Plasmid Conjugation in an Activated Sludge Microbial Community

By Ruoting Pei and Claudia K. Gunsch

ENVIRONMENTAL ENGINEERING SCIENCE,

Vol 26, No 4, 2009, p. 825

This is jet another paper describing plasmid transfer and their role in so called bioaugmentation in natural kind of environments.

Bioaugmentation involves the addition of exogenous micro-organisms that have the ability of degrading the compound of interest. Using this process, new biodegradation pathways can be added, which inherently improve the metabolic conversion of the contaminants. The main problem of this method is that the laboratory strains used to introduce new genetic traits usually cannot grow in natural environmental conditions. This means that bioaugmentation can fail due to the poor establishment and=or survival of new strains under field environmental conditions. The second process used for enhancing natural biodegradation capabilities is biostimulation which consists of adding nutrients (e.g., carbon, nitrogen, electron acceptor, etc.) to promote the growth of indigenous microorganisms. This method is preferred over bioaugmentation because no additional bacterial strains are introduced but it requires the presence of indigenous microorganisms, which are capable of breaking down the contaminant of interest. To overcome the limitations associated with biostimulation and bioaugmentation, it might be possible to combine these two strategies into a technique called genetic bioaugmentation. This technique would consist of adding bacteria with the needed genes and inducing their horizontal gene transfer (HGT) to indigenous bacterial species utilizing natural prokaryotic adaptation mechanisms.

In this paper authors described pWWO plasmid transfer from Pseudomonas putida strain to bacterial population present in activated sludge. The pWWO plasmid is well known for its toluene degradation capability. The plasmid DNA was tagged with transposone containing gfp gene driven by lac promoter. In the donor strain which overproduce LacI repressor gfp is not expressed. In other bacterial hosts gfp is expressed and gives bright green fluorescence which can be measured. Using flow cytometry authors showed that plasmid was actually transferred into bacterial community of activated sludge with number of bacteria expressing GFP protein up to 6%. They showed that the highest number of transconjugants is reached at the third day of experiment and it depends on the donor to recipient ratio with the best results at the 1:20 ratio. Authors tried to connect the plasmid transfer with the actual capability of activated sludge to toluene degradation. This experiment did not really work as the level of toluene degradation remained unchanged during the time of the experiment.

The paper stays in a main stream of microbial ecology and engineering facing the problem of global pollutions and waists utilization. It is very important to use new methods for detecting plasmid transfer in natural environment. Authors were facing the same problem as in previously published papers that there are some limitations in using fluorescent proteins as a plasmid infection marker. The true plasmid bearing cells are not known until gfp gene is expressed and protein is properly matured to give strong fluorescence, which can be detected by flow cytometry. Authors cannot also link the presence of TOL phenotype (toluene degradation) with the number of plasmid bearing cells as the sludge microbial community shows already high toluene degradation activity.

Perhaps, it is not a very “big” paper, but it is a small step toward understanding how the plasmid transfer occurs in natural environment and how can we use this to save our polluted world.

Jaroslaw Krol, PhD

UofI

The SOS Response Controls Integron Recombination

Émilie Guerin, Guillaume Cambray, Neus Sanchez-Alberola, Susana Campoy, Ivan Erill, Sandra Da Re, Bruno Gonzalez-Zorn, Jordi Barbé, Marie-Cécile Ploy, and Didier Mazel

Science, May 2009 p 1034, Vol. 324, No. 5930.

An ever-growing concern in the medical world is the rise of antibiotic-resistant pathogens. One source of comfort that we have had in this fight against the spread of resistance is that the expression of resistance genes confers a cost to bacteria. In the absence selection for antibiotics it is thought that this cost will give a selective advantage to non-resistant bacteria, and so bacterial populations will loose resistance over time in the absence of antibiotics. The authors of this paper suggest that this is unfortunately not always the case.

In the words of Jurassic Park, “Nature will find a way.” For bacteria, one of the weapons in their arsenal against antibiotics is the SOS response. Under normal conditions the protein LexA binds to the operator region controlling SOS genes to repress their expression. In the event of DNA damage LexA is cleaved from this region and a series of DNA repair mechanisms are activated, some of which are low- fidelity and therefore lead to an increase of mutations, some of which may be beneficial in resisting further DNA damage.

In terms of antibiotic resistance, this paper explains that LexA repression and SOS expression also influences recombination of genes cassettes (which often code for antibiotic resistance. During SOS expression cassette recombination is induced. This can result in either silencing or reactivation of cassettes. In other words, a cassette carrying resistance genes can be reactivated through recombination under times of stress and then revert to a “dormant” form that provides no cost to the bacterium once the SOS response is again repressed.


