- Email: [email protected]
Searching for clues
354
News & Comment
TRENDS in Microbiology Vol.10 No.8 August 2002
Microbial Genomics
Searching for clues The analysis of genome sequences is often likened to molecular-genetic detective work, with clues to the biology of a bacterium often found within the chromosome. It is therefore fitting that we start with a recent paper by Read et al. [1] that represents a real-life bacterial ‘Who done it?’. Continuing the theme, this month’s genomes also provide evidence of stolen genes and some clues to the past. In a landmark investigative case, bacterial genomics has been used as a forensic tool to trace the source of the Bacillus anthracis spores used in the bioterrorism attacks on the USA late last year. Genetic fingerprinting using multiplelocus variable-number tandem repeat analysis (MLVA) established that the B. anthracis used in the bioterrorism attack, the Florida isolate, is a descendant of the Ames strain, which was isolated from a dead cow in Texas in 1981 and has since been distributed to several laboratories around the world. Prior to the bioterrorist attacks, one of the Ames strains, the Porton isolate, was being sequenced at The Institute for Genomic Research (TIGR). This proved to be crucial as MLVA failed to distinguish the Florida isolate from the different Ames isolates, mainly owing to the limited number of variable-number tandem repeat (VNTR) markers selected [2] and the highly homogenous nature of B. anthracis strains. The availability of the DNA sequence of the Porton isolate therefore presented an excellent opportunity to use comparative genomics for this investigation. TIGR sequenced the whole genome of the Florida isolate and compared the assembled sequences with the complete sequence of the Porton isolate. Like all virulent B. anthracis isolates, the Florida isolate carries two virulence plasmids, pXO1 and pXO2. The Porton isolate has been cured of these plasmids, therefore the Florida isolate plasmids were compared with pXO1 and pXO2 plasmids from the B. anthracis Stern and Pasteur strains, respectively. Comparative genomic analysis revealed four differences at the chromosomal level – two single-nucleotide polymorphisms (SNPs) and two insertions/deletions (indels) – and 49 differences between the plasmids – 38 SNPs, eight VNTRs and three indels. http://tim.trends.com
Based on these findings, the scientists tested further the usefulness of these newly found polymorphisms as genetic markers by analyzing seven other isolates: four Ames laboratory isolates, one Ames isolate from a dead goat in Texas and two non-Ames isolates from cattle. The status of the 53 polymorphic markers was determined for each of these isolates. Although the chromosomal markers were unable to distinguish between the isolates, the plasmid markers allowed some distinction and clustered the nine isolates into six distinct groups.
Although the publication does not deliver an answer as to the identity of the B. anthracis strain used in the attacks, some important conclusions emerged from this study. First, it highlights the potential of whole-genome sequencing as a powerful forensic and epidemiological tool for the identification and monitoring of pathogens; and second, it underlines the importance of complete and accurate whole-genome sequences, particularly for the identification of minute strain-specific differences. Hot on the heels of the Methanosarcina acetivorans genome paper comes the second genome for the genus, that of Methanosarcina mazei [3,4]. Archaea of this genus inhabit freshwater and marine sediments and, as methanogens, they are crucial players in the carbon cycle. Particularly significant is their ability to ferment acetate, the precursor for 60% of the earth’s methane, in environments such as rice paddies. The M. acetivorans and M. mazei genomes both have an unusually high proportion of non-coding DNA with predictions of only 74% and 75.15%,
