine 19, 2303–2308 Schuler, G.D. et al. (1991) A workbench for multiple alignment construction and analysis. Proteins 9, 180–190 Batzoglou, S. et al. (2000) Human and mouse gene structure: comparative analysis and application to exon prediction. Genome Res. 10, 950–958
Patrick F.M. Meeus Anthony F. Barbet* Dept of Pathobiology, College of Veterinary Medicine, PO Box 110880, University Of Florida, Gainesville, FL 32611-0880, USA. *e-mail: [email protected]
Ingenious gene generation Response from Brayton Meeus and Barbet have highlighted recent work on how bacteria with small genomes can efficiently generate an extensive repertoire of antigenic variants. They gave an excellent overview of the organization of the msp2/msp3 pseudogenes and the biological significance of rearrangement into the expression site. In addition, they have speculated about sequences flanking these pseudogenes. Meeus and Barbet suggest that the 600 bp repeat element that we described flanking the msp2 and msp3 pseudogenes1 exists because it is a functional gene repeat. This might be correct; however, we are cautious in accepting this proposed role until further research can establish the true function of this repeat. Our findings concerning this repeat are as follows: http://tim.trends.com
• Much of the repeat (309/625 bp) is not part of gene X. • The repeat exists in partial form. Not all occurrences of the repeat contain gene X. Primers specific for the 3′ end of gene X will amplify sequences that do have similarity to gene X, but do not encode an open reading frame (K. Brayton, unpublished). • Gene X encodes a potential signal peptide, indicating that it has an intact 5′ end; this is also consistent with gene X being full length. Pseudogenes such as those described for msp2 and msp3 are used as reservoirs for antigenic variation, and are not as likely to encode a full-length protein. By contrast, multigene families like those described for omp1/p28 (Refs 2–5) are full length and each has its own promoter. There
does not seem to be a consensus promoter in the repeat 5′ to gene X. • The similarity in the signal peptide between gene X and msp3–12 occurs because the 5′ end of msp3–12 is created by a fusion of the repeat to an msp3 gene. • Gene X is not predicted to be an outer membrane protein using PSORT, and therefore might not fit the definition of the MSP2/3 superfamily of surface proteins. Furthermore, Meeus and Barbet suggest that the full-length msp3 gene would encode sequences corresponding to the carboxyl terminus of msp2 as the msp3–2 pseudogene is fused to the 3′ end of the msp2 gene. When a complete pseudogene (for msp3 or msp2) is recombined into the expression site, the regions of conserved sequence are used as ‘anchors’ for the recombination event. As
356
Research Update
the msp2 3′ end sequence occurs after the msp3 3′ conserved region as Meeus and Barbet have shown, it is unlikely that the msp2 sequences would end up in functional MSP3 protein. We believe that the fusion of these two related genes is more likely to be an artifact of promiscuous recombination. However, the final determination of what is encoded by a full-length msp3 gene will be elucidated by the discovery of the expression site for this gene, which is, as yet, unknown.
TRENDS in Microbiology Vol.9 No.8 August 2001
References 1 Brayton, K.A. et al. (2001) Efficient use of a small genome to generate antigenic diversity in tickborne ehrlichial pathogens. Proc. Natl. Acad. Sci. U. S. A. 98, 4130–4135 2 McBride, J.W. et al. (2000) A conserved, transcriptionally active p28 multigene locus of Ehrlichia canis. Gene 254, 245–252 3 Yu, X. et al. (2000) Characterization of the complete transcriptionally active Ehrlichia chaffeensis 28 kDa outer membrane protein multigene family. Gene 248, 59–68 4 Ohashi, N. et al (2001) Analysis of transcriptionally active gene clusters of major outer membrane protein multigene family in
Ehrlichia canis and E. chaffeensis. Infect. Immun. 69, 2083–2091 5 Ohashi, N. et al. (1998) Immunodominant major outer membrane proteins of Ehrlichia chaffeensis are encoded by a polymorphic multigene family. Infect. Immun. 66, 132–139
Kelly A. Brayton Program in Vector Borne Diseases, Dept of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA. e-mail: [email protected]
Meeting Report
Microbial fitness and genome dynamics Tone Tønjum and Erling Seeberg ‘Microbial Genome Maintenance’ was held at the Institute of Microbiology, Dept of Molecular Microbiology, University of Oslo/Rikshospitalet, 20–21 April 2001.
