Horizontal gene transfer (HGT) is thought to play an important role in the evolution of species and innovation of genomes. Different researchers examined functional propensity in HGT of protein families.
First, Jain et al. (1999) proposed under the complexity hypothesis that HGT may have occurred preferentially among operational genes (those that maintain cell growth such as metabolism-related genes) than among informational genes (those involved in DNA replication, transcription, and translation) which are part of more complex protein-interaction networks.
Second, Nakamura et al. (2004) observed that only parts of genes in functional categories such as mobile element, cell surface, DNA binding, and pathogenicity-related, were preferred.
Third, Beiko et al. (2005) found extensive evidence for the preferential transfer of metabolic genes, while informational genes (e.g. ribosomal proteins, and proteins involved in DNA replication and repair, cell wall synthesis, and cell division) are susceptible or resistant to HGT.
Recently, Choi et al. (2007) suggested that there is no strong preference of HGT for protein families of particular cellular or molecular functions. They reconfirmed previous findings that HGT was biased toward cell surface and DNA binding functions (Nakamura et al., 2004), but the biases are marginal. They suggest that HGT is nearly neutral to all genes and that a random HGT process is followed by selection due to environment or other factors.
These discrepancies may be due to differences in the methods (e.g. phylogenetic versus compositional methods) and databases used, the genome samples tested, and possibly other reasons. For example, functions were assigned to protein families by using different databases: that is, (i) The Institute for Genomic Research role categories database (Peterson et al., 2001), (ii) The NCBI clusters of orthologous groups (COG) database (http://www.ncbi.nlm.nih.gov/COG/), and (iii) Gene Ontology (GO) terms (Camon et al., 2003).
This has inspired us to examine functional correlates of the vertically versus horizontally transferred genes using uniform approaches (methods and databases).
REFERENCES:
Jain R, Rivera MC, Lake JA. Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3801-6. Horizontal gene transfer among genomes: the complexity hypothesis.
Nakamura Y, Itoh T, Matsuda H, Gojobori T. Nat Genet. 2004 Jul;36(7):760-6. Biased biological functions of horizontally transferred genes in prokaryotic genomes.
Beiko RG, Harlow TJ, Ragan MA. Proc Natl Acad Sci U S A. 2005 Oct 4;102(40):14332-7. Highways of gene sharing in prokaryotes.
Choi IG, Kim SH. Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4489-94. Global extent of horizontal gene transfer.
Dr. Haruo Suzuki
University of Idaho
Monday, December 8, 2008
Monday, December 1, 2008
Host roles in plasmid partitioning
In order to gain a clear idea of how plasmids or bacteria function on their own, it is necessary to have some understanding of how the two interact. There are, for example, obvious ways in which one influences the other, such as the conference of useful phenotypic traits (e.g. antibiotic resistance) to the host and the molecular maintenance (e.g. DNA replication) of plasmids by the host. There are also much more subtle interactions between the two, which are just now being elucidated for the first time. One such interaction is described by Kolatka et. al. in their recent publication.
This article describes the interactions of the broad-host-range IncP-1 plasmid RK2 and the partitioning systems of Pseudomonas putida and Escherichia coli. The authors found that the subcellular location of a given type of plasmid in a given strain of host will have a particular location within the cell. Also, and even more interestingly, this position depends on the protein products of both the host and plasmid.
Comparing the subcellular location of an RK2 mini-derivative in E. coli and P. putida showed this interaction between host and plasmid. Because the plasmid lacked an active partitioning system, the partitioning machinery of its host determined its position. In the case of E. coli, the plasmid was found to cluster at the cell poles, whereas in P. putida it was located either at a mid-cell or one quarter of the way into the cell on either side. The location within P. putida was explained by the interactions between the centromere-like sequences on the plasmid and the ParB protein encoded by the host’s chromosome. Indeed, when the parB gene was inserted into the E. coli chromosome the resultant partitioning mirrored that of P. putida. Conversely, when the par genes in P. putida where made nonfunctional then the plasmids were found at the poles. The position of RK2 itself showed similar locational disruptions when its position was determined in the P. putida par mutants. In all cases, plasmid location was determined by fluorescence in situ hybridization (FISH) and fluorescence microscopy. Protein interactions between the plasmids and bacteria were determined by formaldehyde cross-linking and chromatin immunoprecipitation.
It is shown here that, as always, the actions and activity of an individual organism (or even a mobile genetic element) are by no means completely independent, but rather that they are constantly influenced by the organisms and environments that they come into contact with. Indeed, the basic tenets of evolution involve, not isolated individuals, but the interaction between individuals. Therefore, in the case of bacteria and plasmids, it is necessary to not only study one and then the other, but also the influence that they have on one another.
Primary Article:
Kolatka, K., M. Witosinska, M. Pierechod, and I. Konieczny. 2008. Bacterial partitioning proteins affect the subcellular location of broad-host-range plasmid RK2. Microbiology. 154:2847-56.
Additional References:
Ebersbach, G. & Gerdes, K. (2005). Plasmid segregation mechanisms. Annu Rev Genet 39, 453–479.
Funnell, B. E. (2005). Partition-mediated plasmid pairing. Plasmid 53, 119–125.
Gordon, S., Rech, J., Lane, D. & Wright, A. (2004). Kinetics of plasmid segregation in Escherichia coli. Mol Microbiol 51, 461–469.
Julie Hughes
Graduate Student
Department of Biological Sciences
University of Idaho
This article describes the interactions of the broad-host-range IncP-1 plasmid RK2 and the partitioning systems of Pseudomonas putida and Escherichia coli. The authors found that the subcellular location of a given type of plasmid in a given strain of host will have a particular location within the cell. Also, and even more interestingly, this position depends on the protein products of both the host and plasmid.