respectively, coding for proteins. Another interesting feature they have in common is the presence of a high proportion of TAG amber codons within coding sequences. It has recently been shown that Methanosarcinae can use this codon to incorporate pyrrolysine, the so-called 22nd natural amino acid, into translated peptides [5,6]. The mechanism by which in-frame amber codons are distinguished from TAG stop codons remains a mystery. In their analysis of M. mazei, the authors have focused on the phenomenon of lateral gene transfer, particularly from Bacteria to Archaea. When searched against the ERGO database, 1043 of the 3371 M. mazei coding sequences have their best match to a bacterial protein. Furthermore, 544 had a significant match only to a bacterial protein and were designated ‘bacterial-like’. Metabolic reconstruction revealed that M. mazei has two acetate-activation pathways, one bacterial-like and the other archaeal. Other methanogenesis pathways are described as having a ‘mosaic-type of origin’ with a mix of bacterial and archaeal proteins. Intriguingly, M. mazei seems to be equipped to fold both bacterial and archaeal proteins. Another interesting observation is that, in comparison to their thermophilic relatives, mesophilic archaea contain a much higher proportion of bacterial-like genes. It seems likely that this is because the mesophiles live in a genetically diverse niche alongside bacteria. This is further implied by the fact that many of the M. mazei bacterial-like proteins are most similar to those from obligate and facultative anaerobes. The evidence does not give a clear picture of the mechanism by which M. mazei acquired bacterial genes but it is noted that the genome contains a high proportion of IS elements and the bacterial-like genes are dispersed throughout the genome rather than being clustered in islands. Comparative genomic analysis is often used to provide insights into the ancestry of many bacterial species. However, unlike higher organisms, the overall interpretation of the timescale of prokaryotic species divergence is limited by the absence of palaeontological records. The recent paper by Tamas et al. goes some way to
0966-842X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0966-842X(02)02417-4
News & Comment
remedying this by presenting a comparison of Buchnera aphidocola genomes that uncovers evidence of an ancient past [7]. Buchnera species are taxonomically related to enteric species such as Escherichia coli, but are distinguished both by having a small genome and by occupying a unique ecological niche. Buchnera are mutualistic intracellular symbionts of aphids, residing in large specialized cells called bacteriocytes, which are transmitted maternally from egg to embryo. The benefit of having a population of ‘free-loading’ prokaryotes is that they can provide additional vital nutrients not found in the aphid’s natural food of sap. The interdependence of these two organisms has led to a close evolutionary relationship. The phylogeny of the Buchnera spp. mirrors that of aphids, for which there are fossil records, thus allowing a relatively accurate prediction of the evolutionary timescale. Tamas et al. compared the 0.64 Mb genome of B. aphidocola with a previously published Buchnera spp. sequence; both species originate from different insect hosts [7,8]. By comparing the evolutionary distance of both the symbionts and their hosts, they estimate that the two Buchnera species diverged from a common ancestor between 50 and 70 million years ago. Surprisingly, the two genomes have
TRENDS in Microbiology Vol.10 No.8 August 2002
remained remarkably similar over such a large timescale, both in size and in gene content. Although the two genomes exhibit sequence divergence at the nucleotide level, they show high levels of conservation of genomic architecture, suggesting that the genomes have been in stasis rather than flux for the past 50 million years. This contrasts markedly with those of related enteric bacteria, which exhibit large numbers of deletions and insertions. Any differences between the two genomes tend to be caused by the presence of pseudogenes. The mutation of key metabolic enzymes alters the range of nutrients available to the aphid, and thus affects the ecological range of the organism. The authors point to the lack of repeats and mobile elements, as well as the absence of recombination functions, as factors contributing to the stability of Buchnera gene order and conservation. Interestingly, although a comparison of the two genome sequences suggests overall stability, a recent study of Buchnera spp. genomes from five different aphid lineages shows that the genome can be reduced in size further [9]. This is significant as it shows that while genome reduction might have taken place early on in the evolution of this genus, Buchnera spp. still have the capacity, in certain niches, to strip their genomes further.
