Sunday, December 28, 2008

Chromosomal Toxin-Antitoxin Systems May Act as Antiaddiction Modules

De Bast M. S., Mine N. and Van Melderen L. (2008) J. Bacteriol. 190:4603-4609

Plasmids are known as carriers of pathogenic determinants and antibiotic resistance genes that help bacteria survive in specific circumstances. However, if the circumstances changed, the bacteria would no longer need such genes, so that the plasmid would become just a burden for bacteria. As a result, plasmid-free bacterial cells would increase their number more rapidly than plasmid-carrying cells. Most of naturally occurring plasmids have special systems to prevent such an event from happening. One of the systems is the Toxin-Antitoxin (TA) system, in which one gene encodes a stable toxin protein and the other gene encodes an unstable antitoxin protein that counteracts the toxin activity. If the plasmid carrying the TA system was lost from a cell, the cell would be immediately killed or damaged by the more stable toxins which persist in the cell; this phenomenon is called postsegregational killing [PSK]. Gene pairs comprising TA systems are called addiction modules. Addiction modules were originally discovered on a plasmid (Hiraga et al., 1986), but recently they have also been discovered on chromosomes (reviewed by Kobayashi I., 2004). Here, we have a question: What is the biological function of chromosomally-located addiction modules?

The first hypothesis proposed is the so-called programmed cell death (PCD) hypothesis: the addiction module induces cell death under stress conditions and the dead cells release nutrients for other cells to remain viable (Aizenman et al., 1996). Recently, this hypothesis has been shown to be unlikely by several research groups (Tsilibaris et al., 2007; Szekeres et al., 2007; Dudde et al., 2007; Pedersen et. al., 2002). The second and more reasonable hypothesis is that addiction modules contribute to stabilize a genome: the toxin reduces the number of bacteria that have lost the chromosomal DNA segment containing the addiction modules, which ensures that the DNA in the region of the addiction module is maintained in the bacterial population (Szekeres et al., 2007).

In this paper, the authors propose a third theory: the "anti-addiction module" hypothesis. In this hypothesis, addiction modules on a chromosome protect bacteria against PSK induced by orthologous addiction modules on a plasmid, which confers selective advantage on a host bacterium under PSK conditions.

To test the anti-addiction module hypothesis, the authors used the CcdB(F)/CcdA(F) TA system of F-plasmid (Hiraga et al., 1986) and its homologous system [CcdB(Ech)/CcdA(Ech) TA system] found in the Escherichia chrysanthemi chromosome; CcdB(F) toxin is 61% identical to CcdB(Ech) while CcdA(F) antitoxin is 65% identical to CcdA(Ech). In this article, the authors first showed that the ccdB(Ech) and ccdA(Ech) genes indeed act as toxin and antitoxin genes in E. coli MG1655 where the ccdB(F)/ccdA(F) homologous genes are absent. However, unlike F-plasmid's ccdB(F)-ccdA(F) gene pair, the ccdB(Ech)-ccdA(Ech) gene pair could not mediate PSK when it was cloned on a plasmid. This suggests that the two homologous TA systems have evolved for different purposes. The authors also showed that CcdA(Ech) can antagonize CcdB(F) toxic activity as efficiently as CcdA(F) can. Furthermore, they showed that the E. coli MG1655 derivative that carries the ccdB(Ech)-ccdA(Ech) gene pair on its chromosome (designated MG1655ccdEch) are more viable than the original MG1655 after the induction of PSK mediated by F-plasmid's ccdB(F)-ccdA(F) gene pair on a plasmid. The following competition assays between MG1655 and MG1655ccdEch in PSK conditions indicated that MG1655ccdEch has a 25% fitness advantage over MG1655. Therefore, all experiments performed in this article support anti-addiction module hypothesis. A related idea was also proposed by Takahashi et al. (2002), using a restriction-modification TA system, in which restriction enzymes act as toxins and modification methyltransferases act as antitoxins. They showed that dcm methyltransferase gene located on the E. coli chromosome protected cells against PSK mediated by a restriction enzyme and DNA modification gene pair on a plasmid.

If the anti-addiction module hypothesis is valid, there would be few cases in nature that counteracting addiction modules are found on both plasmid and chromosome in the same cell, because PSK does not happen in such a situation and consequently there would be no advantage for plasmids to carry the addiction module. However, in the genome of E. coli O157:H7, two homologous ccdB-ccdA gene pairs exist. One ccdB-ccdA gene pair is located on plasmid pO157 and the other is located on the chromosome. The ccd genes of pO157 are identical to F-plasmid's counterparts. Chromosomal ccdB and ccdA gene products, CcdB(O157) and CcdA(O157), are 35% and 30% identical to CcdB(F) and CcdA(F), respectively. As we can expect, the chromosomal ccdA-ccdB gene pair of O157:H7 does not counteract CcdB(F) toxicity and O157:H7 is susceptible to PSK mediated by the CcdB(F)/CcdA(F) TA system (Wibaux et al., 2007).

The integration of addiction modules into the chromosome can protect bacteria from plasmids that may have a high cost under some conditions. However, as the authors state in this article, that in turn drives the evolution of plasmid TA systems so as not to be counteracted by chromosomal TA systems. It thus appears to me that the primal role of chromosomal TA systems is maintaining the integrity of chromosomes in bacterial populations and the secondary role may be protecting bacteria against PSK mediated by invader DNA elements such as phages and plasmids. Do you have another hypothesis? If so, please let me know.

Aizenman E, Engelberg-Kulka H, Glaser G. (1996) An Escherichia coli chromosomal "addiction module" regulated by guanosine 3',5'-bispyrophosphate: a model for programmed bacterial cell death. Proc. Natl. Acad. Sci. 93:6059-6063.

Budde PP, Davis BM, Yuan J, Waldor MK. (2007) Characterization of a higBA toxin-antitoxin locus in Vibrio cholerae. J Bacteriol. 189:491-500

Hiraga S, Jaffé A, Ogura T, Mori H, Takahashi H. (1986) F plasmid ccd mechanism in Escherichia coli. J. Bacteriol. 166:100-104.

Kobayashi I. (2004) Genetic Addition: a principle of Gene symbiosis in a Genome, in Plasmid Biology (Funnell B. E. and Phillips G. J. eds), pp.105-144 ASM press, Washigton D.C.