Comparing the subcellular location of an RK2 mini-derivative in E. coli and P. putida showed this interaction between host and plasmid. Because the plasmid lacked an active partitioning system, the partitioning machinery of its host determined its position. In the case of E. coli, the plasmid was found to cluster at the cell poles, whereas in P. putida it was located either at a mid-cell or one quarter of the way into the cell on either side. The location within P. putida was explained by the interactions between the centromere-like sequences on the plasmid and the ParB protein encoded by the host’s chromosome. Indeed, when the parB gene was inserted into the E. coli chromosome the resultant partitioning mirrored that of P. putida. Conversely, when the par genes in P. putida where made nonfunctional then the plasmids were found at the poles. The position of RK2 itself showed similar locational disruptions when its position was determined in the P. putida par mutants. In all cases, plasmid location was determined by fluorescence in situ hybridization (FISH) and fluorescence microscopy. Protein interactions between the plasmids and bacteria were determined by formaldehyde cross-linking and chromatin immunoprecipitation.
It is shown here that, as always, the actions and activity of an individual organism (or even a mobile genetic element) are by no means completely independent, but rather that they are constantly influenced by the organisms and environments that they come into contact with. Indeed, the basic tenets of evolution involve, not isolated individuals, but the interaction between individuals. Therefore, in the case of bacteria and plasmids, it is necessary to not only study one and then the other, but also the influence that they have on one another.
Primary Article:
Kolatka, K., M. Witosinska, M. Pierechod, and I. Konieczny. 2008. Bacterial partitioning proteins affect the subcellular location of broad-host-range plasmid RK2. Microbiology. 154:2847-56.
Additional References:
Ebersbach, G. & Gerdes, K. (2005). Plasmid segregation mechanisms. Annu Rev Genet 39, 453–479.
Funnell, B. E. (2005). Partition-mediated plasmid pairing. Plasmid 53, 119–125.
Gordon, S., Rech, J., Lane, D. & Wright, A. (2004). Kinetics of plasmid segregation in Escherichia coli. Mol Microbiol 51, 461–469.
Julie Hughes
Graduate Student
Department of Biological Sciences
University of Idaho
Wednesday, November 26, 2008
Piggery manure used for soil fertilization is a reservoir for
transferable antibiotic resistance plasmids
Chu Thi Thanh Binh, Holger Heuer, Martin Kaupenjohann & Kornelia Smalla
FEMS MICROBIOL. ECOL. 66:25-37
Overuse of antibiotics has been responsible for the spread of antibiotic resistance among bacteria all over the world. Continual use of antibiotics has maintained a strong selective pressure for the persistence of antibiotic resistance genes, while horizontal gene transfer has resulted in the spread of these genes across phylogenetically diverse bacteria (Witte, 1998; Rhodes et al., 2000; Schmidt et al., 2001; Tennstedt et al., 2003). Studies on plasmid content from manures have shown the presence of transferable plasmids carrying antibiotic resistance genes (Gotz et al., 1996; Smalla et al., 2000; Heuer et al., 2002; van Overbeek et al., 2002). This study looks at manures from 15 pig farms, where each farm represents a different size of herd or different quantity of meat production.
16 manure samples were taken from 15 different farms across Germany. Exogenous biparental matings were carried out in the laboratory by using E. coli CV601 as the recipient and manure as donor. Mixtures of recipient and donor were incubated overnight and then plated on agar supplemented with either amoxicillin, sulfadiazine or tetracycline.
A total of 228 transconjugants were picked. Eight antibiotics were tested on all transconjugants using the disc diffusion method (Barry et al.,). Based on different combinations of antibiotic resistance phenotypes, 37 unique patterns were found. 204 transconjugants showed sulfadiazine resistance. The frequent use of sulfadizine in animal husbandry may be the reason for this observation. 40 transconjugants showed resistance to six antibiotics and 4 were resistant to all 8 antibiotics used. This is a frightening scenario, since only 8 antibiotics were tested and many more resistance genes may be present on these plasmids. A previous study (Normark & Normark, 2002) had shown that selection for one antibiotic might co-select other antibiotics. The authors hypothesize that this may be the reason for the appearance of multiple antibiotic resistances on these plasmids. In order to make their study simpler, they decided to use a subset of the 228 transconjugants. Hence, one transconjugant was chosen per manure for each antibiotic resistance pattern. This gave them 81 plasmids which they decided to analyse further.
Plasmids extracted from transconjugants were dot-blotted and hybridized with probes specific for replicon sequences of the broad-host-range (BHR) plasmid classes IncN, IncW, IncP-1 and IncQ. 28 plasmids were found to be IncN, 1 was IncW, 13 were IncP-1, 19 were similar to the recently discovered pHHV216-like plasmids (Heuer et al., 2008) and 20 plasmids could not be assigned to any of the known Inc groups. Next the authors wanted to see which genes were conferring resistances to amoxicillin and sulfadiazine in these plasmids. Dot-blotted plasmid DNA was hybridized with labeled probes for bla-TEM, sul1, sul2 and sul3 genes. While bla-TEM genes are most often associated with resistance to amoxicillin, a combination of sul1, sul2 and sul3 genes may be responsible for sulfadiazine resistance. From this experiment they saw that all transconjugants with the amoxicillin resistance phenotype carried the bla-TEM gene, confirming the findings of Binh et al., who showed the frequent occurrence of bla-TEM genes in manure and amoxicillin resistance soils. An interesting observation was the repeated occurrence of these genes on similar plasmids, for example the occurrence of bla-TEM genes on all 28 IncN plasmids. The authors conclude that IncN plasmids that were captured from 10 different manures could be responsible for the dissemination of bla-TEM genes. Similarly, the sul2 gene was found on all 19 pHHV216-type plasmids captured from 6 manures and the sul1 gene was found on 12 of 13 IncP-1 plasmids. The authors state that their work shows that antibiotic resistance genes are associated preferably with BHR plasmids. Next the authors tested the transferability of the 81 plasmids by carrying out matings where they used their transconjugants as donors and E. coli J53 as the recipient. They found that 73 could be transferred to the recipient and only 8 could not. 6 of these 8 were the pHHV216-like plasmids.
In order to compare the method of direct PCR-based detection of plasmids in total DNA of manure to the method of plasmid capture, they used primers specific to repA for IncN, trfA2 for IncP-1, or oriV for IncQ and IncW for PCR of total DNA of manure.