355
Matthew Holden Mohammed Sebaihia Stephen Bentley Julian Parkhill e-mail: [email protected] References 1 Read, T.D. et al. (2002) Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis. Science 296, 2028–2033 2 Keim, P. et al. (2000) Multiple-locus variablenumber tandem repeat analysis reveals genetic relationships within Bacillus anthracis. J. Bacteriol. 182, 2928–2936 3 Galagan, J.E. et al. (2002) The genome of M. acetivorans reveals extensive metabolic and physiological diversity. Genome Res. 12, 532–542 4 Deppenmeier, U. et al. (2002) The genome of Methanosarcina mazei: Evidence for lateral gene transfer between Bacteria and Archaea. J. Mol. Microbiol. Biotechnol. 4, 453–461 5 Srinivasan, G. (2002) Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. Science 296, 1459–1462 6 Hao, B. et al. (2002) A new UAG-encoded residue in the structure of a methanogen methyltransferase. Science 296, 1462–1466 7 Tamas, I. et al. (2002) 50 million years of genomic stasis in endosymbiotic bacteria. Science 296, 2376–2379 8 Shigenobu, S. et al. (2000) Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407, 81–86 9 Gil, R. et al. (2002) Extreme genome reduction in Buchnera spp.: toward the minimal genome needed for symbiotic life. Proc. Natl. Acad. Sci. U. S. A. 99, 4454–4458
Journal Club
FRET probes toxin activity in situ For a virulence factor secreted by a pathogen, identification of the active conformation is complicated by the fact that the protein is often translocated into the host cell cytosol. Using a variety of techniques, the vacuolating cytotoxin VacA, which is produced by Helicobacter pylori, has been shown to exist as monomers as well as large oligomeric structures. The amino- and carboxy-terminal regions of VacA are connected by a protease-sensitive loop. However, until recently, it remained unclear whether the independent fragments of the toxin were active in the host cell cytosol, or whether the toxin acted as a complex. Willhite and colleagues [1] tagged VacA with cyan and yellow fluorescent proteins and transfected these into HeLa cells to examine protein–protein http://tim.trends.com
interactions in the cytosol using fluorescence resonance energy transfer (FRET) microscopy. This method can detect protein–protein interactions because only if two labelled species are proximal does the excitation of one fluorophore result in an emission that excites the second fluorophore causing a detectable, distinct emission. They performed numerous controls to demonstrate the lack of interference of the tags and the specificity of the signal to the fusion constructs rather than the tags alone. Using FRET, they determined that both the 37- and 58-kDa proteolytic fragments and full-length VacA associate in the cytosol of vacuolating cells. In addition, they were able to use this technique to identify point mutations that affected the multimeric interaction.
The number of virulence determinants found to be secreted and active in the host cell cytosol is increasing. These toxic factors either work as complexes, dissociate to form active species, and/or interact with host proteins to disturb native cellular processes. As has now been demonstrated for vacuolating toxin, FRET will be a valuable tool to probe these dynamics in the context of the live cell. 1 Willhite, D.C. et al. (2002) Fluorescence resonance energy transfer microscopy of the Helicobacter pylori vacuolating cytotoxin within mammalian cells. Infect. Immun. 70, 3824–3832
Meta J. Kuehn [email protected]
0966-842X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
News & Comment
TRENDS in Microbiology Vol.10 No.8 August 2002
Microbial Genomics
Searching for clues The analysis of genome sequences is often likened to molecular-genetic detective work, with clues to the biology of a bacterium often found within the chromosome. It is therefore fitting that we start with a recent paper by Read et al. [1] that represents a real-life bacterial ‘Who done it?’. Continuing the theme, this month’s genomes also provide evidence of stolen genes and some clues to the past. In a landmark investigative case, bacterial genomics has been used as a forensic tool to trace the source of the Bacillus anthracis spores used in the bioterrorism attacks on the USA late last year. Genetic fingerprinting using multiplelocus variable-number tandem repeat analysis (MLVA) established that the B. anthracis used in the bioterrorism attack, the Florida isolate, is a descendant of the Ames strain, which was isolated from a dead cow in Texas in 1981 and has since been distributed to several laboratories around the world. Prior to the bioterrorist attacks, one of the Ames strains, the Porton isolate, was being sequenced at The Institute for Genomic Research (TIGR). This proved to be crucial as MLVA failed to distinguish the Florida isolate from the different Ames isolates, mainly owing to the limited number of variable-number tandem repeat (VNTR) markers selected [2] and the highly homogenous nature of B. anthracis strains. The availability of the DNA sequence of the Porton isolate therefore presented an excellent opportunity to use comparative genomics for this investigation. TIGR sequenced the whole genome of the Florida isolate and compared the assembled sequences with the complete sequence of the Porton isolate. Like all virulent B. anthracis isolates, the Florida isolate carries two virulence plasmids, pXO1 and pXO2. The Porton isolate has been cured of these plasmids, therefore the Florida isolate plasmids were compared with pXO1 and pXO2 plasmids from the B. anthracis Stern and Pasteur strains, respectively. Comparative genomic analysis revealed four differences at the chromosomal level – two single-nucleotide polymorphisms (SNPs) and two insertions/deletions (indels) – and 49 differences between the plasmids – 38 SNPs, eight VNTRs and three indels. http://tim.trends.com
Based on these findings, the scientists tested further the usefulness of these newly found polymorphisms as genetic markers by analyzing seven other isolates: four Ames laboratory isolates, one Ames isolate from a dead goat in Texas and two non-Ames isolates from cattle. The status of the 53 polymorphic markers was determined for each of these isolates. Although the chromosomal markers were unable to distinguish between the isolates, the plasmid markers allowed some distinction and clustered the nine isolates into six distinct groups.