Additional Reading:
Erill, S. Campoy, J. Barbe, FEMS Microbiol. Rev. 31, 637 (2007).
C. M. Collis, R. M. Hall, Antimicrob. Agents Chemother. 39, 155 (1995).
A. Aertsen, C. W. Michiels, Trends Microbiol. 14, 421 (2006).
D. I. Andersson, Curr. Opin. Microbiol. 9, 461 (2006).


Julie Hughes,
Graduate Student, University of Idaho

A rapid method to construct gene cassette libraries enriched with first gene cassettes and an associated screening method for the clone selection

First Gene Cassettes of Integrons as Targets in Finding Adaptive Genes in Metagenomes
Lionel Huang, Christine Cagnon, Pierre Caumette, and Robert Duran
Applied and Environmental Microbiology, 2009, 75（11）：3823–3825

Here, I will introduce a short publication focusing on a very narrow specific problem. This paper propose a rapid method for the selection of clones carrying an integron first gene cassette that is useful for finding adaptive genes in environmental metagenomic libraries.

Integrons are genetic structures capable of capturing and excising gene cassettes, which usually encode adaptive proteins in different environmental contexts, such as genes for degradation of pollutants, and resistance to antibiotics or heavy metals. Thus, environmental pressures may favor the propagation of cassettes conferring a selective advantage.

Integrons are not a new and hot topic, but there is an increasing interest in finding of adaptive genes associated with integrons among the huge metagenomes. The integration of a new gene cassette, catalyzed by the integrase, occurs by recombination between the attC site and the attI site of the integron. The first gene cassette of an integron is, therefore, the last one integrated. In previous studies, the determinations of gene cassette collection from environmental metagenomes did not target first gene cassettes, since they were performed by PCR methods targeting attC sites. As the first gene cassette is the closest gene to the promoter, its expression level is the highest in the integron. Thus, it is a good target to find new adaptive genes in metagenomes.

The method was developed by using a pure strain isolate, Xanthomonas campestris ATCC 33913T, which carry an integron possessing 23 gene cassettes. One environmental sample, coastal sediment, was used to validate the method. There are two key points in developing this method, one is the construction of first gene cassettes libraries. To amplify the first gene cassettes, a forward primer targeting the intI gene or attI site must be used. Forward primer AJH72 was used for PCR of X. campestris DNA, and primer ICC48 (intB-inverted primer), targeting the class 1 integron intI, was used for PCR of sediment metagenome. A less-degenerated primer ICC21, was designed to target the attC sites from class 1 and 2 integrons. The other key point is the trick for clone selection. Due to the particular structure of the attC site with inverted repeat sequences, the reverse primer was also used in the forward direction. As a result, several amplified fragments were obtained, and the sequence analyses revealed that most of them were gene cassettes other than the first one. Here, the authors develop a triplex PCR method by labeling the forward primer with HEX (6-carboxyhexafluorescein). The fluorescent PCR fragments were selected for further sequencing.

This method was then applied to coastal mud metagenomes, and 23 fluorescent fragments were detected and sequenced. A total of 29 open reading frames (ORF) were characterized as potentially transcribed by an integron promoter. The first-gene cassettes of integrons appear to be good candidates to find gene cassettes, which aid bacteria in effecting a rapid adaptive response. We are now able to reveal integron last gene acquisitions of environmental bacterial communities submitted to stressful conditions. The PCR method combined with the screening method leads to 100% of clones carrying a first gene cassette. Thus, this new method allows the focus to be on spreading first gene cassettes in metagenomes after a specific stress.

In this paper, the authors propose a good idea to construct gene cassette libraries enriched with first gene cassettes and an associated screening method for the clone selection. However, the only one drawback in this paper is that the relationship between the function of ORF and the oil degradation should be discussed further. Since the first gene cassettes was enriched by adding oil into the coastal sediment, it should be served as the environmental pressure for selecting adaptive genes. I have checked the information from EMBL, where the author deposited the sequences, but still no related information in the database.



Hui Li, PhD
University of Idaho

DNA transfer proteins of broad-host-range plasmid can mediate chromosomal DNA transfer

The R1162 mob proteins can promote conjugative transfer from cryptic origins in the bacterial chromosome. Richard Meyer (2009) J. Bacteriol. 191: 1574-1580


It is well-known that plasmids mediate horizontal gene transfer among bacteria and play major roles in the rapid spread of antibiotic resistance. Generally, plasmid-mediated horizontal gene transfer from one bacterial chromosome to another requires the help of transposons as follows; in the first step, a transposon that has captured chromosomal genes moves into the plasmid; in the second step, the plasmid moves into a recipient cell by conjugative transfer; and in last step the transposon on the plasmid jumps into the chromosome of the recipient cell.