No correlation was observed between the frequency of plasmid capture and plasmid abundance as noted from total DNA of manure. For example, although one third of the plasmids captured from 15 manures were characterized as IncN, this class of plasmid was detected in only 5 manures by PCR and Southern blot hybridization. The authors attribute this to the low abundance of IncN plasmids in manure, which could have resulted in making PCR based detection difficult. Using the exogenous plasmid isolation method, they were able to capture IncN plasmids from these soils. Thus, they suggest that the exogenous isolation method captures plasmids even when they are not abundant and PCR-based detection of plasmid types may not be as efficient.
This study is important because it shows how prevalent broad host range plasmids are. Moreover, association of antibiotic resistance genes with such plasmids ensures their rapid spread in an environment with antibiotics that maintain a strong selection. We get some idea of the prevalence of resistance to antibiotics in bacteria. All transconjugants were found to confer resistance to one or more antibiotics. Co-selection of antibiotics is also a phenomenon that we should be looking at closely.
References:
Witte W. (1998): Medical consequences of antibiotic use in agriculture. Science 279: 996–997.
Rhodes G., Huys G., Swings J., McGann P., Hiney M., Smith P. & Pickup R.W.
(2000): Distribution of oxytetracycline resistance plasmids between Aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant Tet A. Appl Environ Microbiol 66: 3883–3890.
Schmidt A.S., Bruun M.S., Dalsgaard I. & Larsen J.L. (2001): Incidence, distribution, and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish farming environment. Appl Environ Microbiol 67: 5675–5682.
Tennstedt T., Szczepanowski R., Braun S., Puhler A. & Schlüter A. (2003): Occurrence of integron-associated resistance gene cassettes located on antibiotic resistance plasmids isolated from a wastewater treatment plant. FEMS Microbiol Ecol 45:239–252.
Gotz A., Pukall R., Smit E., Tietze E., Prager R., Tschape H., van Elsas J.D. & Smalla K. (1996) Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR. Appl Environ Microbiol 62: 2621–2628.
Smalla K., Heuer H., Gotz A., Niemeyer D., Krogerrecklenfort E. & Tietze E., 2000: Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity of IncQ-like plasmids. Appl Environ Microbiol 66: 4854–4862.
Heuer H., Krogerrecklenfort E., Egan S. et al. (2002): Gentamicin resistance genes in environmental bacteria: prevalence and transfer. FEMS Microbiol Ecol 42: 28-302.
Van Overbeek L.S., Wellington E.M.H., Egan S., Smalla K., Heuer H., Collard J.M., Guillaume G., Karagouni A.D., Nikolakopoulou T.L. & van Elsas J.D. (2002): Prevalence of streptomycin-resistance genes in bacterial populations in European habitats. FEMS Microbiol Ecol 42: 277–288.
Normark B.H. & Normark S (2002): Evolution and spread of antibiotic resistance. J Intern Med 252: 91–106.
Heuer H., Kopmann C., Binh C.T. T., Top E.M., Smalla K.(2008): Spreading antibiotic resistance through spread manure: characteristics 1 of a novel 2 plasmid type with low %G+C content. In press.
Barry A. L., Garcia F., and Thrupp L.D. (1970): An improved single-disk method for testing the antibiotic susceptibility of rapidly-growing pathogens. Am. J. Clin. Pathol. 53:149-158.
Diya Sen
Graduate Student,
Department of Biological Sciences,
University of Idaho
transferable antibiotic resistance plasmids
Chu Thi Thanh Binh, Holger Heuer, Martin Kaupenjohann & Kornelia Smalla
FEMS MICROBIOL. ECOL. 66:25-37
Overuse of antibiotics has been responsible for the spread of antibiotic resistance among bacteria all over the world. Continual use of antibiotics has maintained a strong selective pressure for the persistence of antibiotic resistance genes, while horizontal gene transfer has resulted in the spread of these genes across phylogenetically diverse bacteria (Witte, 1998; Rhodes et al., 2000; Schmidt et al., 2001; Tennstedt et al., 2003). Studies on plasmid content from manures have shown the presence of transferable plasmids carrying antibiotic resistance genes (Gotz et al., 1996; Smalla et al., 2000; Heuer et al., 2002; van Overbeek et al., 2002). This study looks at manures from 15 pig farms, where each farm represents a different size of herd or different quantity of meat production.
16 manure samples were taken from 15 different farms across Germany. Exogenous biparental matings were carried out in the laboratory by using E. coli CV601 as the recipient and manure as donor. Mixtures of recipient and donor were incubated overnight and then plated on agar supplemented with either amoxicillin, sulfadiazine or tetracycline.
A total of 228 transconjugants were picked. Eight antibiotics were tested on all transconjugants using the disc diffusion method (Barry et al.,). Based on different combinations of antibiotic resistance phenotypes, 37 unique patterns were found. 204 transconjugants showed sulfadiazine resistance. The frequent use of sulfadizine in animal husbandry may be the reason for this observation. 40 transconjugants showed resistance to six antibiotics and 4 were resistant to all 8 antibiotics used. This is a frightening scenario, since only 8 antibiotics were tested and many more resistance genes may be present on these plasmids. A previous study (Normark & Normark, 2002) had shown that selection for one antibiotic might co-select other antibiotics. The authors hypothesize that this may be the reason for the appearance of multiple antibiotic resistances on these plasmids. In order to make their study simpler, they decided to use a subset of the 228 transconjugants. Hence, one transconjugant was chosen per manure for each antibiotic resistance pattern. This gave them 81 plasmids which they decided to analyse further.