Pederson K, Christensen SK, and Gerdes K (2002) Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins. Mol. Microbiol. 45:501-510.

Szekeres S, Dauti M, Wilde C, Mazel D, Rowe-Magnus DA. (2007) Chromosomal toxin-antitoxin loci can diminish large-scale genome reductions in the absence of selection. Mol. Microbiol. 63:1588-1605.

Tsilibaris V, Maenhaut-Michel G, Mine N, Van Melderen L (2007) What is the benefit to Escherichia coli of having multiple toxin-antitoxin systems in its genome? J. Bacteriol. 189:6101-6108.

Takahashi N, Naito Y, Handa N, Kobayashi I. (2002) A DNA methyltransferase can protect the genome from postdisturbance attack by a restriction-modification gene complex. J. Bacteriol. 184:6100-6108.

posted by Hirokazu Yano, University of Idaho

Tuesday, December 16, 2008

Who will win this war…….?

High in vitro antimicrobial activity of synthetic antimicrobial
peptidomimetics against staphylococcal biofilms.

Kristina Flemming, Claus Klingenberg, Jorun Pauline Cavanagh, Merethe Sletteng, Wenche Stensen, John Sigurd Svendsen and Trond Flægstad

Journal of Antimicrobial Chemotherapy (2009) 63, 136–145

It is more than 80 years when the first antibiotic was discovered by Sir Alexander Fleming (September 28, 1928). Since the mid 40’s antibiotics have been widely used to prevent bacterial outbreaks in humans, but they also play a role as growth promoting agents in agriculture. The years of positive selection pressure have caused the global spread of antibiotic resistance in the microbial population. Special threats are bacteria that form a biofilm. Biofilm is defined as microbial-derived sessile communities attached to a surface and embedded in a self-produced polymeric matrix. Bacteria in biofilms are usually less susceptible to antimicrobial agents than rapidly growing planktonic cells. There are several hypotheses to explain the strong antimicrobial tolerance of biofilm cells such as the limitation of agent penetration, the existence of dormant cells, phenotypic variations, a quorum sensing system, and multidrug efflux pumps. So there is always need to develop new effective antimicrobial agents that can kill bacteria.

Cationic antimicrobial peptides (CAPs) are widespread in nature and play an important role as part of innate immunity. In general, CAPs are fairly large molecules that carry a net positive charge and contain ~50% hydrophobic residues. Their mode of action involves binding to negatively charged structural molecules on the microbial membrane. Once bound, CAPs form pores that increase the cell membrane permeability and ultimately lead to cell lysis. There is also evidence for other antimicrobial mechanisms such as interaction with intracellular targets or activation of autolytic enzymes in the bacteria, or induction of the immune response in the host. CAPs have a broad spectrum of antimicrobial activity and development of resistance is rare. Unfortunately, CAPs are difficult and expensive to produce in large quantities and are usually sensitive to protease digestion. Modifications of CAPs have resulted in the development of extremely short synthetic antimicrobial peptidomimetics, also called SAMPs. SAMPs mimic the effect of CAPs, but have improved pharmacokinetic properties and are thus a promising new group of antimicrobial substances.

In this study the authors investigated the antimicrobial activity of clinically relevant antibiotics like linezolid, tetracycline, rifampicin and vancomycinand, and newly designed SAMPs against biofilms of three different staphylococcal species (six strains). They also evaluate a simple screening method to quantify the metabolic activity of biofilms before and after the biofilm had been subjected to treatment with antimicrobial agents.

Two methods were used for quantify the biofilm metabolic activity. The first method used Alamar Blue (AB) to measure metabolic activity. AB is a redox indicator that both fluoresces and changes color in response to chemical reduction that can be measured by monitoring absorbances at 570 and 600nm. The AB method showed excellent applicability and it is simple, fast, non-toxic and suitable for high-throughput quantification, at least for biofilms grown in microtitre plates. It shows also great reproducibility and good sensitivity which is very important in antibiotic effect studies. To confirm the killing properties of the antibiotics used, the second method based on LIVE/DEAD biofilm staining was used. This use two stains: Syto9 (green fluorescence) and propionium iodide (PI – red fluorescence). Syto9 stains DNA in living cells while PI reduces green fluorescence only in dead cells. Fluorescence is observed with a confocal laser scanning microscope (CLSM).

Using those two methods authors showed that all SAMPs were clearly more effective in reducing metabolic activity in staphylococcal biofilms at low concentrations compared with antibiotics, even though they generally had higher MICs under planktonic growth conditions. In general, antibiotics were rarely able to cause a complete suppression of metabolic activity. In contrast, SAMPs were frequently able to suppress metabolic activity completely, indicating effective killing. It seems that SAMPs caused damage of the bacterial cell membranes even in slow growing or dormant bacteria embedded in a biofilm. In contrast, the antimicrobial agents used in this study predominantly affected growing bacteria by inhibiting their cell wall development (vancomycin) or by inhibition of their protein synthesis (linezolid, rifampicin and tetracycline).

In conclusion the SAMPs are potential new therapeutic agents in biofilm-associated infections. They could be especially attractive for topical treatment of chronic wound infections. The possible clinical applicability of SAMPs to prevent medical device-associated staphylococcal infections warrants future in vivo studies.

So maybe we can win this war…….

dr Jaroslaw E. Krol

UoI, Moscow

Monday, December 8, 2008

Functions of horizontally transferred genes

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 (, 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).

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 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

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

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.


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

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.

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 β-lactam antibiotics, such as ampicilin. Apart from multidrug resistance, pOLA52 plasmid carries 5.6 kb operon consisting of five genes, homologous to mrkABCDF genes contained in the mrk operon of Klebsiella pneumoniae, encoding type 3 fimbriae, which are known to be involved in attachment of bacteria to different kinds of biotic and abiotic surfaces, and thus increased biofilm formation.
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 …….


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

Monday, September 22, 2008

Contribution of horizontal gene transfer to microbial evolution

“Modular networks and cumulative impact of lateral transfer in prokaryote genome evolution” by Dagan et al. (2008)

A horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the process by which genetic material is transferred between distinct evolutionary lineages, through (plasmid-mediated) conjugation, (virus-mediated) transduction, and transformation (extracellular DNA uptake). Although HGT may occur at the cellular level frequently, transferred genes cannot be always inherited to the subsequent generations.

