The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair. Fineran PC, Blower TR, Foulds IJ, Humphreys DP, Lilley KS, and Salmond GP. (2009) Proc Natl Acad Sci U S A. 106:894-899.
Recently it has become easy to determine the complete sequence of 100 kb long plasmids. However, the ensuing annotation process, assigning a function to a DNA sequence, is still a frustrating process for people who are working on sequence analysis; often, the newly sequenced DNA segment does not show homology to well-known genes which makes difficult to judge whether or not there is a gene in the segment.
Let's hope that no homology is a good sign for a big discovery. Here, I introduce a discovery of the novel gene module on Erwinia carotovora plasmid pECA1039, which protect host bacterium from phage infection.
Erwinia carotovora is a plant pathogenic bacterium that causes rot in diverse vegetables. The author's group has been studying virulence mechanisms of E. carotovora (Barnard A.M. and Bowden S.D. et al., 2007). They identified a probable protein coding sequence, named toxN, through the complete sequence analysis of E. carotovora's 5,620-bp cryptic plasmid., whose product shows 31% amino-acid identity to a protein associated with phage abortive infection (Abi) in gram-positive bacteria (Emond E. and Shirley E.D. et al., 1998). Genes related to Abi generally exert a cellular process that shuts down the phage lytic cycle or that kills phage-infected cells to prevent the phage particle production (Chopin M.C. and Chopin A. et al., 2005). The toxN gene on pECA1039 is adjacent to a potential coding sequence toxI that includes five copies of a 36-bp sequence tandem repeat, followed by an inverted repeat sequence. The potential gene product ToxI exhibits no similarity to proteins in databases. Since the Abi system was not so common among gram-negative bacteria, the authors focused the study on the toxI-toxN region and experimentally showed that the toxI-toxN region confers phage-resistance to the host.
The authors have two major subject to be addressed: one is the mechanism of phage resistance, and another is whether or not toxI encodes a protein.
The authors showed that toxN encodes a toxic protein that inhibits the host's growth, which means that the mechanism of phage resistance might be a growth inhibition induced by phage infection. The authors also found that the co-expression of toxI with toxN can counteract the toxic activity of ToxN. But, the hypothetical protein ToxI seemed to not be produced from the toxI gene region according to Western blotting analysis, using the modified toxI gene fused with a sequence coding for hexa-histidine tag.
It is possible that toxI RNA itself has an activity to counteract ToxN. To test this hypothesis, the authors introduced a translational stop codon into the toxI coding sequence and found that the ToxN-counteracting activity was still retained in the mutant toxI region. Furthermore, point mutations that do not change the amino-acid sequence of ToxI but do change the transcript sequence resulted in the loss of ToxN-counteracting activity. These results suggest that the toxI RNA is responsible for the antitoxin function. They also pointed out that similar gene modules that are comprised of the tandem repeat region and the toxN homologous gene are present in diverse Eubacteria and Archea.
Detailed mechanisms of toxin (ToxN) induction and the interaction between the toxI RNA and the ToxN protein are still unclear. However, it is obvious that the authors identified a novel type of functional RNA. The authors' work proved that we can still discover new biological concepts from plasmid sequences.
References:
Fineran PC, Blower TR, Foulds IJ, Humphreys DP, Lilley KS, Salmond GP. (2009) The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair. Proc Natl Acad Sci U S A. 106:894-899.
Barnard AM, Bowden SD, Burr T, Coulthurst SJ, Monson RE, Salmond GP. (2007) Quorum sensing, virulence and secondary metabolite production in plant soft-rotting bacteria. Philos Trans R Soc Lond B Biol Sci. 362:1165-1183.
Chopin MC, Chopin A, Bidnenko E. (2005) Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8:473-479.