Plasmids extracted from transconjugants were dot-blotted and hybridized with probes specific for replicon sequences of the broad-host-range (BHR) plasmid classes IncN, IncW, IncP-1 and IncQ. 28 plasmids were found to be IncN, 1 was IncW, 13 were IncP-1, 19 were similar to the recently discovered pHHV216-like plasmids (Heuer et al., 2008) and 20 plasmids could not be assigned to any of the known Inc groups. Next the authors wanted to see which genes were conferring resistances to amoxicillin and sulfadiazine in these plasmids. Dot-blotted plasmid DNA was hybridized with labeled probes for bla-TEM, sul1, sul2 and sul3 genes. While bla-TEM genes are most often associated with resistance to amoxicillin, a combination of sul1, sul2 and sul3 genes may be responsible for sulfadiazine resistance. From this experiment they saw that all transconjugants with the amoxicillin resistance phenotype carried the bla-TEM gene, confirming the findings of Binh et al., who showed the frequent occurrence of bla-TEM genes in manure and amoxicillin resistance soils. An interesting observation was the repeated occurrence of these genes on similar plasmids, for example the occurrence of bla-TEM genes on all 28 IncN plasmids. The authors conclude that IncN plasmids that were captured from 10 different manures could be responsible for the dissemination of bla-TEM genes. Similarly, the sul2 gene was found on all 19 pHHV216-type plasmids captured from 6 manures and the sul1 gene was found on 12 of 13 IncP-1 plasmids. The authors state that their work shows that antibiotic resistance genes are associated preferably with BHR plasmids. Next the authors tested the transferability of the 81 plasmids by carrying out matings where they used their transconjugants as donors and E. coli J53 as the recipient. They found that 73 could be transferred to the recipient and only 8 could not. 6 of these 8 were the pHHV216-like plasmids.
In order to compare the method of direct PCR-based detection of plasmids in total DNA of manure to the method of plasmid capture, they used primers specific to repA for IncN, trfA2 for IncP-1, or oriV for IncQ and IncW for PCR of total DNA of manure.
No correlation was observed between the frequency of plasmid capture and plasmid abundance as noted from total DNA of manure. For example, although one third of the plasmids captured from 15 manures were characterized as IncN, this class of plasmid was detected in only 5 manures by PCR and Southern blot hybridization. The authors attribute this to the low abundance of IncN plasmids in manure, which could have resulted in making PCR based detection difficult. Using the exogenous plasmid isolation method, they were able to capture IncN plasmids from these soils. Thus, they suggest that the exogenous isolation method captures plasmids even when they are not abundant and PCR-based detection of plasmid types may not be as efficient.
This study is important because it shows how prevalent broad host range plasmids are. Moreover, association of antibiotic resistance genes with such plasmids ensures their rapid spread in an environment with antibiotics that maintain a strong selection. We get some idea of the prevalence of resistance to antibiotics in bacteria. All transconjugants were found to confer resistance to one or more antibiotics. Co-selection of antibiotics is also a phenomenon that we should be looking at closely.
References:
Witte W. (1998): Medical consequences of antibiotic use in agriculture. Science 279: 996–997.
Rhodes G., Huys G., Swings J., McGann P., Hiney M., Smith P. & Pickup R.W.
(2000): Distribution of oxytetracycline resistance plasmids between Aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant Tet A. Appl Environ Microbiol 66: 3883–3890.
Schmidt A.S., Bruun M.S., Dalsgaard I. & Larsen J.L. (2001): Incidence, distribution, and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish farming environment. Appl Environ Microbiol 67: 5675–5682.
Tennstedt T., Szczepanowski R., Braun S., Puhler A. & Schlüter A. (2003): Occurrence of integron-associated resistance gene cassettes located on antibiotic resistance plasmids isolated from a wastewater treatment plant. FEMS Microbiol Ecol 45:239–252.
Gotz A., Pukall R., Smit E., Tietze E., Prager R., Tschape H., van Elsas J.D. & Smalla K. (1996) Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR. Appl Environ Microbiol 62: 2621–2628.
Smalla K., Heuer H., Gotz A., Niemeyer D., Krogerrecklenfort E. & Tietze E., 2000: Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity of IncQ-like plasmids. Appl Environ Microbiol 66: 4854–4862.
Heuer H., Krogerrecklenfort E., Egan S. et al. (2002): Gentamicin resistance genes in environmental bacteria: prevalence and transfer. FEMS Microbiol Ecol 42: 28-302.
Van Overbeek L.S., Wellington E.M.H., Egan S., Smalla K., Heuer H., Collard J.M., Guillaume G., Karagouni A.D., Nikolakopoulou T.L. & van Elsas J.D. (2002): Prevalence of streptomycin-resistance genes in bacterial populations in European habitats. FEMS Microbiol Ecol 42: 277–288.
Normark B.H. & Normark S (2002): Evolution and spread of antibiotic resistance. J Intern Med 252: 91–106.
Heuer H., Kopmann C., Binh C.T. T., Top E.M., Smalla K.(2008): Spreading antibiotic resistance through spread manure: characteristics 1 of a novel 2 plasmid type with low %G+C content. In press.
Barry A. L., Garcia F., and Thrupp L.D. (1970): An improved single-disk method for testing the antibiotic susceptibility of rapidly-growing pathogens. Am. J. Clin. Pathol. 53:149-158.
Diya Sen
Graduate Student,
Department of Biological Sciences,
University of Idaho
Saturday, November 22, 2008
Lessons from the first comprehensive survey of prokaryote genomics
Genomics of bacteria and archaeal: the emerging dynamic view of the prokaryotic world. Eugene V. Koonin & Yuri I. Wolf, Nucleic Acids Research (2008)
The first bacterial genome (Haemophilus influenzae) was published in 1995, ushering in the so-called age of genomics. Since then, exponentially increasing numbers of whole-genome sequencing projects have generated a huge amount of raw data. While this holds great promise for developing our understanding of how prokaryotic genomes are formed and function, extracting meaningful observations from that mountain of data is a big challenge.
Drs Eugene Koonin and Yuri Wolf recently tackled this daunting task and embarked on a comprehensive survey of the genomic data produced to date by microbial sequencing projects. Their latest paper, titled “Genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world”, presents their findings in a dense but rich monograph that offers deep insight into processes of genome evolution and defines general principles of prokaryotic genome organization.
Although there can be no substitute for reading the original paper, a point-by-point summary of the paper that may interest readers is provided here.