Generally, a gene is thought to be acquired by HGT if gene tree conflicts or unusual nucleotide composition is observed. The major caveat of these approaches is that the observations can also be explained by other reasons, such as inaccurate phylogenetic reconstruction methods, gene loss in multiple lineages, novel sequences arising from the divergence of gene duplications, and varying mutation rates for different proteins (Kechris et al., 2006).

HGT is an important source of genetic diversity among microorganisms, but the degree of its contribution on microbial genome evolution is still debated. Dagan et al. (2008) conducted a network analysis of shared gene content across prokaryotic genomes to estimate the contribution of HGT to microbial evolution. Their result suggests that on average, 81 ± 15% of the genes in each genome were involved in HGT at some point in their history. Once acquired, genes can be vertically inherited within a group, and their result suggests that this has occurred for the vast majority of genes.

The Dagan's work have inspired us to estimate relative contributions of different mechanisms (conjugation, transduction, and transformation) on horizontal gene transfer among prokaryotes.

Dagan T, Artzy-Randrup Y, Martin W. Proc Natl Acad Sci U S A. 2008 Jul 22;105(29):10039-44. Modular networks and cumulative impact of lateral transfer in prokaryote genome evolution.

Choi IG, Kim SH. Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4489-94. Global extent of horizontal gene transfer.

Dagan T, Martin W. Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):870-5. Ancestral genome sizes specify the minimum rate of lateral gene transfer during prokaryote evolution.

Kechris KJ, Lin JC, Bickel PJ, Glazer AN. Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9584-9. Quantitative exploration of the occurrence of lateral gene transfer by using nitrogen fixation genes as a case study.

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.

Kunin V, Goldovsky L, Darzentas N, Ouzounis CA. Genome Res. 2005 Jul;15(7):954-9. The net of life: reconstructing the microbial phylogenetic network.

Dr. Haruo Suzuki
University of Idaho

Tuesday, September 16, 2008

Impact of conjugal transfer on the stability of IncP-1 plasmid pKJK5

Bahl MI, Hansen HL & Sørensen SJ (2007. FEMS Microbiol Lett 266:250-6.

“With great power comes great responsibility”. As strange as it may seem, Churchill’s famous quote can be applicable to the bacterial as well as the human world. Many bacteria contain plasmids that confer to them the “power” to do many impressive things: resist antibiotics or heavy metals, break down toxins in the environment, become virulent, and some even give bacteria the power to conjugate (mate) with other bacteria. All this power comes at a cost, however. Having a plasmid that gifts a bacterium with novel traits also means that the bacterium has to invest in maintaining the plasmid. A variable amount of a bacterium’s resources will have to be diverted to keeping the plasmid and its various functions up and running. Thus, plasmids often affect bacterial growth rates, causing the plasmid bearing bacteria to grow and reproduce more slowly than their plasmid free neighbors. Therefore, it is commonly thought that a plasmid is only truly beneficial if there’s an immediate selective advantage to having it (for instance, having a plasmid that keeps you alive in the presence of antibiotics is great when being actively doused in antibiotics, but the costs of the plasmid might not be worth it when the flow of antibiotics stops). In times where such selective pressures are removed the percentage of plasmid harboring cells will often decrease due to factors such as the slower growth rate and the occasional loss of plasmids in one of the daughter cells during segregation (Bergstrom et. al., 2000).

All this said, the authors of a recent paper suggest that plasmid loss in the absence of selective pressures may not be such a sure thing after all. They propose that when bacteria have a plasmid and frequent access to each other (as is the case in bacterial biofilms or microcolonies) conjugation can more than compensate for plasmid loss in a population. By constructing fluorescing bacteria the authors of this paper were able to see and quantify plasmid stability in bacterial populations that could and could not conjugate. They therefore were able to study the impact of conjugal transfer on the stability of an IncP-1 plasmid in bacterial populations, as the name of their article implies.

The authors carried out this study using Escherichia coli MC4100 and Kluyveria sp. MB101. A gene cassette coding for kanamycin (Km) and streptomycin (Sm) resistance, as well as a green fluorescing protein (GFP) was inserted into the chromosome of each of the above-mentioned bacteria. In liquid broth the constitutively expressed gfp can be detected by flow cytometry using an argon ion laser, whereas an epi-flouresence microscope was used for visual detection of fluorescing colonies on solid media. The production of GFP is regulated by a lac operon, and so is repressed in the presence of a functional lacI gene (which is not present in either of the constructed strains of bacteria). In order to test the importance of conjugation on plasmid stability, the authors inserted an entranceposon containing a lacIq1 gene into plasmid pKJK5. The entire genome of pKJK5 had been previously sequenced, and so the authors were able to use PCR to screen for neutral insertions (e.g. pMIB4) and insertions that disrupted conjugation (e.g. pMIB8) (Haase et al., 1997). The authors introduced these lacI-containing plasmids into E. coli MC4100 and Kluyveria sp. MB101. Therefore, any bacterium containing pKJK5 or one of its derivatives would not fluoresce, due to the lacI suppression of GFP production. Thus, this method allowed the authors to quantify the percentage of plasmid harboring and plasmid free cells.

Using this system, the authors were also able to compare the stability of plasmid pKJK5 in the presence and absence of conjugation. In a culture initially containing 100% pMIB4, three days (and many generations) later more than 99.99% of the cells still contained the lacI plasmid even without selection for it. On the other hand, in bacteria that couldn’t conjugate (those containing pMIB8) only around 99.43% or 99.13% of the E. coli and Kluyvera sp., respectively, still were plasmid harboring in bacterial mats. As with similar experiments involving stability of conjugation-deficient bacteria conducted by Sia et al (1995), this suggests that conjugation plays a significant role in sustaining an IncP-1 plasmid in bacterial mats. But that’s not all. Not only can conjugation promote plasmid persistence in a population, but according to the authors it can also account for the infectious spread of plasmids throughout a mat population within three days, even when starting from only an initial 25% of the population containing the plasmid. Again, this is only true if conjugation is possible. With the pMIB8 plasmid the total plasmid-containing population actually decreased, likely due to segregational loss.