Emond E, Dion E, Walker SA, Vedamuthu ER, Kondo JK, Moineau S. (1998) AbiQ, an abortive infection mechanism from Lactococcus lactis. Appl Environ Microbiol. 64:4748-4756.
posted by H. Yano (University of Idaho)
Tuesday, March 31, 2009
Wednesday, March 25, 2009
Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase
Scott A. Lujan, Laura M. Guogas, Heather Ragonese, Steven W. Matson, Matthew R. Redinbo,
PNAS 2007 vol. 104 no. 30 12282-12287
Since mid sixties when so-called “R factors” were found to spread antibiotic resistance between different bacterial strains, lots of effort has been put on finding the simplest and universal method to stop horizontal plasmid transfer. Many papers described the effect of various substances on conjugation. Many substrates have a detrimental effect on plasmid transfer just by influencing the bacterial propagation and general life functions. Sometimes the results could be quite surprising and unexpected, but some ordinary everyday products like green tea (epigallocatechin gallate), coffee (caffeine), and papaya seed macerate can inhibit conjugation and ipso facto spreading of an antibiotic resistance. The most common approach to this kind of study is just to add the material under investigation to the conjugation mixture and evaluate plasmid transfer efficiency over a time. Conjugation is a very complicated, multistep process. The functions necessary for conjugation are encoded by plasmids and do not depend on the host. Many studies have been done to establish the function of particular genes. One of the most important proteins involved in plasmid transfer is a DNA relaxase. The conjugative relaxase initiates DNA transfer with a site- and strand-specific ssDNA nick in the transferred strand (T-strand) at the origin of transfer (oriT), forming a covalent 5′-phosphotyrosine intermediate. The nicked T-strand moves from the donor cell to the recipient cell via an intercellular junction mediated by a type IV secretion system. The relaxase completes DNA transfer by reversing the covalent phosphotyrosine linkage and releasing the T-strand. In the F plasmid, this relaxase is located in the N-terminal domain of a large multifunctional protein, TraI (DNA helicase I). The relaxase active site contains one or several tyrosine residues: F-like relaxases (found in IncF, IncN, IncP9 and IncW plasmids) contain 2 to 5 tyrosines while relaxases of IncQ, IncP, IncI plasmids and Ti plasmid of Agrobacterium sp. possess only one. F-like relaxases encoded by the traI gene shares significant sequence identity with relaxases of many R plasmids (e.g., 98% with R100 TraI); thus, the F plasmid serves as a model system for examining conjugative plasmids and the inhibition of conjugative transfer.
In this study the authors first describe the role that the relaxase enzyme plays in the initiation and termination of DNA conjugation and then use that information to identify potent relaxase-specific inhibitors. This is the first paper which described a bottom-up approach to identify the first small molecule inhibitors of conjugative DNA transfer.
The authors determined the 2.4-Å crystal structure of the 300-residue N-terminal relaxase domain of F plasmid TraI and found it similar to other, previously described relaxase domains. They found that the tyrosine at the active site is responsible for binding the oriT thymidine. Based on electron density they found presence of a divalent cation in the active site and identified it as Mg2+. A survey of magnesium-binding proteins in the Protein Data Bank revealed that the chelation of Mg2+ by neutral amino acid residues is diagnostic of a site that simultaneously binds to multiple phosphate groups. Mutation of the metal-chelating residue histidine-159 to glutamic acid eliminated relaxase activity. These data indicated that the 2+ charge on the bound metal ion is critical to relaxase function. The proposed models for relaxase role in binding DNA strands during conjugation suggested that relaxase binds two phosphate groups. To prove this theory authors used a simple and relatively stable bisphosphonate – imidobisphosphate (PNP) molecule and found that at nanomolar concentration PNP inhibited relaxase activity in vitro. Further studies established that relaxase can be effectively inhibited by substrates where two phosphonate residues are separated by three or fewer atoms and have no additional negative charge at pH 7.4. Five additional inhibitors were found: methylenediphosphonic acid (PCP), iminobis(methylphosphonic acid) (PCNCP), etidronic acid (ETIDRO), clodronic acid (CLODRO), and 1,2-bis(dimethoxyphosphoryl)benzene (PBENP). ETIDRO and CLODRO are bisphosphonates clinically approved as drugs used to treat bone loss by inhibiting farnesyl diphosphate synthase Two other inhibitors identified, PCP and PNP, have been used as radioisotope carriers in humans. The simplest inhibitors, PCP, ETIDRO, and CLODRO, were then characterized further by using a kinetic assay and exhibited purely competitive inhibition, with Kic,app values ranging from 3 to 145 nM. Taken together with the PNP results, these data validate the prediction that F-like conjugative relaxases can accommodate two phosphotyrosine intermediates simultaneously within their active sites. Significantly, these data also establish that bisphosphonates (including clinically approved compounds) potently inhibit the in vitro relaxase activity of F TraI with Ki values in the nanomolar range.
