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