Whereas conjugation may compensate for segregational loss in high-density bacterial mats, the same cannot be said of lower density, well mixed liquid broth cultures. It appears that the percentage of plasmid containing cells decreased in populations harboring either pMIB4 or pMIB8, although the decline was less dramatic in those populations that could conjugate. So what does it matter if plasmid stability in bacterial mats differs from that in liquid media is different? Well, for one, other than the thermos of chicken soup that’s been rolling around in the back of your car for a week, bacterial populations in nature may not be accurately modeled by the perfectly mixed broth cultures common to most labs. This means that in general, we may be underestimating the role of conjugation in plasmid stability due to unrepresentative experimental systems.

There is a vast range of applications of studies in horizontal gene transfer in general. In some cases we may want to limit plasmid stability in populations such as in the fight against antibiotic resistant strains. In other cases, as with bioremediation, we may want to encourage plasmid stability so that plasmids that we introduce into bacteria allow the bacteria to do our clean-up work for us. In either case, we need a solid understanding of how, when, and under what conditions plasmids are more or less stable. That’s not to say that conjugation is the only important factor in stability. As mentioned above, and in the author’s paper, segregational loss, relative growth rates, and transfer frequency all contribute to overall plasmid stability. This article doesn’t discount the importance of these other factors, but rather emphasizes the need to respect conjugation as a major player that can, given the right conditions, act parasitically in its spread through a population, even when it doesn’t benefit the host bacterium. So maybe the bacteria aren’t as “responsible” for the process as we originally thought. Maybe the plasmids themselves are the ones with the real power after all.

This study also opens up a question for the philosophers of science out there (although it’s a question much too broad for one blog, so an answer won’t be attempted here). That question is one raised by Richard Dawkins, and pertains to the idea of the selfish gene. If plasmids behave parasitically, does that support the selfish gene idea? Could the results of this article be applied to an argument that the population isn’t always the level that we should think about when considering evolution, if it’s the plasmids and not their host bacteria that run the show? Maybe, maybe not, but it’s a fun debate either way, and something to think about.


Bahl MI, Sørensen SJ &Hansen HL (2004) Impact of conjugal transfer on the stability of IncP-1plasmid pKJK5 in bacterial populations. FEMS Microbiol Lett 232:45-49.

Bergstrom CT, Lipsitch M & Levin BR (2000) Natural selection, infectious transfer and the existence conditions for bacterial plasmids. Genetics 155: 1505–1519.

Haase J & Lanka E (1997) A specific protease encoded by the conjugative DNA transfer systems of IncP and Ti plasmids is essential for pilus synthesis. J Bacteriol 179.

Sia EA, Roberts RC, Easter C, Helinski DR & Figurski DH (1995) Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2. J Bacteriol 177: 2789–2797. 5728–5735.

Julie Hughes, Ph.D. student
Department of Biological Sciences, University of Idaho

Thursday, September 4, 2008

Transfer of antimicrobial resistance plasmids from Klebsiella pneumoniae to Escherichia coli in the mouse intestine

Schjørring, S., Struve, C., & Krogfelt, K.A. (2008). (e-publ. Aug. 13, 2008) Journal of Antimicrobial Chemotherapy doi: 10.1093/jac/dkn323.

Nosocomial infections, commonplace in health care systems including intensive care units (Çaatay et al., 2007), are becoming more of a pressing issue as bacteria continue to exhibit multiple antimicrobial resistances. Such cases have been reported in hospitals as well as extended care facilities such as nursing homes (Wiener et al., 1999). While the development of antibiotics are important in our fight against pathogens, it is equally important to focus on the mechanisms involved in developing increased resistance. In this paper by Schjørring, et al., one of the most common nosocomial pathogens was studied: Klebsiella pneumoniae, gram-negative bacteria shown to exhibit multiple antimicrobial resistances. The authors studied the effects of introducing antimicrobial genes and monitoring the colonization of K. pneumonia in mice intestines. The findings reveal several pieces of information about K. pneumonia, including the nature of the pathogen as well as its’ ability to transfer resistance genes to other bacteria (Schjørring, et al., 2008).

The authors of this article, created an intestinal colonizational model in order to observe this transfer more readily, so the plasmid transfer procedure was observed both in vitro and in vivo (Schjørring, et al., 2008). K. pneumoniae strain MGH75875 was used to follow the transfer process including colonization, and horizontal gene transfer (Schjørring, et al., 2008). This strain was originally isolated from an intensive care unit (ICU) patient with pneumonia. K. pneumoniae MGH75875 is currently known to be resistant to ampicillin, streptomycin, tetracycline, nalidixic acid, ticarcillin, trimethoprim/ sulfamethoxazole, cefotaxime and gentamicin, and is susceptible to imipenem (Schjørring, et al., 2008).

The plasmids monitored in this experiment presented interesting results on the basis of in vitro and in vivo examination. Several of the plasmids monitored (only named by their relative size) showed that environmental conditions do influence the nature of transfer. For example, the in vitro experiments showed transfer of the 108 or 157 kb plasmid, while in vivo only showed transfer of the 89 kb plasmid (Schjørring, et al., 2008).

The mice used in this study were individually caged and had unlimited access to resources, including food and water; antibiotics were administered through the water, at dosages described in the protocol. To begin, mice were first inoculated with the strain K. pneumoniae. This was done by growing up overnight cultures and resuspending the cultures in a 20% sucrose solution. Each mouse was given 100μL of this solution orally and subsequently their fecal matter was measured for bacteria; up to 109 cfu/g feces was found (Schjørring, et al., 2008). To determine the effects of antimicrobial treatment on the intestinal flora, three mice were treated with only K. pneumoniae and later treated with ampicillin added to their drinking water to represent treatment of infection. To determine the colonization of the intestine, two mice per experiment were treated with 0.5g/L ampicillin prior to exposure to the strain. Finally, to monitor gene transfer in the intestine three mice per experiment were treated with 0.5 g/L streptomycin sulphate in their drinking water prior to inoculation with the recipient strain as well as during the experiment (Schjørring, et al., 2008). A verification of transconjugants was done, via biochemical marker assays and by DNA isolation to provide a plasmid profile to detect the E. coli transconjugants. Also, a PCR was used to detect the presence of the plasmid-encoded extended spectrum β-lactamases (ESBL) genes, or the genes that code for antimicrobial resistance. In mice without any antimicrobial pretreatment, the inoculated strain quickly dropped below the detection limit due to the competitive nature of the other strains of bacteria present in the intestine. With the introduction of an antimicrobial treatment, there was an immediate increase in the population of the MGH75875 strain up to 109 cfu/g feces (Schjørring, et al., 2008). This experiment thus shows a direct relationship between selection factors and the immediate colonization of the gastrointestinal tract (GI) by the resistant pathogen (Schjørring, et al., 2008). There was also an observable higher transfer frequency of different plasmids into E. coli from K. pneumoniae during colonization of the mouse intestine. K. pneumoniae is thus an excellent colonizer in the GI tract of antibiotic-pretreated mice, and highly promiscuous with respect to numerous plasmids. The observed increase in the number of resistant bacteria, which can inherently lead to an increased risk of spreading resistance genes (Schjørring, et al., 2008).