The in vivo tests confirmed the results obtained in vitro. In addition to conjugation inhibition, micromolar concentrations of PNP caused death of plasmid-containing, but not plasmid free cells by blocking relaxase activity.
The presented results show that the clinically approved bisphosphonates etidronate (Didronel) and clodronate (Bonefos) are potently effective at killing F+ cells and preventing conjugative DNA transfer. These particular compounds could also be combined with existing antibiotics to create potent antimicrobial cocktails. Etidronate and clodronate exhibit low absorption and can be administered at high oral doses. According to the authors, extrapolating from the results, approved doses of etidronate and clodronate would be expected kill >90% of plasmid + cells and to stop >80% of conjugative transfer within the gastrointestinal tract. Such results are relatively mild, given the large bacterial populations present in the gastrointestinal tract or at wound sites, but may be enough shift the balance toward success in a variety of recalcitrant clinical infections, especially given the prevalence of conjugative plasmids within multidrug-resistant bacterial strains. The treatment of skin infections, primary sites of nosocomial antibiotic resistance transfer, using topical applications of bisphosphonates may also be effective. In summary, this study establishes conjugative relaxases as a unique antimicrobial target. The results suggest that approved therapeutics could have an immediate impact, alone or in combination with existing antibiotics, in the prevention of resistance propagation during clinical treatment of bacterial infections, thus extending the lifetime of our antibiotic arsenal.
In conclusion this paper shows us a very important thing: basic research on the conjugation process in plasmid transfer that showed the crucial role of relaxase protein led to more detailed application studies that give us a potential weapon to fight bacterial conjugation and the spread of antibiotic resistance in the world of microorganisms.
Additional papers.
Zhao, W.-H. , Z.-Q. Hu, Y. Hara and T. Shimamura 2001 Inhibition by epigallocatechin gallate (EGCg) of conjugative R plasmid transfer in Escherichia coli. J.Infect. Chemotherapy 7: 195-197
Tiagunenko IuV, Glatman LI, Antsiferova NG., 1975. Caffeine as an inhibitor of the conjugation transfer of R-factors. A study of certain aspects of the mechanism of action of caffeine on the conjugation transfer of R-factors], Antibiotiki 20: 253-257.
Leite A.A.M., Nardi R.M.D., Nicoli J.R., Chartone-Souza E. and Nascimento A.M.A., 2005. Carica papaya seed macerate as inhibitor of conjugative R plasmid transfer from Salmonella typhimurium to Escherichia coli in vitro and in the digestive tract of gnotobiotic mice. Gen. Appl. Microbiol. 51: 21-26
Fernandez-Lopez R., Machón C., Longshaw C.M., Martin S., Molin S., Zechner E.L., Espinosa M., Lanka E. and de la Cruz F. 2005. Unsaturated fatty acids are inhibitors of bacterial conjugation. Microbiology 151: 3517–3526
dr Jaroslaw E. Krol
UofI
Friday, March 13, 2009
Genome's barcodes
It is important to assign short sequence fragments generated by metagenomic studies to original sources (genomes). Zhou et al. (2008) investigated the oligonucleotide (k-mer) frequencies termed 'barcode' for this purpose.