Microbial genomes are constantly challenged by environmental agents that induce DNA damage, but the detrimental effects of these challenges are normally counteracted by mechanisms of DNA repair. Furthermore, genome alterations and mutations also occur by horizontal gene transfer and various recombinational events. Changing environments and selective pressures demand mechanisms for rapid genome variability and adaptability. Pathogenic microorganisms experience serious counter-attack from the human host by, for example, the oxidative burst; these counter-attacks require appropriate repair responses from the microorganism. Moreover, host immune responses necessitate mechanisms for fast genome variation and diversification. Consequently, there is a need for concurrent genome variability and maintenance, which potentially seems conflicting. However, if DNA repair and recombination are regulated in response to appropriate signals, a finely tuned balance between variability and stability according to changing needs can be achieved (Fig. 1). It is clear, however, that each microorganism has its own profile in terms of genes for repair and recombination1, and that this profile is important for microbial fitness, survival and virulence. http://tim.trends.com
Mechanisms of mutagenesis and DNA repair
The keynote lecture by Philip C. Hanawalt (Stanford University, CA, USA) reviewed DNA repair mechanisms in general, and addressed genomic stress responses in Escherichia coli as revealed by the microarray analysis of global genome regulation. More than 40 genes appear to be induced by the SOS response in E. coli and several genes are also downregulated2. No additional groups of genes are remarkably upregulated (greater than twofold) in an SOS-independent manner. Hanawalt proposed that DNA repair enzymes present in some pathogens could be new drug targets. He also pointed out that the order of discovery has a profound effect on the way we think about the function(s) of a gene, as exemplified by recA, which has evolved from being a specific recombination factor to a multifunctional regulatory protein in E. coli. Highlights of a new class of DNA polymerases, the Y-family of polymerases, which was recently discovered in all three forms of life, were presented by Robert Fuchs (University of Strasbourg, France)3. The Y-polymerases enable the replication process to continue past damaged areas of DNA. This process, referred to as translesion synthesis (TLS), is responsible for the induction of base substitutions as well as small frameshifts. Recently, in E. coli all three stress-inducible DNA polymerases (DNA Pol II, IV and V) were found to be involved in mutagenesis. Martin G. Marinus (University of Massachussetts, Worcester, MA, USA)
elaborated on the role of recombination in survival of E. coli dam mutants, as double mutants carrying dam in combinations with mutations in genes affecting recombination are not viable4. However, when MutHLS mismatch repair (MMR) is inactivated, recombination is no longer required for survival. The high sensitivity of dam mutants to methylating agents and cisplatin can be explained by MutHSL-mediated formation of doublestrand breaks requiring recombinational repair. The specific adducts produced by these agents that are recognized by the MMR system have now been identified. Magnar Bjørås (University of Oslo, Norway) focused on the importance of base-excision-repair mechanisms in the repair of endogenous DNA damage, such as oxidized (e.g. 8-oxoguanine) and alkylated base residues5. The repertoire of DNA glycosylases present in different bacteria varies widely, reflecting the environmental habitat of each organism. For example, Mycobacterium tuberculosis has four different DNA glycosylases of the MutM/Fpg family for removal of oxidized residues (Tone Tønjum, University of Oslo, Norway) whereas the soil bacteria Bacillus anthracis and Bacillus cereus both have five different enzymes for removal of alkylation damage. The irregular landscape of strain diversity
The population genetics of different bacterial species can be highly variable. For example, co-colonization of the stomach by multiple isolates of Helicobacter pylori results in horizontal