Dr. Geraldine A. Van der Auwera
Harvard Medical School
The first bacterial genome (Haemophilus influenzae) was published in 1995, ushering in the so-called age of genomics. Since then, exponentially increasing numbers of whole-genome sequencing projects have generated a huge amount of raw data. While this holds great promise for developing our understanding of how prokaryotic genomes are formed and function, extracting meaningful observations from that mountain of data is a big challenge.
Drs Eugene Koonin and Yuri Wolf recently tackled this daunting task and embarked on a comprehensive survey of the genomic data produced to date by microbial sequencing projects. Their latest paper, titled “Genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world”, presents their findings in a dense but rich monograph that offers deep insight into processes of genome evolution and defines general principles of prokaryotic genome organization.
Although there can be no substitute for reading the original paper, a point-by-point summary of the paper that may interest readers is provided here.
Dr. Geraldine A. Van der Auwera
Harvard Medical School
Wednesday, October 15, 2008
Bacteriophages SPP1 contains two tail tube proteins produced by programmed translational frameshift
Origin and function of the two major tail proteins of bacteriophage SPP1. Auzat et al., Molecular Microbiology (2008) 70, 557-569
Bacteriophages are viruses that infect bacteria and are known to play roles in horizontal gene transfer. The majority of known bacteriophages have a head and long non-contractile tail that serves as a pipeline for phage genome delivery into bacterial cell. Here, the authors report that the tail tube of Bacillus subtilus bacteriaphage SPP1 is comprised of two proteins, gp17.1 and gp17.1* , that are produced by a translational frameshift. This mosaic construction of tail tube was found to be important for assembly of the functional tail tube, but its significance is not fully uncovered yet.
Previously, it was shown that the key event of phage DNA injection in bacterial cell is a rearrangement of the inner wall of the tail tube (EMBO J, (2007) 26, 3720-3728). In this article, the authors separated SSP1 tail proteins by SDS-PAGE and found a protein band which was not expected from phage DNA sequence. Protein sequencing analysis indicated that the unexpected protein designated gp17.1* has the same amino-terminal sequence as that of tail protein gp17.1. gp17.1* is 10 kDa larger than gp17.1. The tail tube is made up of these two proteins at the ratio of 1:3. Based on the molecular size of gp17.1* and the sequences of gene 17.1, authors postulated that 17.1* is produced by a translational frameshift. Using site-directed mutagenesis of coding sequence 17.1 with protein profile analysis of the mutant phages, authors found out that 5'-CCCUAA-3' sequence located at the end of coding sequence 17.1 was the frameshift position.
To get insight into the function of gp17.1*, the authors constructed mutant phages that have tail tubes comprised exclusively of gp17.1 or gp17.1* and analyzed their structures by electron microscopy. When phages are assembled under the condition which either gp17.1 or gp17.1* are exclusively expressed, significant numbers of tailless heads (capsids) are made. This suggests the 3:1 ratio of gp17.1 and gp17.1* is important for correct phage assembly. Interestingly, both mutant phages had infection activity. The length and flexibility of mutant tails composed of either gp17.1 or gp17.1* were identical to SSP1 wild-type tails. gp17.1*-specific tail has protrusions on surface while gp17.1-specific tail has smooth surface. Authors postulate that carboxyl-terminus of gp17.1* causes protrusions on tail surface which facilitate initial contact of phages and attachment to the bacterial surface, while gp17.1 is ensures correct assembly of tail tube.
The potential translational frameshift site producing a carboxyl-terminus extension in a protein are also found in other phage surface protein genes (Mol Microbiol. (2003) 50, 303-317). Why do phages need two types of surface proteins which are identical except the carboxyl end extension of one of the proteins? What is the significance of strict ratio of the two proteins? Why is this the best strategy for phages? What is the target of the protrusion on tail tube surface? These are still interesting mysteries. It might be interesting to compare the host ranges of mutant phages that have either one of the two surface proteins.
References
Origin and Function of the Two Major Tail Proteins of Bacteriophage SPP1. Auzat I, Dröge A, Weise F, Lurz R, Tavares P. Molecular Microbiology (2008) 70: 557-569
Structure of bacteriophage SPP1 tail reveals trigger for DNA ejection. Plisson C, White HE, Auzat I, Zafarani A, São-José C, Lhuillier S, Tavares P, Orlova EV,
EMBO J (2007) 26:3720-8.
Genome and proteome of Listeria monocytogenes phage PSA: an unusual case for programmed + 1 translational frameshifting in structural protein synthesis. Zimmer M, Sattelberger E, Inman RB, Calendar R, Loessner MJ., Molecular Microbiology. (2003) 50:303-317.
Hirokazu Yano (university of idaho)
Bacteriophages are viruses that infect bacteria and are known to play roles in horizontal gene transfer. The majority of known bacteriophages have a head and long non-contractile tail that serves as a pipeline for phage genome delivery into bacterial cell. Here, the authors report that the tail tube of Bacillus subtilus bacteriaphage SPP1 is comprised of two proteins, gp17.1 and gp17.1* , that are produced by a translational frameshift. This mosaic construction of tail tube was found to be important for assembly of the functional tail tube, but its significance is not fully uncovered yet.
Previously, it was shown that the key event of phage DNA injection in bacterial cell is a rearrangement of the inner wall of the tail tube (EMBO J, (2007) 26, 3720-3728). In this article, the authors separated SSP1 tail proteins by SDS-PAGE and found a protein band which was not expected from phage DNA sequence. Protein sequencing analysis indicated that the unexpected protein designated gp17.1* has the same amino-terminal sequence as that of tail protein gp17.1. gp17.1* is 10 kDa larger than gp17.1. The tail tube is made up of these two proteins at the ratio of 1:3. Based on the molecular size of gp17.1* and the sequences of gene 17.1, authors postulated that 17.1* is produced by a translational frameshift. Using site-directed mutagenesis of coding sequence 17.1 with protein profile analysis of the mutant phages, authors found out that 5'-CCCUAA-3' sequence located at the end of coding sequence 17.1 was the frameshift position.