From the barcodes (Figure 1), 4-mer frequency distrubitions are stable along the chromosomes, and phylogenetically closely related species have more similar barcodes than distantly related species. This is completely agreement with previous studies by Karlin and colleagues, using 2-mer frequeinces (dinucleotide relative abundance, termed 'genomic signature').
Concerning plasmids, the author stated that 'The barcodes of all plasmid genomes also tend to have similar characteristics among themselves, possibly due to being under similar selection pressure caused by their frequent transferring among cell cultures.' It isn't clear what the selection pressure is.
One interesting observation is that different classes of genomes (prokaryotes, eukaryotes, plastids, plasmids, and mitochondria) were separated by two features derived from their barcodes (Figure 4). One feature (x-axis) is the average variation of 4-mer frequencies (across a whole genome across all 4-mers), and the other (y-axis) is the overall similarity (in 4-mer frequencies) among all fragments of the genome. Note that the neighboring genomes in this feature space do not necessarily have similar barcodes. Although the feature space clearly separated these five different classes of genomes, biological implications of the separations were not described.
This has inspired us to investigate factors contributing variations in barcodes (oligonucleotide frequencies) among different genomes. The possible factors include restriction site (Abe et al., 2003), synonymous codon usage, amino acid usage, G+C content (Sandberg et al., 2003), and mosaic structure of the genome.
PRIMARY ARTICLE:
Zhou F, Olman V, Xu Y. BMC Bioinformatics. (2008) 9:546. Barcodes for genomes and applications.
ADDITIONAL REFERENCES:
Karlin S, Campbell AM, Mrázek J. Annu Rev Genet. 1998;32:185-225. Comparative DNA analysis across diverse genomes.
Abe T, Kanaya S, Kinouchi M, Ichiba Y, Kozuki T, Ikemura T. Genome Res. 2003 Apr;13(4):693-702. Informatics for unveiling hidden genome signatures.
Sandberg R, Bränden CI, Ernberg I, Cöster J. Gene. 2003 Jun 5;311:35-42. Quantifying the species-specificity in genomic signatures, synonymous codon choice, amino acid usage and G+C content.
Dr. Haruo Suzuki
University of Idaho
From the barcodes (Figure 1), 4-mer frequency distrubitions are stable along the chromosomes, and phylogenetically closely related species have more similar barcodes than distantly related species. This is completely agreement with previous studies by Karlin and colleagues, using 2-mer frequeinces (dinucleotide relative abundance, termed 'genomic signature').
Concerning plasmids, the author stated that 'The barcodes of all plasmid genomes also tend to have similar characteristics among themselves, possibly due to being under similar selection pressure caused by their frequent transferring among cell cultures.' It isn't clear what the selection pressure is.
One interesting observation is that different classes of genomes (prokaryotes, eukaryotes, plastids, plasmids, and mitochondria) were separated by two features derived from their barcodes (Figure 4). One feature (x-axis) is the average variation of 4-mer frequencies (across a whole genome across all 4-mers), and the other (y-axis) is the overall similarity (in 4-mer frequencies) among all fragments of the genome. Note that the neighboring genomes in this feature space do not necessarily have similar barcodes. Although the feature space clearly separated these five different classes of genomes, biological implications of the separations were not described.
This has inspired us to investigate factors contributing variations in barcodes (oligonucleotide frequencies) among different genomes. The possible factors include restriction site (Abe et al., 2003), synonymous codon usage, amino acid usage, G+C content (Sandberg et al., 2003), and mosaic structure of the genome.
PRIMARY ARTICLE:
Zhou F, Olman V, Xu Y. BMC Bioinformatics. (2008) 9:546. Barcodes for genomes and applications.