After reading this paper I became very interested in the concept of evolution in our everyday lives. Most of us imagine evolution as a long and gradual process; however, in microbiology a normal 24-hour period can consist of several generations of bacteria. In this way, evolution can be easily observed and measured especially in the presence of selection factors. As a future physician, I recognize the importance of studying the relative effects of antibiotic use, including those associated with overuse, underuse and more recently the effects associated with resistant strains of bacteria in medicine. While studying antibiotic use is important, equally important is gaining a better understanding of what mechanisms are associated with resistance. Through this research and others, we all may come to appreciate what role evolution plays in our everyday lives, including our health care.

Related Articles of Interest:

Çaatay, A.A., Özcan PE, Gulec L, et al. Risk Factors for Mortality of Nosocomial Bacteraemia in Intensive Care Units. Med Princ Pract 2007;16:187-192.

Wiener, J., Quinn, J.P., Bradford, P.A., Goering, R.V., Nathan, C., Bush, K., Weinstein, R.A. JAMA 1999; 281: 517-523.

Nick Hardin, Undergraduate Researcher
University of Idaho

Monday, August 25, 2008

Heterogeneous selection in a spatially structured environment affects fitness tradeoffs of plasmid carriage in Pseudomonads

Slater, Frances R.; Bruce, Kenneth D.; Ellis, Richard J.; Lilley, Andrew K.; Turner, Sarah L.
Applied & Environmental Microbiology-74 (10). 3189-3197. doi:10.1128/AEM.02383-07

Bacteria live in natural environments that have a spatial structure where their movement is restricted by the surrounding soil or water. Such a spatial structure may create physicochemical gradients leading to heterogeneous patches. This heterogeneity is very important to the biodiversity of microorganisms as studied by Rainey and Travisano (1998). The present study by Slater et al aims to compare the effects of uniform and heterogeneous mercuric chloride HgCl(2) on a model community of plasmid-carrying and plasmid-free pseudomonads. The authors describe a novel experimental system for quantification of the different spatially variable selection pressures that are present in natural environments.

Plasmids may carry a variety of genes for antibiotic and heavy metal resistance that benefit their hosts. These genes are beneficial only in the presence of selection for those particular substances. In their absence the plasmid-carrying bacteria has a reduced growth rate due to the metabolic cost of maintaining the plasmid. Thus a tradeoff exists between benefit and burden resulting in persistence or loss of the plasmid as described by Lilley and Bailey (1997).

To study this phenomenon of plasmid persistence in heterogeneous environments, the authors investigated the effects of spatially heterogeneous Hg pollution on selection for P. fluorescens SBW25 carrying the Hg(r) plasmid pQBR103 relative to the plasmid-free strain.

1) The authors labeled the plasmid-free SBW25 chromosome with an RFP cassette and the plasmid pQBR103 with a GFP cassette.
2) They calculated maximum specific growth rates in liquid cultures of SBW25::rif and SBW25 (pQBR103::gfp).
3) Liquid monocultures of SBW25::rif and SBW25 (pQBR103::gfp) were prepared. Cells were harvested and resuspended in PBS (O.D=0.5). Mixtures having 1:1 ratio of Hg(r)-to-Hg(s) were diluted in PBS (to approximately 2 x 104 CFU /ml) and used to inoculate membranes.
4) Black Isopore polycarbonate membrane filters were pre-washed with sterile distilled water (SDW). The filters were incubated with inoculum on 0.7 R2A for 3 days at 21-23°C.
5) For heterogeneous treatment, the authors soaked 5 gm of carboxymethyl cellulose fibers with either 30 ml SDW or 1.15 or 7.66 mM HgCl(2) to have a final concentration of 0, 1,875 or 12,500 μg HgCl(2) g / cellulose. The cellulose mixtures were dried and crushed to separate the fibers. For random distribution of Hg foci, the cellulose fibers were sprayed down a sealed pressurized container onto the pre-grown bacterial culture on the membrane filter.
6) For uniform treatment, the authors placed the membranes on R2A supplemented with HgCl(2) to give a final concentration of 0, 2.5, 5 or 7.5 μM.
7) Visualization of the colonies was done by using an Eclipse E600 microscope. Images of at least 10 fields of view were captured.
8) Calculation of a(Hg)(r) gives the area of each field of view (FOV) occupied by Hg(r) relative to Hg(s) ( where Hg(r) is strain resistant to Hg and Hg(s) is strain sensitive to Hg) . W(Hg)(r) which is the relative fitness of Hg(r) populations was also calculated.