To get insight into the function of gp17.1*, the authors constructed mutant phages that have tail tubes comprised exclusively of gp17.1 or gp17.1* and analyzed their structures by electron microscopy. When phages are assembled under the condition which either gp17.1 or gp17.1* are exclusively expressed, significant numbers of tailless heads (capsids) are made. This suggests the 3:1 ratio of gp17.1 and gp17.1* is important for correct phage assembly. Interestingly, both mutant phages had infection activity. The length and flexibility of mutant tails composed of either gp17.1 or gp17.1* were identical to SSP1 wild-type tails. gp17.1*-specific tail has protrusions on surface while gp17.1-specific tail has smooth surface. Authors postulate that carboxyl-terminus of gp17.1* causes protrusions on tail surface which facilitate initial contact of phages and attachment to the bacterial surface, while gp17.1 is ensures correct assembly of tail tube.
The potential translational frameshift site producing a carboxyl-terminus extension in a protein are also found in other phage surface protein genes (Mol Microbiol. (2003) 50, 303-317). Why do phages need two types of surface proteins which are identical except the carboxyl end extension of one of the proteins? What is the significance of strict ratio of the two proteins? Why is this the best strategy for phages? What is the target of the protrusion on tail tube surface? These are still interesting mysteries. It might be interesting to compare the host ranges of mutant phages that have either one of the two surface proteins.
References
Origin and Function of the Two Major Tail Proteins of Bacteriophage SPP1. Auzat I, Dröge A, Weise F, Lurz R, Tavares P. Molecular Microbiology (2008) 70: 557-569
Structure of bacteriophage SPP1 tail reveals trigger for DNA ejection. Plisson C, White HE, Auzat I, Zafarani A, São-José C, Lhuillier S, Tavares P, Orlova EV,
EMBO J (2007) 26:3720-8.
Genome and proteome of Listeria monocytogenes phage PSA: an unusual case for programmed + 1 translational frameshifting in structural protein synthesis. Zimmer M, Sattelberger E, Inman RB, Calendar R, Loessner MJ., Molecular Microbiology. (2003) 50:303-317.
Hirokazu Yano (university of idaho)
Tuesday, October 7, 2008
Type 3 fimbriae, encoded by the conjugative plasmid pOLA52, enhance biofilm formation and transfer frequencies in Enterobacteriaceae strains
Mette Burmølle, Martin Iain Bahl, Lars Bogø Jensen, Søren J. Sørensen and Lars Hestbjerg Hansen
Microbiology (2008), 154, 187–195
In this paper researchers from University of Copenhagen and The National Food Institute in Denmark bring our attention to a conjugative plasmid pOLA52 which features and genetic content occurred to be disturbing as they regard human and animal health.
pOLA52 plasmid was first isolated from swine manure and was shown to encode multidrug efflux pump which provides resistance to many antimicrobial agents such as olaquindox (which was or still is commonly used as a growth factor in pig farming), chloramphenicol, ethidium bromide, other antibiotics, detergents and disinfectants. It also carries bla gene conferring resisitance to
Authors of the paper have previously observed that E. coli CSH26 strain harbouring pOLA52 plasmid formed higher amounts of biofilm and thus wanted to investigate if the operon conferring type 3 fimbriae could be responsible for observed feature.
In this study authors randomly introduced entranceposon pENTRANCEPOSON (KanR) into pOLA52 plasmid, electroporated it into E. coli Genehogs, selected tranformants on Kan and checked their ability to form biofilms on urinary catheters. Some clones occured to be biofilm negative and sequencing revealed that inserts were located inside type 3 fimbriae operon. Not surprisingly, biofilm positive clones had the inserts outside the operon.
Researchers have checked expression of mrk genes with RT-PCR and showed with immunoblotting that biofilm positive clones expressed type 3 fimbriae, whereas biofilm negative did not.
Later, they have conducted conjugation of pOLA52 plasmid into potentially pathogenic Enterobacteriaceae strains (such as Klebsiella pneumoniae, Salmonella typhimurium, Kluyvera sp., Enterobacter aerogenes) and tested the ability of transconjugants to form biofilms. It occured that transconjugants harbouring plasmid with transposon inside mrk operon showed significantly lower rate of plasmid transfer comparing to strains carrying wild type plasmid. Futhermore, transconjugants harbouring wild type pOLA52 plasmid formed biofilms, whereas strains with operon mrk mutated plasmid showed much lower biofilm formation.
This study proves how important and potentially dangerous pOLA52 plasmid is, as it can be transferred via conjugation to other bacteria, including pathogenic strains, providing them with new antibiotic resistances and type 3 fimbriae increasing their ability of spread plasmids and to form biofilms. Those newly accuaired features can lead to higher antibiotic persistence and increased spreading of pathogens on biological surfaces, such as tissues, as well as abiotic ones, for example catheters or artificial heart valves.
As pOLA52 plasmid is the first of probably many more plasmids with similar genetic content, there is a risk that one day they could be used as another dangerous weapon in hands of pathogenic bacteria. Let's hope we will be well prepared if this day would come...
Sylwia Deneka
Visiting Scholar
University of Idaho
Microbiology (2008), 154, 187–195
In this paper researchers from University of Copenhagen and The National Food Institute in Denmark bring our attention to a conjugative plasmid pOLA52 which features and genetic content occurred to be disturbing as they regard human and animal health.
pOLA52 plasmid was first isolated from swine manure and was shown to encode multidrug efflux pump which provides resistance to many antimicrobial agents such as olaquindox (which was or still is commonly used as a growth factor in pig farming), chloramphenicol, ethidium bromide, other antibiotics, detergents and disinfectants. It also carries bla gene conferring resisitance to
Authors of the paper have previously observed that E. coli CSH26 strain harbouring pOLA52 plasmid formed higher amounts of biofilm and thus wanted to investigate if the operon conferring type 3 fimbriae could be responsible for observed feature.
In this study authors randomly introduced entranceposon pENTRANCEPOSON (KanR) into pOLA52 plasmid, electroporated it into E. coli Genehogs, selected tranformants on Kan and checked their ability to form biofilms on urinary catheters. Some clones occured to be biofilm negative and sequencing revealed that inserts were located inside type 3 fimbriae operon. Not surprisingly, biofilm positive clones had the inserts outside the operon.