ADDITIONAL REFERENCES:
Karlin S, Campbell AM, Mrázek J. Annu Rev Genet. 1998;32:185-225. Comparative DNA analysis across diverse genomes.
Abe T, Kanaya S, Kinouchi M, Ichiba Y, Kozuki T, Ikemura T. Genome Res. 2003 Apr;13(4):693-702. Informatics for unveiling hidden genome signatures.
Sandberg R, Bränden CI, Ernberg I, Cöster J. Gene. 2003 Jun 5;311:35-42. Quantifying the species-specificity in genomic signatures, synonymous codon choice, amino acid usage and G+C content.
Dr. Haruo Suzuki
University of Idaho
Tuesday, March 10, 2009
The Diversity of Nature
With the advent of next-generation sequencing capabilities, many questions that were previously beyond our capabilities to explore are now well within reach. Metagenomic approaches in particular have recently experienced a burst of recognition. One such question that can no be addressed is the extent to which isolated cultures used in laboratory settings represent the true genetic diversity apparent in nature (Tettelin 2005). Pyrosequencing is especially important for such studies in that it is culture-independent. That is, genome sequences can be obtained from a sample even if the species in that sample cannot typically be grown in a laboratory setting. The authors of this paper applied 454 pyrosequencing to marine and coastal samples of cyanobacteria of the genus Synechococcus. This allowed them to identify the levels of diversity present in natural Synechococcus populations and acknowledge the importance of horizontal gene transfer in marine and coastal populations of cyanobacteria.
The authors were able to enrich their samples with Synechococcus by capitalizing on the genus’ natural fluorescence and thereby sorting out the bacteria in the samples using a high-speed flow cytometer. After this enrichment, 454 pyrosequencing was conducted on the two coastal and one open-ocean cyanobacterial populations. These sequences were then compared to the four complete Synechococcus genome sequences available (including representatives from the two most abundant clades-I and IV- as well as clades II and III)( Fuller 2003). Up to 25% of the reads could be mapped to the previously sequenced genomes when the authors used lenient cutoffs (70% identity, 70% sequence length). Of the gene models that had no hits, ca. 48% had atypical trinucleotide content, suggesting that they may have been recently acquired through horizontal gene transfer. Indeed, at least three plasmid families were found in the samples sequenced, again pointing to the potential contribution of horizontal gene transfer to the diversity within marine Synechoccoccus. This is the first reported incidence of plasmid detection in marine, rather than freshwater, cyanobacteria.
In summary, the authors provided this evidence that plasmids play a part in marine cyanobacterial diversity and indicated that, while backbone genes were highly conserved across the metagenome sample, accessory genes were widely varied. Therefore, nature is much more diverse than would be indicated if we only took into account those few cultured representatives that we use in the lab.
Primary article:
B. Palenik, Q. Ren, V. Tai and I. T. Paulsen. (2009) Coastal Synechococcus metagenome reveals major roles for horizontal gene transfer and plasmids in population diversity. Environmental Microbiology 11(2):349-359.
Additional Reading:
Fuller, N.J., Marie, D., Partensky, F., Vaulot, D., Post, A.F., and Scanlan, D.J. (2003) Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea. Appl Environ Microbiol 69: 2430–2443.
Glover, H.E. (1985) The physiology and ecology of the marine cyanobacterial genus Synechococcus. Adv Aquat Microbiol 3: 49–107.
Tettelin, H., Masignan, I.V., Cieslewicz, M.J., Donati, C., Medini, D., Ward, N.L., et al. (2005) Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial ‘pan-genome’. Proc Natl Aca Sci USA 102: 13950–13955.