0966-842X/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0966-842X(01)02099-6
of membrane proteins of any of these rickettsiae that are only expressed in ticks. It is possible that, as with the OspA vaccine against the tick-transmitted spirochete that causes Lyme disease17, such proteins could be targets for blocking transmission from ticks. The rickettsiae might not have evolved mechanisms for dealing with immune responses against these molecules. Sequencing projects are in progress for several of these genomes, and indeed the description by Brayton et al. of the msp2/msp3 pseudogene structure is the first result from A. marginale genome sequencing. It will not be long before we have a full catalog of genes and their stage-specific expression. This should identify many new vaccine targets and help our understanding of how each pseudogene family contributes to immune evasion. References 1 Dumler, J.S. et al. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales; unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia, and Ehrlichia with Neorickettsia; descriptions of five new species combinations; and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. Int. J. Syst. Evol. Bacteriol. (in press) 2 Palmer, G.H. et al. (2000) Antigenic variation in the persistence and transmission of the ehrlichia Anaplasma marginale. Microbes. Infect. 2, 167–176
TRENDS in Microbiology Vol.9 No.8 August 2001
3 Alleman, A.R. et al. (1997) Anaplasma marginale major surface protein 3 is encoded by a polymorphic, multigene family. Infect. Immun. 65, 156–163 4 Palmer, G.H. et al. (1994) The immunoprotective Anaplasma marginale major surface protein 2 is encoded by a polymorphic multigene family. Infect. Immun. 62, 3808–3816 5 Brown, W.C. et al. (1998) The repertoire of Anaplasma marginale antigens recognized by CD4(+) T-lymphocyte clones from protectively immunized cattle is diverse and includes major surface protein 2 (MSP-2) and MSP-3. Infect. Immun. 66, 5414–5422 6 Brayton, K.A. et al. (2001) Efficient use of a small genome to generate antigenic diversity in tickborne ehrlichial pathogens. Proc. Natl. Acad. Sci. U. S. A. 98, 4130–4135 7 Eid, G. et al. (1996) Expression of major surface protein 2 antigenic variants during acute Anaplasma marginale rickettsemia. Infect. Immun. 64, 836–841 8 French, D.M. et al. (1998) Expression of Anaplasma marginale major surface protein 2 variants during persistent cyclic rickettsemia. Infect. Immun. 66, 1200–1207 (Erratum: Infect. Immun. (1998) 66, 2400) 9 French, D.M. et al. (1999) Emergence of Anaplasma marginale antigenic variants during persistent rickettsemia. Infect. Immun. 67, 5834–5840 10 Barbet, A.F. et al. (2000) Antigenic variation of Anaplasma marginale by expression of MSP2 mosaics. Infect. Immun. 68, 6133–6138 11 Barbet, A.F. et al. (2001) Antigenic variation of Anaplasma marginale: major surface protein 2 diversity during cyclic transmission between ticks and cattle. Infect. Immun. 69, 3057–3066 12 Ohashi, N. et al. (2001) Analysis of transcriptionally active gene clusters of major
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17
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outer membrane protein multigene family in Ehrlichia canis and E. chaffeensis. Infect. Immun. 69, 2083–2091 Yu, X. et al. (2000) Characterization of the complete transcriptionally active Ehrlichia chaffeensis 28 kDa outer membrane protein multigene family. Gene 248, 59–68 Gilbert, S.C. et al. (1998) Association of malaria parasite population structure, HLA, and immunological antagonism. Science 279, 1173–1177 de la Fuente, J. et al. (2001) Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells. Int. J. Parasitol. 31, 145–153 McGarey, D.J. et al. (1994) Putative adhesins of Anaplasma marginale: major surface polypeptides 1a and 1b. Infect. Immun. 62, 4594–4601 Poland, G.A. and Jacobson, R.M. (2001) The prevention of Lyme disease with vaccine. Vaccine 19, 2303–2308 Schuler, G.D. et al. (1991) A workbench for multiple alignment construction and analysis. Proteins 9, 180–190 Batzoglou, S. et al. (2000) Human and mouse gene structure: comparative analysis and application to exon prediction. Genome Res. 10, 950–958