Researchers have checked expression of mrk genes with RT-PCR and showed with immunoblotting that biofilm positive clones expressed type 3 fimbriae, whereas biofilm negative did not.
Later, they have conducted conjugation of pOLA52 plasmid into potentially pathogenic Enterobacteriaceae strains (such as Klebsiella pneumoniae, Salmonella typhimurium, Kluyvera sp., Enterobacter aerogenes) and tested the ability of transconjugants to form biofilms. It occured that transconjugants harbouring plasmid with transposon inside mrk operon showed significantly lower rate of plasmid transfer comparing to strains carrying wild type plasmid. Futhermore, transconjugants harbouring wild type pOLA52 plasmid formed biofilms, whereas strains with operon mrk mutated plasmid showed much lower biofilm formation.
This study proves how important and potentially dangerous pOLA52 plasmid is, as it can be transferred via conjugation to other bacteria, including pathogenic strains, providing them with new antibiotic resistances and type 3 fimbriae increasing their ability of spread plasmids and to form biofilms. Those newly accuaired features can lead to higher antibiotic persistence and increased spreading of pathogens on biological surfaces, such as tissues, as well as abiotic ones, for example catheters or artificial heart valves.
As pOLA52 plasmid is the first of probably many more plasmids with similar genetic content, there is a risk that one day they could be used as another dangerous weapon in hands of pathogenic bacteria. Let's hope we will be well prepared if this day would come...
Sylwia Deneka
Visiting Scholar
University of Idaho
Wednesday, October 1, 2008
What a man can make …….
RESEARCH ARTICLES
Genome Transplantation in Bacteria: Changing One Species to Another
Carole Lartigue, John I. Glass, Nina Alperovich, Rembert Pieper, Prashanth P. Parmar, Clyde A. Hutchison III, Hamilton O. Smith, and J. Craig Venter 2007. Science 317, 632.
Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasmagenitalium Genome.
Daniel G. Gibson, Gwynedd A. Benders, Cynthia Andrews-Pfannkoch, Evgeniya A. Denisova, Holly Baden-Tillson, JayshreeZaveri, Timothy B. Stockwell, AnushkaBrownley, David W. Thomas, Mikkel A. Algire, Chuck Merryman, Lei Young, Vladimir N. Noskov, John I. Glass, J. Craig Venter, Clyde A. Hutchison III, Hamilton O. Smith 2008Published Online January 24, 2008 Science DOI: 10.1126/science.1151721
In these two papers, researchers from J. Craig Venter Institute in Rockville Maryland showed us the possibility of changing the entire genetic information of a living microorganism to create a new artificial organism.
In first paper they used two closely related Mycoplasmas: Mycoplasma mycoides large colony (LC) and M. capricolum. They used mycoplasma because of its specificity. Mycoplasmas do not have a cell wall, which makes them resistant to some therapeutic antibiotics like B-lactams. This also makes them more susceptible for uptake of different substances from the environment. One also cannot underestimate the biological importance of Mycoplasmas as pathogenic microorganisms, causing some nasty diseases in human and animals. Another interesting fact is that Mycoplasmas, compared to other bacteria, have relatively small genome, ranging from 0.6 to 1.4Mb. Up to now, 12 Mycolasma genomes have been completely sequenced and 11 more are in progress.
Small genome size and lack of the cell wall makes them ideal candidates for the genome “switching” experiment.
So, Carole Lartigue with coworkers took the large colony of M. mycoides and prepared intact genomic DNA in the way usually used for PFGE (pulsed field gel electrophoresis). All proteins were removed by proteinase treatment. This purified DNA was used for transformation of M. capricolum strain. After a few days some transformants grew on selective media. These transformants had genetic markers specific for donor strain but no sign of host specific markers was detected. That means that it is possible to replace a whole genome, at least using closely relative bacteria.
Cloning large genomic fragments is not a technical problem. Some bacterial artificial chromosome (BAC) clones already contain inserts of about 300kb in size. Such clones can be easily introduced into E. coli cells by electroporation. Using some specific recombination systems it could be possible to join two or three large clones inside E. coli cells to generate single DNA molecule, then isolate such a genome and introduce it into a new bacterial host.
But there is another way to build an entire genome. In second of the presented papers, the authors described this alternative way. They split the whole Mycoplasma genitalium genome into 101 cassettes, each of about 5000-7000bp. Those cassettes were chemically synthesized by three companies. All cassettes have specific overlapping sequences so they can be stuck together. The in vitro recombination event yielded intermediate assembliesof approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4genome"), which were all cloned as bacterial artificial chromosomes(BACs) in The complete synthetic genomewas assembled by transformation-associated recombination (TAR)cloning in the yeast Saccharomycescerevisiae, then isolatedand sequenced. This method allowed construction of an entire “artificial” bacterial genome.
Mycoplasmas have small genomes, but we already know some bacteria with even smaller genomes. The amphid endosymbiont Buchnera aphidicola genome consists of ~422kb, but a psyllidendosymbiont, Carlsonella ruddii, has even smaller genome (~160kb). Such small genomes are specific for pathogenic and especially endosymbiotic style of life. These bacteria reduce their genomes by deleting genes that are not necessary, like some anabolic pathways. On the other hand, they keep genes that are useful for themselves and for their hosts. These genes encode basic functions like replication, transcription and translation, as well as some of the biosynthetic pathways encoding for amino acids, cofactors and other essential compounds that their host cannot obtain from their diet. Surprisingly, also lots of genes encoding transport (uptake) systems have been eliminated.
Studying such small genomes we can learn which genes are really core genes and cannot be removed from the genome and which genes are not necessary. Furthermore, we can induce such big reductions in larger bacterial genomes. Analysis of these data as well as data obtained from analysis of the eukaryotic organelle (mitochondria and plastids) genomes could be used to construct an artificial endosymbiont.