By Julie Hughes
Graduate Student
University of Idaho
The authors were able to enrich their samples with Synechococcus by capitalizing on the genus’ natural fluorescence and thereby sorting out the bacteria in the samples using a high-speed flow cytometer. After this enrichment, 454 pyrosequencing was conducted on the two coastal and one open-ocean cyanobacterial populations. These sequences were then compared to the four complete Synechococcus genome sequences available (including representatives from the two most abundant clades-I and IV- as well as clades II and III)( Fuller 2003). Up to 25% of the reads could be mapped to the previously sequenced genomes when the authors used lenient cutoffs (70% identity, 70% sequence length). Of the gene models that had no hits, ca. 48% had atypical trinucleotide content, suggesting that they may have been recently acquired through horizontal gene transfer. Indeed, at least three plasmid families were found in the samples sequenced, again pointing to the potential contribution of horizontal gene transfer to the diversity within marine Synechoccoccus. This is the first reported incidence of plasmid detection in marine, rather than freshwater, cyanobacteria.
In summary, the authors provided this evidence that plasmids play a part in marine cyanobacterial diversity and indicated that, while backbone genes were highly conserved across the metagenome sample, accessory genes were widely varied. Therefore, nature is much more diverse than would be indicated if we only took into account those few cultured representatives that we use in the lab.
Primary article:
B. Palenik, Q. Ren, V. Tai and I. T. Paulsen. (2009) Coastal Synechococcus metagenome reveals major roles for horizontal gene transfer and plasmids in population diversity. Environmental Microbiology 11(2):349-359.
Additional Reading:
Fuller, N.J., Marie, D., Partensky, F., Vaulot, D., Post, A.F., and Scanlan, D.J. (2003) Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea. Appl Environ Microbiol 69: 2430–2443.
Glover, H.E. (1985) The physiology and ecology of the marine cyanobacterial genus Synechococcus. Adv Aquat Microbiol 3: 49–107.
Tettelin, H., Masignan, I.V., Cieslewicz, M.J., Donati, C., Medini, D., Ward, N.L., et al. (2005) Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial ‘pan-genome’. Proc Natl Aca Sci USA 102: 13950–13955.
By Julie Hughes
Graduate Student
University of Idaho
Tuesday, March 3, 2009
Prevalence of tetracycline resistance genes in Greek seawater habitats
Theodora L. Nikolakopoulou, Eleni P. Giannoutsou, Adamandia A. Karabatsou, and Amalia D. Karagouni
Tetracyclines are antibiotics that have been used for over 40 years as a therapeutic agent in human and veterinary science and also as growth promoters in animal husbandry. Tetracyclines inhibit bacterial growth by interfering with protein synthesis. Overuse of such antibiotics has lead to the rapid spread of antibiotic resistance genes among bacteria. The most common mechanisms of resistance are tetracycline efflux, ribosome protection and tetracycline modification. Since these resistance genes are often found on mobile genetic elements such as transposons, they can be spread rapidly across bacterial species (Schmidt et al., 2001; Roberts, 2005).
The goal of the research presented in this paper was to analyze the presence of 12 tetracycline resistance (tet A,B,C,D,E,G,H,K,L,M,O,T) genes in seawater sampled from different locations in Greece. The broader goal of this research is to study a complex ecosystem such as the marine environment, which acts like a reservoir of antimicrobial compounds and resistant bacteria (Aoki, 1992; Chee-Sanford et al., 2001).