Although the publication does not deliver an answer as to the identity of the B. anthracis strain used in the attacks, some important conclusions emerged from this study. First, it highlights the potential of whole-genome sequencing as a powerful forensic and epidemiological tool for the identification and monitoring of pathogens; and second, it underlines the importance of complete and accurate whole-genome sequences, particularly for the identification of minute strain-specific differences. Hot on the heels of the Methanosarcina acetivorans genome paper comes the second genome for the genus, that of Methanosarcina mazei [3,4]. Archaea of this genus inhabit freshwater and marine sediments and, as methanogens, they are crucial players in the carbon cycle. Particularly significant is their ability to ferment acetate, the precursor for 60% of the earth’s methane, in environments such as rice paddies. The M. acetivorans and M. mazei genomes both have an unusually high proportion of non-coding DNA with predictions of only 74% and 75.15%,
respectively, coding for proteins. Another interesting feature they have in common is the presence of a high proportion of TAG amber codons within coding sequences. It has recently been shown that Methanosarcinae can use this codon to incorporate pyrrolysine, the so-called 22nd natural amino acid, into translated peptides [5,6]. The mechanism by which in-frame amber codons are distinguished from TAG stop codons remains a mystery. In their analysis of M. mazei, the authors have focused on the phenomenon of lateral gene transfer, particularly from Bacteria to Archaea. When searched against the ERGO database, 1043 of the 3371 M. mazei coding sequences have their best match to a bacterial protein. Furthermore, 544 had a significant match only to a bacterial protein and were designated ‘bacterial-like’. Metabolic reconstruction revealed that M. mazei has two acetate-activation pathways, one bacterial-like and the other archaeal. Other methanogenesis pathways are described as having a ‘mosaic-type of origin’ with a mix of bacterial and archaeal proteins. Intriguingly, M. mazei seems to be equipped to fold both bacterial and archaeal proteins. Another interesting observation is that, in comparison to their thermophilic relatives, mesophilic archaea contain a much higher proportion of bacterial-like genes. It seems likely that this is because the mesophiles live in a genetically diverse niche alongside bacteria. This is further implied by the fact that many of the M. mazei bacterial-like proteins are most similar to those from obligate and facultative anaerobes. The evidence does not give a clear picture of the mechanism by which M. mazei acquired bacterial genes but it is noted that the genome contains a high proportion of IS elements and the bacterial-like genes are dispersed throughout the genome rather than being clustered in islands. Comparative genomic analysis is often used to provide insights into the ancestry of many bacterial species. However, unlike higher organisms, the overall interpretation of the timescale of prokaryotic species divergence is limited by the absence of palaeontological records. The recent paper by Tamas et al. goes some way to
0966-842X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0966-842X(02)02417-4
News & Comment
remedying this by presenting a comparison of Buchnera aphidocola genomes that uncovers evidence of an ancient past [7]. Buchnera species are taxonomically related to enteric species such as Escherichia coli, but are distinguished both by having a small genome and by occupying a unique ecological niche. Buchnera are mutualistic intracellular symbionts of aphids, residing in large specialized cells called bacteriocytes, which are transmitted maternally from egg to embryo. The benefit of having a population of ‘free-loading’ prokaryotes is that they can provide additional vital nutrients not found in the aphid’s natural food of sap. The interdependence of these two organisms has led to a close evolutionary relationship. The phylogeny of the Buchnera spp. mirrors that of aphids, for which there are fossil records, thus allowing a relatively accurate prediction of the evolutionary timescale. Tamas et al. compared the 0.64 Mb genome of B. aphidocola with a previously published Buchnera spp. sequence; both species originate from different insect hosts [7,8]. By comparing the evolutionary distance of both the symbionts and their hosts, they estimate that the two Buchnera species diverged from a common ancestor between 50 and 70 million years ago. Surprisingly, the two genomes have
TRENDS in Microbiology Vol.10 No.8 August 2002
remained remarkably similar over such a large timescale, both in size and in gene content. Although the two genomes exhibit sequence divergence at the nucleotide level, they show high levels of conservation of genomic architecture, suggesting that the genomes have been in stasis rather than flux for the past 50 million years. This contrasts markedly with those of related enteric bacteria, which exhibit large numbers of deletions and insertions. Any differences between the two genomes tend to be caused by the presence of pseudogenes. The mutation of key metabolic enzymes alters the range of nutrients available to the aphid, and thus affects the ecological range of the organism. The authors point to the lack of repeats and mobile elements, as well as the absence of recombination functions, as factors contributing to the stability of Buchnera gene order and conservation. Interestingly, although a comparison of the two genome sequences suggests overall stability, a recent study of Buchnera spp. genomes from five different aphid lineages shows that the genome can be reduced in size further [9]. This is significant as it shows that while genome reduction might have taken place early on in the evolution of this genus, Buchnera spp. still have the capacity, in certain niches, to strip their genomes further.