The results showed that when starting with 1:1 ratio of SBW25::rif and SBW25 (pQBR103::gfp), in the absence of Hg, a(Hg)(r) was 0.33 and W(Hg)(r) was 0.78 where mean area of FOVS occupied by Hg(s) was 74.42%. In the presence of uniform and heterogeneous Hg, a(Hg)(r) was found to increase with Hg concentration. However the value of W(Hg)(r) which is calculated over the entire population, increased only for the uniform Hg treatment and not for the heterogeneous treatment. They explain this by localized selection for Hg(r) in the heterogeneous condition, which did not increase the value of W(Hg)(r) considerably. To test this, they repeated the experiment with either a heterogeneous treatment of 9,375 μg HgCl(2) g/cellulose or a no-Hg control treatment and a variety of starting ratios of Hg(r) to Hg(s) bacteria. Changes in a(Hg)(r) between no-Hg control and heterogeneous treatments were proportional to the starting inoculum of Hg(r) bacteria. Starting with a smaller inoculum of Hg(r) resulted in a greater increase in selection. This lead them to conclude that negative frequency-dependent selection for the plasmid carrying bacteria was taking place in heterogeneously distributed spatial environments. Thus when the number of Hg(r) cells is low there is a higher selection for Hg(r) bacteria than when the number of Hg(r) cells is high.
Their conclusion supports previous work by Ellis, R. J., et al (2007) that demonstrated negative frequency-dependent selection in structured and unstructured environments under uniform Hg conditions. This study goes a step ahead and proves that negative frequency-dependent selection is taking place when Hg is distributed heterogeneously in a spatial environment. In order to determine the time periods over which coexistence of plasmid-bearing and plasmid-free cells takes place and other variables, the authors indicate that further work is to be carried out.
This paper is important because the authors carry out experimental work to demonstrate plasmid persistence in heterogeneous environments, which would be more typical of the “real-world” growth conditions for bacteria, rather than the usually more homogeneous laboratory conditions. A novel method was developed for creating a spatially heterogeneous environment using cellulose fibers imbued with HgCl(2) sprayed onto preinoculated membranes.

Primary Article
Heterogeneous selection in a spatially structured environment affects fitness tradeoffs of plasmid carriage in Pseudomonads

Additional references:

Rainey, P.B., and M. Travisano. 1998. Adaptive radiation in a heterogeneous environment. Nature 394:69-72.

Ellis, R. J., A. K. Lilley, S.J. Lacey, D. Murrell, and H.C.J. Godfray.2007.
Frequency-dependent advantages of plasmid carriage by Pseudomonas sp in homogeneous and spatially structured environments. ISME J. 1:92-95

Lilley, A.K., and M.J. Bailey.1997. Impact of plasmid pQBR103 acquisition and carriage on the phytosphere fitness of Pseudomonas fluorescens SBW25: burden and benefit. Appl. Environ. Microbiol.63:1584-1587.

Diya Sen, Graduate Student
University of Idaho

Friday, August 8, 2008

Acclimation of Subsurface Microbial Communities to Mercury
De Lipthay, J.R., Rasmussen, L.D., Oregaard, G., Simonsen, K., Bahl, M.I., Kroer, N., & Sørensen, S.J. (2008). FEMS Microbiology Ecology, 65, 145-155.

As contamination of our oceans, freshwater sources, and soils continues to be an increasing concern, the need for research addressing the potential of remediating contaminated environments also becomes increasingly evident. Mercury (Hg) contamination of soil and water is a particular concern, since mercury is toxic even at very low concentrations. In this article by Lipthay, et al., the authors studied the adaptation of microbial communities to mercury in both mercury-contaminated and non-contaminated subsurface soils. The findings in this study point to the importance of the role of plasmids in the acclimation to mercury of the soil bacteria communities and, on the other hand, their importance in bioremediation efforts.
The authors’ analysis of contaminated and non-contaminated soils included diverse microbiological techniques:
(1) the appearance of colonies on R2A agar plates with or without Hg(II) amendment (to test the abundance of culturable bacteria),
(2) substrate utilization patterns in Biolog ECOplates with or without Hg(II) amendment (to measure the ability of soil bacteria to utilize single carbon sources),
(3) substrate utilization patterns in Biolog mt-2 plates amended with increasing concentrations of Hg(II) (to estimate the minimal inhibitory concentration of Hg(II) in the soil communities),
(4) merA PCR (to test for the presence of merA genes in whole community soil DNA extracts),
(5) IncP trfA1 PCR (to test for the presence of trfA1 genes of the IncP-1 incompatibility group of plasmids in whole community soil DNA extracts), and
(6) 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) analysis (to analyze the genetic diversity of the various bacterial soil communities).
As expected, the abundance of culturable, mercury-resistant bacteria was higher in the mercury-contaminated soils than in the non-contaminated soils and the soil communities from the contaminated site were better adapted to mercury. Results also suggested a higher diversity of mercury-resistant bacteria in the surface soil communities than in the subsurface soil communities. Treatment of the soils with mercury though, resulted in an equally diverse mercury-resistant population in both subsurface and surface soil communities. This increase in diversity following mercury “stimulation” is similar to results found in another study (Muller et al., 2001a), and the authors point out that it is probably the result of either 1) growth of previously low abundance resistant bacteria or 2) transfer of the mer operon by horizontal gene exchange, leading to elevated mercury resistance in the bacterial soil community.
The mercury tolerance of the soil bacteria communities decreased with depth in both the contaminated and non-contaminated soils, with tolerance levels being greater overall in microbial communities from the contaminated soils. Mercury tolerance increased significantly after mercury amendment, indicating a shift in community composition. This increase in tolerance is also most likely due to horizontal gene transfer of the mer operon. Before mercury was added to soils, PCR targeting merA genes resulted only in PCR products from the contaminated soils (cultivation-based methods were used to show that mercury-resistant bacteria were present in non-contaminated soils). After mercury-amendment merA genes were found in all soils, thus demonstrating the adaptive potential of the soil bacteria communities.
IncP-1 trfA1 genes were detected only in DNA extracted from the contaminated soils, not from the non-contaminated soils, and were also more abundant in the surface soil than in subsurface soils.
Another interesting finding of this study to note here is that the microbial diversity did not generally decrease with soil depth, thus contradicting one of the authors’ original hypotheses.
Through biparental exogenous plasmid isolation, the authors found four different mercury-resistance plasmids (pCPH-001, -002, -003, and -004). Further analysis of these plasmids confirmed that all four had identical partial trfA sequences (and belonged to the IncP-1B group), yet had different merA genes. All four plasmid types were isolated from the contaminated surface soil; however, only plasmid pCPH-001 was isolated from the two subsurface soils. This finding of a higher diversity of mercury-resistance plasmids in the surface soil compared with the subsurface soils corresponds well with the findings of a higher diversity of mercury-resistant bacteria in the contaminated surface soil. In contrast, no mercury-resistance plasmids were retrieved from the non-contaminated soils. The observations in this study, that IncP-1 plasmids are present in contaminated but not in non-contaminated soils, are similar to those in other studies (Smalla et al., 2000; Campbell et al., 1995) and are evidence of the importance of IncP-1 plasmids in the dissemination of adaptive functional traits in microbial communities.
The authors suggest that the findings of this study are important to bear in mind when considering bioremediation of mercury-polluted environments.