Water was sampled from 4 different habitats: a) seawater near a wastewater treatment facility b) seawater near a fish farm c) sea water near a tourist spot and d) sea water from an uninhabited location. These water samples were plated onto a nutrient medium and then incubated. Colonies were picked and grown individually. Dilutions were made of these cultures and plated onto Agar containing tetracycline for 3-7 days at 20° C. Distinct colonies having different morphologies from each sample were isolated in pure culture. A total of 89 TcR colonies were picked: 36 from the fish farm, 23 from wastewater, 14 from the tourist place and 16 from the uninhabited location. These 89 colonies were analyzed for the presence of genes conferring TcR by polymerase chain reaction (PCR) using primers specific for the 12 kinds of TcR genes and then southern blotted. This showed that 60 colonies had more than one TcR (tet) gene and the remaining 29 had only tetK which encodes a tetracycline efflux pump. Thus, tetK was clearly the most dominant of the 12 genes. Plasmid extraction from the 60 colonies that had more than one tet gene followed by gel electrophoresis revealed the presence of plasmids (around 40 kb in size) in 37 of the colonies. Ten of these 60 colonies had IncP-type plasmids as revealed by PCR using primers specific to sequences of IncP, IncQ, IncW and IncN plasmids. Thus, it is possible that the TcR genes were carried by these large plasmids. To confirm the presence of plasmids in all seawater samples, exogenous plasmid isolations were performed (Hills et al., 1996; Smalla et al., 2000) using tetracycline as a selective agent. TcR plasmids ranging in size between 40 and 80 kb were isolated from 80 transconjugants. 59 of 80 plasmids possessed one or more tet gene. The tetA gene was dominant as it was found in 36 plasmids as the only tet gene and was found with tetK and tetC on a smaller number of plasmids. PCR showed that 27 of the 59 plasmids belonged to the IncP group of broad host range plasmids.
To summarize, this study showed the presence of TcR bacteria in all seawater 4 samples. However, when the community composition of each sample was analyzed, the samples were found to vary from each other in the content of bacterial species. Thus despite the fact that TcR genes were found in these samples they were probably found on different bacteria. Of the 12 kinds of tet genes, only 4 genes (tet K,A,M,C) were found in these samples while others (such as tet B, D, E, G, H, L, O, and T) were not found at all. This is the first study that reported the presence of tetK in the bacterial strains identified from the seawater samples. Moreover many of the tet genes were found on IncP- type broad host range plasmids. This suggests that the spread of TcR genes in marine environments could be because of their association with IncP-type broad host range plasmids.
References:
Schmidt, A.S., M.S. Bruun, I. Dalsgaard, and J.L. Larsen. 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.
Roberts, M.C. 2005. Update on acquired tetracycline resistance genes. FEMS Microbiol. Lett. 245, 195-203.
Aoki, T. 1992. Present and future problems concerning the development of antibiotic resistance in aquaculture, p. 254-262. In C. Michael and D.J. Alderman (eds.), Chemotherapy in aquaculture: from theory to reality-1992. Office International des Epizooties, Paris, France.
Chee-Sanford, J.C., R.I. Aminov, I.J. Krapac, N. Garrigues-Jeanjean, and R.I. Mackie. 2001. Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities. Appl. Environ. Microbiol. 67, 1494-1502.
Hill, K.E., J.R. Marchesi, and J.C. Fry. 1996. Conjugation and mobilization in the epilithon, p. 5.2.2/1-5.2.2/28. In D.L. Akkermans, J.D. Van Elsas, and F.J. De Bruijn (eds.). Molecular Microbial Ecology Manual. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Smalla, K., H. Heuer, A. Götz, D. Niemeyer, E. Krögerrecklenfort, and E. Tietze. 2000a. 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.
Diya Sen
Graduate student, University of Idaho
Theodora L. Nikolakopoulou, Eleni P. Giannoutsou, Adamandia A. Karabatsou, and Amalia D. Karagouni
Tetracyclines are antibiotics that have been used for over 40 years as a therapeutic agent in human and veterinary science and also as growth promoters in animal husbandry. Tetracyclines inhibit bacterial growth by interfering with protein synthesis. Overuse of such antibiotics has lead to the rapid spread of antibiotic resistance genes among bacteria. The most common mechanisms of resistance are tetracycline efflux, ribosome protection and tetracycline modification. Since these resistance genes are often found on mobile genetic elements such as transposons, they can be spread rapidly across bacterial species (Schmidt et al., 2001; Roberts, 2005).
The goal of the research presented in this paper was to analyze the presence of 12 tetracycline resistance (tet A,B,C,D,E,G,H,K,L,M,O,T) genes in seawater sampled from different locations in Greece. The broader goal of this research is to study a complex ecosystem such as the marine environment, which acts like a reservoir of antimicrobial compounds and resistant bacteria (Aoki, 1992; Chee-Sanford et al., 2001).