Patrick F.M. Meeus Anthony F. Barbet* Dept of Pathobiology, College of Veterinary Medicine, PO Box 110880, University Of Florida, Gainesville, FL 32611-0880, USA. *e-mail: [email protected]
Ingenious gene generation Response from Brayton Meeus and Barbet have highlighted recent work on how bacteria with small genomes can efficiently generate an extensive repertoire of antigenic variants. They gave an excellent overview of the organization of the msp2/msp3 pseudogenes and the biological significance of rearrangement into the expression site. In addition, they have speculated about sequences flanking these pseudogenes. Meeus and Barbet suggest that the 600 bp repeat element that we described flanking the msp2 and msp3 pseudogenes1 exists because it is a functional gene repeat. This might be correct; however, we are cautious in accepting this proposed role until further research can establish the true function of this repeat. Our findings concerning this repeat are as follows: http://tim.trends.com
• Much of the repeat (309/625 bp) is not part of gene X. • The repeat exists in partial form. Not all occurrences of the repeat contain gene X. Primers specific for the 3′ end of gene X will amplify sequences that do have similarity to gene X, but do not encode an open reading frame (K. Brayton, unpublished). • Gene X encodes a potential signal peptide, indicating that it has an intact 5′ end; this is also consistent with gene X being full length. Pseudogenes such as those described for msp2 and msp3 are used as reservoirs for antigenic variation, and are not as likely to encode a full-length protein. By contrast, multigene families like those described for omp1/p28 (Refs 2–5) are full length and each has its own promoter. There
does not seem to be a consensus promoter in the repeat 5′ to gene X. • The similarity in the signal peptide between gene X and msp3–12 occurs because the 5′ end of msp3–12 is created by a fusion of the repeat to an msp3 gene. • Gene X is not predicted to be an outer membrane protein using PSORT, and therefore might not fit the definition of the MSP2/3 superfamily of surface proteins. Furthermore, Meeus and Barbet suggest that the full-length msp3 gene would encode sequences corresponding to the carboxyl terminus of msp2 as the msp3–2 pseudogene is fused to the 3′ end of the msp2 gene. When a complete pseudogene (for msp3 or msp2) is recombined into the expression site, the regions of conserved sequence are used as ‘anchors’ for the recombination event. As
356
Research Update
the msp2 3′ end sequence occurs after the msp3 3′ conserved region as Meeus and Barbet have shown, it is unlikely that the msp2 sequences would end up in functional MSP3 protein. We believe that the fusion of these two related genes is more likely to be an artifact of promiscuous recombination. However, the final determination of what is encoded by a full-length msp3 gene will be elucidated by the discovery of the expression site for this gene, which is, as yet, unknown.
TRENDS in Microbiology Vol.9 No.8 August 2001
References 1 Brayton, K.A. et al. (2001) Efficient use of a small genome to generate antigenic diversity in tickborne ehrlichial pathogens. Proc. Natl. Acad. Sci. U. S. A. 98, 4130–4135 2 McBride, J.W. et al. (2000) A conserved, transcriptionally active p28 multigene locus of Ehrlichia canis. Gene 254, 245–252 3 Yu, X. et al. (2000) Characterization of the complete transcriptionally active Ehrlichia chaffeensis 28 kDa outer membrane protein multigene family. Gene 248, 59–68 4 Ohashi, N. et al (2001) Analysis of transcriptionally active gene clusters of major outer membrane protein multigene family in
Ehrlichia canis and E. chaffeensis. Infect. Immun. 69, 2083–2091 5 Ohashi, N. et al. (1998) Immunodominant major outer membrane proteins of Ehrlichia chaffeensis are encoded by a polymorphic multigene family. Infect. Immun. 66, 132–139
Kelly A. Brayton Program in Vector Borne Diseases, Dept of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA. e-mail: [email protected]
Meeting Report
Microbial fitness and genome dynamics Tone Tønjum and Erling Seeberg ‘Microbial Genome Maintenance’ was held at the Institute of Microbiology, Dept of Molecular Microbiology, University of Oslo/Rikshospitalet, 20–21 April 2001.