Additional References:
Campbell, J.I.A., Jacobsen, C.S., & Sørensen, J. (1995). Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils. FEMS Microbiology Ecology, 18, 51–62.

Muller, A.K., Rasmussen, L.D., & Sørensen, S.J. (2001a). Adaptation of the bacterial community to mercury contamination. FEMS Microbiology Letters, 204, 49–53.

Smalla, K., Krogerrecklenfort, E., Heuer, H., et al. (2000) PCR-based detection of mobile genetic elements in total community DNA. Microbiology, 146, 1256–1257.

Matt Bauer, B.S.
University of Idaho

Friday, July 25, 2008

The Role of Plasmid-encoded H-NS-like Protein

"An H-NS-like stealth protein aids horizontal DNA transmission in bacteria" Doyle M et al., Science 315: 251-252 (2007)

H-NS is one of the abundant DNA-binding proteins that are found in gram-negative bacterial cells. H-NS is known to bind to A+T-rich sequences and regulate expression of a large number of chromosomal genes (Fang FC and Rimsky S, 2008) . Recently, it has become clear that some narrow-host-range plasmids derived from gram-negative bacteria encode H-NS-like proteins. Yet, their roles were still obscure.

So far, it has been shown that Sfh, an H-NS paralog encoded by IncHI1 group plasmid pSf-R27, interacts with host H-NS, and both proteins are functionally exchangeable (Deighan P et al., 2003; Beloin C et al., 2003). People thus might think that plasmid-encoded H-NS-like proteins influence global gene expression of host bacteria. Interestingly, the results shown in this article look to be in the contrary: the absence of Shf disturbed global gene expression of transconjugants. In this article, authors proposed that plasmid-encoded H-NS is a stealth protein that allows host bacteria to carry A+T-rich plasmids with minimal effect on global gene expression and "fitness", by preventing plasmids from titrating cellular pool of H-NS.

Authors introduced pSf-R27, with or without the sfh gene, from original host Shigella flexeneri into Salmonella Typhimurium, and analyzed the transcriptome as well as several phenotypes of transconjugants. Interestingly, the transfer of wild-type pSf-R27 resulted in a few change in the recipient, but transfer of pSf-27Δsfh resulted in the drastic changes in expression of a wide range of genes. Noteworthy phenotypes of the recipient carrying sfh mutant were increased resistance to UV, increased virulence (persistence in macrophage) and reduced motility. These phenotypes are reminiscent of the chromosomal hns mutant (Navarre WW et al., 2006). Authors then showed that the sfh mutation significantly reduced the fitness of recipient (this phenotype was completely complemented by supplying Sfh in trans from another plasmid). To figure out if the reduction of fitness resulted from the titration of "host" H-NS by A+T-rich sequence on the plasmid, authors constructed a pUC18 derivative that carried chromosome-derived A+T-rich DNA fragment and introduced it into the recipient cells, instead of pSf-R27Δsfh. The recipient that carries the pUC18 derivative caused reduction in the fitness, and this reduction was complemented in the presence of Sfh, as in the case of pSf-R27.

Based on these observations authors proposed that sfh is a "stealth" gene that allows the A+T-rich pSf-R27 to invade a new bacterial host with a minimal impact on global gene expression patterns and fitness. They added that the positive effects of sfh on the fitness can be applied to biotechnology to construct more stable cloning vectors.

Doyle M, Fookes M, Ivens A, Mangan MW, Wain J, Dorman CJ.
An H-NS-like stealth protein aids horizontal DNA transmission in bacteria.
Science 2007, 315:251-2.

Fang FC, Rimsky S.
New insights into transcriptional regulation by H-NS.
Curr. Opin. Microbiol. 2008, 11:113-20.

Deighan P, Beloin C, Dorman CJ.
Three-way interactions among the Sfh, StpA and H-NS nucleoid-structuring proteins of Shigella flexneri 2a strain 2457T.
Mol. Microbiol. 2003, 48:1401-16.

Beloin C, Deighan P, Doyle M, Dorman CJ.
Shigella flexneri 2a strain 2457T expresses three members of the H-NS-like protein family: characterization of the Sfh protein.
Mol. Genet. Genomics 2003, 270:66-77.

Navarre WW, Porwollik S, Wang Y, McClelland M, Rosen H, Libby SJ, Fang FC. Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella.
Science. 2006 Jul 14;313(5784):236-8.

Hirokazu Yano (Ph. D.)
University of Idaho

Saturday, July 19, 2008

Direct Visualization of Plasmid Transfer

Direct evidence of extant in situ plasmid transfer in natural environments has typically been obtained by identifying plasmid-encoded phenotypes following the introduction of donor strains. Many plasmids do not encode any known functions (cryptic plasmids); in many unknown plasmids, functions are not determined or there is no easy method to select plasmid-containing cells. So there is the necessity to use known reporter markers, like antibiotic resistance, lacZ, gusA, luxAB or fluorescent proteins genes to label plasmids prior to study. The main advantage of using luciferase and especially fluorescent proteins is that it is possible to detect the presence of plasmid DNA without plating (non-culturable bacteria) and adding additional substrates to the environment. Using of fluorescent proteins enables direct visualization of plasmid transfer but has also some limitations. First is using epifluorescence microscopy or flow-cytometry-based method to detect fluorescent cells, both of which are technically demanding and require expensive equipment. The second limitation arises from the properties of fluorescent proteins. They need some time - from a few to several hours, to produce the strong, detectable signal. So this make impossible to detect precisely the time of plasmid DNA entering the recipient cell.

In the paper “Direct visualization of horizontal gene transfer” by Ana Babic et al. (2008) authors developed an experimental system that enables them to distinguish the transferred donor DNA from both donor and recipient DNA, and to visualize DNA transfer and recombination by means of fluorescence microscopy in real time, at the level of individual living cells. This tool also allowed them to quantify the ongoing transfer of DNA during conjugation and to acquire time-lapse movies that follow the fate of the newly acquired DNA in individual cells through any number of cell divisions.

