Wednesday, May 27, 2009

Plasmid Capture by the Bacillus thuringiensis Conjugative Plasmid pXO16

Plasmid Capture by the Bacillus thuringiensis Conjugative Plasmid pXO16
Sophie Timmery, Pauline Modrie, Olivier Minet, and Jacques Mahillon
Journal of Bacteriology, April 2009, p. 2197-2205, Vol. 191, No. 7

In this article authors describe the ability of Bacillus thuringiensis plasmid pXO16 to transfer as well as mobilize three other plasmids. It is very important issue to study plasmid transfer in Gram positive bacteria as this process is not as well known as plasmid transfer in Gram negative bacteria. Gram positive bacteria are very important as they can be as deadly as Bacillus anthracis – the causative agent of anthrax, but also can be opportunistic pathogens associated with food, like Bacillus cereus. The widely used B. thuringiensis plays very important role as the source of insecticidal toxins. Other Gram positive bacteria like Streptococcus sp. and Staphylococcus sp. are also pathogenic for human. The presence and horizontal transfer of mobile genetic elements that can play a role in spreading an antibiotic resistance as well as pathogenic determinants is very important issue.
So this paper gives us some information about pXO16 plasmid transfer and its ability to mobilize other plasmids. I would like to point out some parts of this paper. First, I was intrigued by the title of this paper. The title could suggest that pXO16 can capture other plasmids DNA molecules and incorporate into its own DNA. From the second sentence we can figured out what authors have on mind talking about capture of other plasmids by pXO16, and that it is the mobilization and retromobilization of other plasmids to the host cells by this Bacillus thuringiensis plasmid.
In the introduction section authors presented very briefly the “state of art” in the plasmid transfer and mobilization field. The special interest is put on retromobilization which is very interesting event. Retromobilization occurs when recipient DNA, either plasmid or chromosomal markers are transferred to the donor during conjugation. This reciprocal DNA transfer is very interesting and it was discussed as it is against the unidirectional DNA transfer rule. The schematic representation of retromobilization models on Fig. 1. is very easy and easy understandable. In the results section we have a very nice story about pXO16 plasmid transfer, and mobilization of “mob” plasmids pUB110, and pE194, as well as no mobilizable plasmid pC194. One thing that bothers me is the way to present conjugation efficiency as the transconjugant to recipient ratio. I know that it is the way, but it does not show overall number of donor, recipient and transconjugant cells in the conjugation mixture and I personally do not like it. In the triparental matings with the plasmid free cells as recipients only two plasmids were used, pUB110 and pC194. Generally lower frequencies of plasmid transfer were observed in triparental than in biparental matings. Authors described also the ability of pXO16 to mobilize pUB110 in three different “media” cow, soy and rice milk and showed the influence of media on mobilizable plasmid transfer. It was also shown that plasmid pXO16 can be used to capture the pUB110 plasmid from other bacteria in all 3 kinds of milk. This is quite interesting that food products can be used as media in this kind of experiments and that biological events can occur in such an environment. We should consider this and look on the food quality especially on expiration date in “milk related” products.
Very interesting results are presented in the section describing plasmid transfer kinetics where it is shown that the plasmid transfer occurs only in a short period of time reaching plateau after certain period of time. It is consistent with other results and proves that plasmid transfer is controlled by specific mechanisms in the cell and these functions are not the only plasmid related.
In discussion authors focused on the mode of retrotransfer and showed that retrotransfer in the case of B. thuringiensis pXO16, and plasmids used in the paper support the two step model. It is interesting as we always thought that we need two kinds of cells, donor and recipient for conjugation to occur. And now we have two kinds of cells but both contain the same conjugative plasmid. Mobilizable plasmid can be transfer only if two cells form mating pair. Mobilizable plasmid encode no functions related to mating pair formation so it means that two “donor” cells can form mating pair and maybe reciprocally transfer DNA molecules. That is really exciting…
To summarize I found this paper quite interesting but some weak points, like poorly written materials and methods, as well as graphical representation of results depreciate the quality of this work and should be corrected before publication.

Jarek Krol, PhD

Sunday, May 17, 2009

A new Family of Gram-positive bacterial plasmids

Weaver, K. E., Kwong, S. M., Firth, N. & Francia, M. V. (2009).The RepA_N replicons of Gram-positive bacteria: a family of broadly distributed but narrow host range plasmids. Plasmid 61, 94–109.

Considering the rate at which sequence databases, and therefore our knowledge of existing plasmids, are growing, it is becoming increasingly important to organize and categorize plasmids into relevant groups. Proper organization of known plasmids would allow us to unravel evolutionary histories and make more efficient the process of integrating our current knowledge. In this article, Weaver et. al. propose to create a family of plasmids characterized by RepA_N, a highly conserved domain in the initiator protein.

Unlike most other plasmid classification systems, this family is not based upon the incompatibility of two plasmids due to similar replication machineryc. Indeed, many of the plasmids within the RepA_N family can stably coexist within a single host bacterium (Kwong et. al. 2008). Rather, the determining characteristic of this plasmid is this conserved initiator protein domain. Phylogenies based on RepA_N matched those of each plasmid’s hosts. What’s more, members of this family are narrow host range plasmids but are found in a diverse range of low G+C gram-positive bacteria (Firth et al., 2000). This suggests that RepA_N family plasmids were present in ancestral gram-positive bacteria of low G+C content and then proceeded to diverge with individual hosts at an early split in the host evolution.