Water was sampled from 4 different habitats: a) seawater near a wastewater treatment facility b) seawater near a fish farm c) sea water near a tourist spot and d) sea water from an uninhabited location. These water samples were plated onto a nutrient medium and then incubated. Colonies were picked and grown individually. Dilutions were made of these cultures and plated onto Agar containing tetracycline for 3-7 days at 20° C. Distinct colonies having different morphologies from each sample were isolated in pure culture. A total of 89 TcR colonies were picked: 36 from the fish farm, 23 from wastewater, 14 from the tourist place and 16 from the uninhabited location. These 89 colonies were analyzed for the presence of genes conferring TcR by polymerase chain reaction (PCR) using primers specific for the 12 kinds of TcR genes and then southern blotted. This showed that 60 colonies had more than one TcR (tet) gene and the remaining 29 had only tetK which encodes a tetracycline efflux pump. Thus, tetK was clearly the most dominant of the 12 genes. Plasmid extraction from the 60 colonies that had more than one tet gene followed by gel electrophoresis revealed the presence of plasmids (around 40 kb in size) in 37 of the colonies. Ten of these 60 colonies had IncP-type plasmids as revealed by PCR using primers specific to sequences of IncP, IncQ, IncW and IncN plasmids. Thus, it is possible that the TcR genes were carried by these large plasmids. To confirm the presence of plasmids in all seawater samples, exogenous plasmid isolations were performed (Hills et al., 1996; Smalla et al., 2000) using tetracycline as a selective agent. TcR plasmids ranging in size between 40 and 80 kb were isolated from 80 transconjugants. 59 of 80 plasmids possessed one or more tet gene. The tetA gene was dominant as it was found in 36 plasmids as the only tet gene and was found with tetK and tetC on a smaller number of plasmids. PCR showed that 27 of the 59 plasmids belonged to the IncP group of broad host range plasmids.
To summarize, this study showed the presence of TcR bacteria in all seawater 4 samples. However, when the community composition of each sample was analyzed, the samples were found to vary from each other in the content of bacterial species. Thus despite the fact that TcR genes were found in these samples they were probably found on different bacteria. Of the 12 kinds of tet genes, only 4 genes (tet K,A,M,C) were found in these samples while others (such as tet B, D, E, G, H, L, O, and T) were not found at all. This is the first study that reported the presence of tetK in the bacterial strains identified from the seawater samples. Moreover many of the tet genes were found on IncP- type broad host range plasmids. This suggests that the spread of TcR genes in marine environments could be because of their association with IncP-type broad host range plasmids.
References:
Schmidt, A.S., M.S. Bruun, I. Dalsgaard, and J.L. Larsen. 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.
Roberts, M.C. 2005. Update on acquired tetracycline resistance genes. FEMS Microbiol. Lett. 245, 195-203.
Aoki, T. 1992. Present and future problems concerning the development of antibiotic resistance in aquaculture, p. 254-262. In C. Michael and D.J. Alderman (eds.), Chemotherapy in aquaculture: from theory to reality-1992. Office International des Epizooties, Paris, France.
Chee-Sanford, J.C., R.I. Aminov, I.J. Krapac, N. Garrigues-Jeanjean, and R.I. Mackie. 2001. Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities. Appl. Environ. Microbiol. 67, 1494-1502.
Hill, K.E., J.R. Marchesi, and J.C. Fry. 1996. Conjugation and mobilization in the epilithon, p. 5.2.2/1-5.2.2/28. In D.L. Akkermans, J.D. Van Elsas, and F.J. De Bruijn (eds.). Molecular Microbial Ecology Manual. Kluwer Academic Publishers, Dordrecht, The Netherlands.
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Diya Sen
Graduate student, University of Idaho
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