This method uses fusion of YFP gene with seqA gene. This translational fusion driven from the native seqA promoter was introduced into E.coli chromosome replacing the wild type seqA allel. SeqA protein has strong affinity for DNA which is hemimethylated by Dam methylase at GATC sequences. Such a hemimethylated DNA usually occurs in the replication forks during replication and SeqA-YFP fusion protein had been found bound to chromosomal DNA previously. When the host strain lacks Dam methylase, the chromosomal DNA is not methylated and SeqA-YFP protein is dispersed in the cytoplasm giving dim background fluorescence.
During conjugation single stranded DNA is transferred from a donor strain to the recipient cells and the second DNA strand is synthesized. When plasmid DNA is methylated by Dam methylase and transferred to a Dam deficient strain, stable hemimethylated duplex is formed. Such a duplex is recognized by SeqA-YFP fusion protein and gives strong fluorescence foci.
Using this technique authors were able to detect presence of transferred plasmid DNA in transconjugants as quickly as 5 minutes after mixing together parental strains. After 30-40 minutes almost all recipient cells in the vicinity of donors showed fluorescent foci. In comparison, the RFP protein expressed from the transferred plasmid from tetracycline promoter showed visible signal 2 hours after transfer. They also showed that in the case of F plasmid direct cell wall contact is not necessary for transfer; this means that single stranded plasmid DNA is transferred from cell to cell thru the sex pili.
To summarize this method allows them to visualize and quantify the DNA of any sequence as it is being transferred from one individual cell to another, and to watch its stable genomic acquisition via genetic recombination (horizontal gene transfer) in real time. This experimental system can be applied to monitor horizontal gene transfer by indefinitely following the fate of DNA acquired in intra- and interspecies crosses.

Ana Babic, Ariel B. Lindner, Marin Vuli, Eric J. Stewart and Miroslav Radman 2008, Direct Visualization of Horizontal Gene Transfer. Science 319, pp. 1533 - 1536.

Sunday, July 13, 2008

Reticulate classification of mobile genetic elements

“Reticulate representation of evolutionary and functional relationships between phage genomes.” by Lima-Mendez et al. (2008)

In this paper, the authors note that mobile genetic elements (MGE) in prokaryotes (such as phages, plasmids, conjugative transposons, and genomic islands) show mosaic structures, indicating the importance of horizontal gene exchange in their evolution. These elements represent unique combinations of modules, each of them with a different phylogenetic history. The traditional classification schemes cannot be applied to these genetic elements in part due to the intrinsic inability of tree-based methods to efficiently deal with mosaicism.

To solve the problem, Lima-Mendez et al. (2008) proposed a framework for a reticulate classification of phages based on gene content; i.e., presence (1) or absence (0) of protein family. First, the authors built a graph, where nodes represent phages and lines represent similarities in gene content between phages. Then, the authors applied a two-step clustering [Markov clustering (MCL) and fuzzy clustering] to this graph to generate a reticulate classification of phages: each phage is represented by a membership vector, which quantitatively characterizes its membership in the set of clusters. Phages within the same MCL cluster are likely descendant from a unique module combination, and one phage could belong to several clusters (Lawrence et al. 2002); for example, phage lambda belongs almost equally to two different clusters. Lima-Mendez et al. (2008) stated that “The weight of the intracluster connections represents 79% of the total weight of the connections of the network. This number can be taken as a rough estimate of the contribution of vertical evolution in this network. However, phages from different MCL clusters may be also be related through vertical evolution, but they might have diverged so much that sequence similarities are no longer recognizable or only some [evolutionary cohesive] modules may have been vertically inherited, whereas others have been replaced through horizontal gene transfer.” Thus, it is still difficult to estimate the contribution of different evolutionary events (i.e., vertical and horizontal gene transfer). Kunin and Ouzounis (2003) suggested a framework for the inference of presence or absence of individual protein families at any node on a phylogenetic tree, and assumed that: (1) A protein family shared by most of the clade members would be vertically transmitted; (2) If a protein family is present in most of the descendants of a particular ancestor, but is not found in some subclade, the observed gene absence would normally result from gene loss; and (3) A protein family interspersed across distantly related clades would be horizontally transferred. This assumption cannot detect horizontal gene transfer (HGT) among closely related species, as is true for most methods used to identify HGT (those based on phylogenetic information and compositional features). However, it is well recognized that phage and plasmid transfers (and consequently HGT) should be more likely among closely related species than among distantly related species.

Phylogenetic profiles have been widely applied to bacterial genomes to predict functional links between proteins on the assumption that proteins interacting in metabolic pathways or physical structure would be required to co-occur in genomes (Pellegrini et al. 1999). Lima-Mendez et al. (2008) clustered genes based on their “phylogenetic profiles” to define “evolutionary cohesive modules.” In virulent phages, evolutionary modules span several functional categories, whereas in temperate phages they correspond better to functional modules, suggesting that the phylogenetic profile method does not work well at predicting protein function in virulent phages. The Lima-Mendez analysis reminds us that we must be careful to consider the total context of the MGE, and not only the genome content.

The Lima-Mendez analysis was implemented using Network Analysis Tools (NeAT) (Brohée et al. 2008), available at

Lima-Mendez G, Van Helden J, Toussaint A, Leplae R. Mol Biol Evol. (2008) 25:762-77. Reticulate representation of evolutionary and functional relationships between phage genomes.

Lawrence JG, Hatfull GF, Hendrix RW. J Bacteriol. (2002) 184:4891-905. Imbroglios of viral taxonomy: genetic exchange and failings of phenetic approaches.

Kunin V, Ouzounis CA. Bioinformatics. (2003) 19:1412-6. GeneTRACE-reconstruction of gene content of ancestral species.

Pellegrini M, Marcotte EM, Thompson MJ, Eisenberg D, Yeates TO. Proc Natl Acad Sci U S A. (1999) 96:4285-8. Assigning protein functions by comparative genome analysis: protein phylogenetic profiles.

Brohée S, Faust K, Lima-Mendez G, Sand O, Janky R, Vanderstocken G, Deville Y, van Helden J. Nucleic Acids Res. (2008) 36(Web Server issue):W444-51. NeAT: a toolbox for the analysis of biological networks, clusters, classes and pathways.

Dr. Haruo Suzuki
University of Idaho