When compared to phylogenies based on other protein domains the modular nature of plasmid evolution becomes apparent. Phylogenies based on RepB, for instance, do not match with host phylogenies or those of RepA_N. RepB is just one among many examples of how plasmids can acquire complete, functional units of DNA from various sources throughout their evolutionary histories. The authors site the specific examples of the replication, partition, and conjugative components of RepA_N plasmids as evolving by “shuffling” between various other plasmids that are found within the same host. Again, RepA_N serves as a good classification marker in that it is conserved within each host and matches its host’s phylogeny.

This article touched on several points that bear particular attention. To begin, the authors point out that this sort of a study cannot be effective without a certain volume of raw data in sequence databases, which was not feasible even a few years ago but is now available and growing. Secondly, with such data evolutionary histories of plasmids and their coevolution with their bacterial hosts can be elucidated and that such information is vital to our understanding of why plasmids are distributed as they are today (e.g., how a family of plasmids can be both broadly distributed and only stably transferred to and maintained in a narrow host range). Finally, this study provides several excellent examples of the modular nature of plasmid evolution. Hopefully in the future available information on previously uncharacterized plasmids will continue to grow and will continue to be organized such that more such evolutionary insights may be made in the future.


Kwong, S.M., Lim, R., LeBard, R.J., Skurray, R.A., Firth, N., 2008. Analysis of the pSK1 replicon, a prototype from the staphylococcal multiresistance plasmid family. Microbiology 154, 3084–3094.

Firth, N., Apisiridej, S., Berg, T., O’Rourke, B.A., Curnock, S., Dyke, K.G.H., Skurray, R.A., 2000. Replication of staphylococcal multiresistance plasmids. J. Bacteriol. 182, 2170–2178.

Julie Hughes
Graduate Student
Department of Biological Sciences
University of Idaho

Monday, May 11, 2009

Bacterial Toxin–Antitoxin Systems: More Than Selfish Entities?
Laurence Van Melderen and Manuel Saavedra De Bast

Bacterial Toxin-Antitoxin systems are diverse and widespread in the prokaryotic world. They are composed of two components, a toxin that can harm the host and its corresponding antitoxin that is needed by the host to prevent cell death. TA systems that are found on chromosomes are hypothesized to have been acquired by horizontal gene transfer. Some bacteria are known to have around 50 putative TA systems such as Nitrosomonas europeae, and Sinorhizobium meliloti. Others have none or a few TA systems. Plasmid encoded TA systems act as addiction modules and help in maintaining plasmid-containing cells. Thus, while the function of plasmid encoded TA systems is well known, those found on chromosomes are not as well understood. There are several theories on the physiological roles of chromosomal TA systems. Following are some of these proposed models:
1) The programmed cell death model: This model is based on the chromosomally located mazEF system of E. coli [1]. Under conditions of stress such as amino acid starvation, high temperature or presence of antibiotics, transcription of mazEF is affected. This is followed by degradation of MazE (antitoxin) by an ATP-dependent protease and subsequent toxification by previously produced MazF (toxin). This in turn leads to cell death.
2) The growth modulation model: This model is based on the relBE system of E coli [2,3]. The primary difference between this model and the previous one is that this model proposes cell growth inhibition under conditions of amino acid starvation and not cell death.
3) The developmental model: This model was proposed for the toxin gene in Myxococcus xanthus [4] an organism that forms fruiting bodies under nutrient starved conditions. The genome of this organism has a homologue of the mazF toxin gene. During fruiting body formation, MazF protein is produced which induces cell death. In fact, nearly 80% of the cells that undergo fruiting body formation die by lysis. However, MazF has also been found to be essential for fruiting body formation.
4) The stabilization model: The model proposes that TA systems could help stabilize some regions of the genome that are unstable and prone to being lost [5]. Such TA systems are often found on structures called super integrons that carry many essential and non-essential genes. The TA systems stabilize the super integrons as well as unstable plasmids or genomic regions.
5) The anti-addiction model: This model proposes that chromosomal TA systems can benefit their hosts during post seggregational killing [6]. The chromosomal TA system of Erwinia chrysanthemi 3937 was found to prevent post seggregational killing of bacterial cell after loss of plasmid.
The above models show the different ways in which TA systems can confer a selective advantage to their hosts.
Thus TA systems on chromosomes are diverse and have evolved multiple roles from being simple addiction modules to more complex systems involved in cell physiology.
[1] Engelberg-Kulka H, Amitai S, Kolodkin-Gal I, Hazan R. Bacterial programmed cell death and multicellular behavior in bacteria. PLoS Genet. 2006;2:e135. doi:10.1371/journal.pgen.0020135.
[2] Christensen SK, Mikkelsen M, Pedersen K, Gerdes K. RelE, a global inhibitor of translation, is activated during nutritional stress. Proc Natl Acad Sci U S A. 2001;98: 14328–14333.
[3] Christensen SK, Pedersen K, Hansen FG, Gerdes K. Toxin-antitoxin loci as stress-response-elements: ChpAK/MazF and ChpBK cleave translated RNAs and are counteracted by tmRNA. J Mol Biol. 2003; 332:809–819.
[4] Nariya H, Inouye M. MazF, an mRNA interferase, mediates programmed cell death during multicellular Myxococcus development. Cell. 2008;132:55–66.
[5] Rowe-Magnus DA, Guerout AM, Biskri L, Bouige P, Mazel D. Comparative analysis of superintegrons: engineering extensive genetic diversity in the Vibrionaceae. Genome Res. 2003;13:428–442.
[6] Saavedra De Bast M, Mine N, Van Melderen L. Chromosomal toxin-antitoxin systems may act as antiaddiction modules. J Bacteriol. 2008;190:4603–4609.