Perhaps, it could be possible to make a new endosymbiont,which could be specific for some body tissue like liver or pancreas. They could live inside the cell,producingspecific proteins like insulin, clotting factors or other factors deficient in genetic diseases. This is fantasy but who knows the future…….
Additional articles:
S. G. E. Andersson 2006. TheBacterial World Gets Smaller. Science 13, pp.: 259 – 260.
A. I. Nilsson,S. Koskiniemi,S. Eriksson,E. Kugelberg,J. C. D. Hinton,D. I. Andersson 2005, Bacterial genome size reduction by experimental evolution. Proc. Natl. Acad.Sci. 102, pp.: 12112-12116.
Quanzhou Tao and Hong-Bin1998 Zhang 1998 Cloning and stable maintenance of DNA fragmentsover 300 kb in Escherichia coli with conventional plasmid-based vectors. NAR 26, 21, pp.: 4901-4909.
dr Jaroslaw Krol
UofI
Genome Transplantation in Bacteria: Changing One Species to Another
Carole Lartigue, John I. Glass, Nina Alperovich, Rembert Pieper, Prashanth P. Parmar, Clyde A. Hutchison III, Hamilton O. Smith, and J. Craig Venter 2007. Science 317, 632.
Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasmagenitalium Genome.
Daniel G. Gibson, Gwynedd A. Benders, Cynthia Andrews-Pfannkoch, Evgeniya A. Denisova, Holly Baden-Tillson, JayshreeZaveri, Timothy B. Stockwell, AnushkaBrownley, David W. Thomas, Mikkel A. Algire, Chuck Merryman, Lei Young, Vladimir N. Noskov, John I. Glass, J. Craig Venter, Clyde A. Hutchison III, Hamilton O. Smith 2008Published Online January 24, 2008 Science DOI: 10.1126/science.1151721
In these two papers, researchers from J. Craig Venter Institute in Rockville Maryland showed us the possibility of changing the entire genetic information of a living microorganism to create a new artificial organism.
In first paper they used two closely related Mycoplasmas: Mycoplasma mycoides large colony (LC) and M. capricolum. They used mycoplasma because of its specificity. Mycoplasmas do not have a cell wall, which makes them resistant to some therapeutic antibiotics like B-lactams. This also makes them more susceptible for uptake of different substances from the environment. One also cannot underestimate the biological importance of Mycoplasmas as pathogenic microorganisms, causing some nasty diseases in human and animals. Another interesting fact is that Mycoplasmas, compared to other bacteria, have relatively small genome, ranging from 0.6 to 1.4Mb. Up to now, 12 Mycolasma genomes have been completely sequenced and 11 more are in progress.
Small genome size and lack of the cell wall makes them ideal candidates for the genome “switching” experiment.
So, Carole Lartigue with coworkers took the large colony of M. mycoides and prepared intact genomic DNA in the way usually used for PFGE (pulsed field gel electrophoresis). All proteins were removed by proteinase treatment. This purified DNA was used for transformation of M. capricolum strain. After a few days some transformants grew on selective media. These transformants had genetic markers specific for donor strain but no sign of host specific markers was detected. That means that it is possible to replace a whole genome, at least using closely relative bacteria.
Cloning large genomic fragments is not a technical problem. Some bacterial artificial chromosome (BAC) clones already contain inserts of about 300kb in size. Such clones can be easily introduced into E. coli cells by electroporation. Using some specific recombination systems it could be possible to join two or three large clones inside E. coli cells to generate single DNA molecule, then isolate such a genome and introduce it into a new bacterial host.
But there is another way to build an entire genome. In second of the presented papers, the authors described this alternative way. They split the whole Mycoplasma genitalium genome into 101 cassettes, each of about 5000-7000bp. Those cassettes were chemically synthesized by three companies. All cassettes have specific overlapping sequences so they can be stuck together. The in vitro recombination event yielded intermediate assembliesof approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4genome"), which were all cloned as bacterial artificial chromosomes(BACs) in The complete synthetic genomewas assembled by transformation-associated recombination (TAR)cloning in the yeast Saccharomycescerevisiae, then isolatedand sequenced. This method allowed construction of an entire “artificial” bacterial genome.
Mycoplasmas have small genomes, but we already know some bacteria with even smaller genomes. The amphid endosymbiont Buchnera aphidicola genome consists of ~422kb, but a psyllidendosymbiont, Carlsonella ruddii, has even smaller genome (~160kb). Such small genomes are specific for pathogenic and especially endosymbiotic style of life. These bacteria reduce their genomes by deleting genes that are not necessary, like some anabolic pathways. On the other hand, they keep genes that are useful for themselves and for their hosts. These genes encode basic functions like replication, transcription and translation, as well as some of the biosynthetic pathways encoding for amino acids, cofactors and other essential compounds that their host cannot obtain from their diet. Surprisingly, also lots of genes encoding transport (uptake) systems have been eliminated.
Studying such small genomes we can learn which genes are really core genes and cannot be removed from the genome and which genes are not necessary. Furthermore, we can induce such big reductions in larger bacterial genomes. Analysis of these data as well as data obtained from analysis of the eukaryotic organelle (mitochondria and plastids) genomes could be used to construct an artificial endosymbiont.
Perhaps, it could be possible to make a new endosymbiont,which could be specific for some body tissue like liver or pancreas. They could live inside the cell,producingspecific proteins like insulin, clotting factors or other factors deficient in genetic diseases. This is fantasy but who knows the future…….
Additional articles:
S. G. E. Andersson 2006. TheBacterial World Gets Smaller. Science 13, pp.: 259 – 260.
A. I. Nilsson,S. Koskiniemi,S. Eriksson,E. Kugelberg,J. C. D. Hinton,D. I. Andersson 2005, Bacterial genome size reduction by experimental evolution. Proc. Natl. Acad.Sci. 102, pp.: 12112-12116.
Quanzhou Tao and Hong-Bin1998 Zhang 1998 Cloning and stable maintenance of DNA fragmentsover 300 kb in Escherichia coli with conventional plasmid-based vectors. NAR 26, 21, pp.: 4901-4909.
dr Jaroslaw Krol
UofI
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