355
Matthew Holden Mohammed Sebaihia Stephen Bentley Julian Parkhill e-mail: [email protected] References 1 Read, T.D. et al. (2002) Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis. Science 296, 2028–2033 2 Keim, P. et al. (2000) Multiple-locus variablenumber tandem repeat analysis reveals genetic relationships within Bacillus anthracis. J. Bacteriol. 182, 2928–2936 3 Galagan, J.E. et al. (2002) The genome of M. acetivorans reveals extensive metabolic and physiological diversity. Genome Res. 12, 532–542 4 Deppenmeier, U. et al. (2002) The genome of Methanosarcina mazei: Evidence for lateral gene transfer between Bacteria and Archaea. J. Mol. Microbiol. Biotechnol. 4, 453–461 5 Srinivasan, G. (2002) Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. Science 296, 1459–1462 6 Hao, B. et al. (2002) A new UAG-encoded residue in the structure of a methanogen methyltransferase. Science 296, 1462–1466 7 Tamas, I. et al. (2002) 50 million years of genomic stasis in endosymbiotic bacteria. Science 296, 2376–2379 8 Shigenobu, S. et al. (2000) Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407, 81–86 9 Gil, R. et al. (2002) Extreme genome reduction in Buchnera spp.: toward the minimal genome needed for symbiotic life. Proc. Natl. Acad. Sci. U. S. A. 99, 4454–4458
Journal Club
FRET probes toxin activity in situ For a virulence factor secreted by a pathogen, identification of the active conformation is complicated by the fact that the protein is often translocated into the host cell cytosol. Using a variety of techniques, the vacuolating cytotoxin VacA, which is produced by Helicobacter pylori, has been shown to exist as monomers as well as large oligomeric structures. The amino- and carboxy-terminal regions of VacA are connected by a protease-sensitive loop. However, until recently, it remained unclear whether the independent fragments of the toxin were active in the host cell cytosol, or whether the toxin acted as a complex. Willhite and colleagues [1] tagged VacA with cyan and yellow fluorescent proteins and transfected these into HeLa cells to examine protein–protein http://tim.trends.com
interactions in the cytosol using fluorescence resonance energy transfer (FRET) microscopy. This method can detect protein–protein interactions because only if two labelled species are proximal does the excitation of one fluorophore result in an emission that excites the second fluorophore causing a detectable, distinct emission. They performed numerous controls to demonstrate the lack of interference of the tags and the specificity of the signal to the fusion constructs rather than the tags alone. Using FRET, they determined that both the 37- and 58-kDa proteolytic fragments and full-length VacA associate in the cytosol of vacuolating cells. In addition, they were able to use this technique to identify point mutations that affected the multimeric interaction.
The number of virulence determinants found to be secreted and active in the host cell cytosol is increasing. These toxic factors either work as complexes, dissociate to form active species, and/or interact with host proteins to disturb native cellular processes. As has now been demonstrated for vacuolating toxin, FRET will be a valuable tool to probe these dynamics in the context of the live cell. 1 Willhite, D.C. et al. (2002) Fluorescence resonance energy transfer microscopy of the Helicobacter pylori vacuolating cytotoxin within mammalian cells. Infect. Immun. 70, 3824–3832
Meta J. Kuehn [email protected]
0966-842X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.