This paper showed that the above-described scheme is not the only way that plasmids can participate in the horizontal transfer of chromosomal genes. The author found that a plasmid can directly transfer donor chromosomal DNA into the recipient chromosome.


In a canonical model, conjugative DNA transfer starts with the DNA cleavage at the origin of transfer "oriT", a unique site on the plasmid, which is mediated by a protein called "relaxase". Previously, the author found that relaxase of the broad-host-range plasmid R1162 (also called RSF1010) can initiate DNA transfer at several sequence variants of oriTR1161. The observed promiscuous activity of the relaxase raised the possibility that plasmids can directly mediate the transfer of chromosomal DNA from oriT-like sequences in a chromosome (Jandal S. and Meyer R., 2006).


Draper et al. (2005) showed that relaxase can mediate recombination between two directly repeated oriTs on the transferred DNA in the recipient cell. This nature of the relaxase was used in the experiment to test the hypothesis that relaxase can initiate DNA transfer from cryptic oriT in a chromosome. A plasmid that has the original oriT and a selectable drug-resistance gene marker was artificially integrated into the downstream region of one of the candidate oriT sites in the chromosome of donor strain Pectobacterium atrosepticum to make directly repeated oriT sites. If single-strand DNA is branched out from the candidate oriT and moves into recipient cells, the transferred DNA would become a plasmid that carries a hybrid oriT comprised of the candidate oriT and the original oriT due to the recombination activity of the relaxase. As the author expected, the plasmid that has a part of the donor chromosome and a hybrid oriT was obtained in the recipient E. coli. This result indicates that the initiation of transfer does happen at the candidate oriT in P. atrosepticum.


The next questions are "What length of DNA is it possible to transfer?" and "Is the transferred chromosomal DNA integrated into chromosome in the recipient cell?" To answer these questions, the author used an E. coli strain as donor strains, that has a drug-resistance gene marker at a particular location in the chromosome. In the presence of helper plasmids that express relaxase and pillus proteins, the drug resistance gene marker was transferred and integrated into the chromosome of recipient E. coli. The candidate oriT closest to the resistance gene was 40 kbp away from the resistance gene in the donor chromosome. It suggests that a chromosomal DNA fragment of at least 40 kbp was transferred in the mating process. Surprisingly, even when the closest oriT was eliminated from the donor chromosome, the transfer of the drug resistance maker was observed with almost the same frequency as it was in the presence of the closest candidate oriT. This result suggests that chromosome transfer can be initiated at multiple cryptic oriT sites in the donor chromosome. Given that the second closest candidate oriT is 708 kbp away from the resistance gene, a DNA fragment of at least 708 kbp was indicated to be transferable in this experiment.

The authors estimated that there are 10 candidate oriT sites in the P. atrosepticum chromosome and 8 in the E. coli chromosome, which could be active in the presence of R1162 relaxase. Although it is still not clear how many plasmids have a potential to mobilize fragments of the chromosome, this article clearly showed that there is a novel manner of horizontal gene transfer.


Bacteriophage are also known to mediate the transfer of host's DNA in the manner called "general transduction", where host's DNA are accidentally packed in phage's capsid and are introduced into new host cells. Relaxase-mediated chromosomal DNA transfer resembles phages' general transduction, but different in that the length of transferable DNA is not limited in the relaxase-mediated chromosomal DNA transfer; the size of transferable DNA is limited in general transduction due to the limited size of phages' capsid. Given the size of transferable DNA, it seems that plasmids play much more important roles in bacterial evolution than bacteriophages.



References:

Meyer R. (2009) The R1162 mob proteins can promote conjugative transfer from cryptic origins in the bacterial chromosome. J. Bacteriol. 191: 1574-1580

Jandle S. and Meyer R. (2006) Stringent and relaxed recognition of oriT by related systems for plasmid mobilization; implications for horizontal gene transfer. J. Bacteriol. 188: 499-506

Draper O., César C. E., Machón C., de la Cruz F., and Llosa M. (2005) Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells. Proc. Natl. Acad. Sci. USA 102: 16385–16390.



H.Yano. University of Idaho

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
UofI

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.

Citations:

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

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.
References:
[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.


DIYA SEN
GRADUATE STUDENT
BIOLOGICAL SCIENCES
UNIVERSITY OF IDAHO