Microbial genomes are constantly challenged by environmental agents that induce DNA damage, but the detrimental effects of these challenges are normally counteracted by mechanisms of DNA repair. Furthermore, genome alterations and mutations also occur by horizontal gene transfer and various recombinational events. Changing environments and selective pressures demand mechanisms for rapid genome variability and adaptability. Pathogenic microorganisms experience serious counter-attack from the human host by, for example, the oxidative burst; these counter-attacks require appropriate repair responses from the microorganism. Moreover, host immune responses necessitate mechanisms for fast genome variation and diversification. Consequently, there is a need for concurrent genome variability and maintenance, which potentially seems conflicting. However, if DNA repair and recombination are regulated in response to appropriate signals, a finely tuned balance between variability and stability according to changing needs can be achieved (Fig. 1). It is clear, however, that each microorganism has its own profile in terms of genes for repair and recombination1, and that this profile is important for microbial fitness, survival and virulence. http://tim.trends.com
Mechanisms of mutagenesis and DNA repair
The keynote lecture by Philip C. Hanawalt (Stanford University, CA, USA) reviewed DNA repair mechanisms in general, and addressed genomic stress responses in Escherichia coli as revealed by the microarray analysis of global genome regulation. More than 40 genes appear to be induced by the SOS response in E. coli and several genes are also downregulated2. No additional groups of genes are remarkably upregulated (greater than twofold) in an SOS-independent manner. Hanawalt proposed that DNA repair enzymes present in some pathogens could be new drug targets. He also pointed out that the order of discovery has a profound effect on the way we think about the function(s) of a gene, as exemplified by recA, which has evolved from being a specific recombination factor to a multifunctional regulatory protein in E. coli. Highlights of a new class of DNA polymerases, the Y-family of polymerases, which was recently discovered in all three forms of life, were presented by Robert Fuchs (University of Strasbourg, France)3. The Y-polymerases enable the replication process to continue past damaged areas of DNA. This process, referred to as translesion synthesis (TLS), is responsible for the induction of base substitutions as well as small frameshifts. Recently, in E. coli all three stress-inducible DNA polymerases (DNA Pol II, IV and V) were found to be involved in mutagenesis. Martin G. Marinus (University of Massachussetts, Worcester, MA, USA)
elaborated on the role of recombination in survival of E. coli dam mutants, as double mutants carrying dam in combinations with mutations in genes affecting recombination are not viable4. However, when MutHLS mismatch repair (MMR) is inactivated, recombination is no longer required for survival. The high sensitivity of dam mutants to methylating agents and cisplatin can be explained by MutHSL-mediated formation of doublestrand breaks requiring recombinational repair. The specific adducts produced by these agents that are recognized by the MMR system have now been identified. Magnar Bjørås (University of Oslo, Norway) focused on the importance of base-excision-repair mechanisms in the repair of endogenous DNA damage, such as oxidized (e.g. 8-oxoguanine) and alkylated base residues5. The repertoire of DNA glycosylases present in different bacteria varies widely, reflecting the environmental habitat of each organism. For example, Mycobacterium tuberculosis has four different DNA glycosylases of the MutM/Fpg family for removal of oxidized residues (Tone Tønjum, University of Oslo, Norway) whereas the soil bacteria Bacillus anthracis and Bacillus cereus both have five different enzymes for removal of alkylation damage. The irregular landscape of strain diversity
The population genetics of different bacterial species can be highly variable. For example, co-colonization of the stomach by multiple isolates of Helicobacter pylori results in horizontal
0966-842X/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0966-842X(01)02099-6