Vanessa Guimarães, Patrick Linder and Peter Redder
Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland
Staphylococcus aureus is an opportunistic pathogen that causes both superficial and invasive infections, such as sepsis, endocarditis, pneumonia and osteomyelitis. Due to the frequent emergence of strains with plasmid-encoded antibiotic-resistance, it is important to understand how S. aureus plasmids are transferred and maintained.
The plasmid pSA564a belongs to the large β-lactamase/heavy-metal resistance plasmid family, which can also be found in feared MRSA strains such as S. aureus N315 and the community-acquired S. aureus USA300. Many plasmids from this family provide resistance to multiple antibiotics, with pSK1 encoding trimethoprim, quaternary ammonium, gentamicin, tobramycin and kanamycin resistance genes. Fortunately, these plasmids all appear to have a very narrow host-range, which limits their occurrence to S. aureus, and suggest the existence of a host-factor that is required for their replication.
A small plasmid-encoded transcript (RNA1) was identified anti-sense to the 5'-UTR of the RepA replication initiation gene in pSK1 and pN315, and this RNA1 was shown to inhibit translation of the RepA protein in pSK1. Our lab recently discovered that deletion of the 5' to 3' exoribonuclease J1 (RNase J1) results in the loss of the otherwise stably maintained pSA564a. By using a vector that replicates independently of the pSA564a origin of replication, we can show that the RNA1 accumulates in the RNase J1 mutant, suggesting that efficient degradation of the RNA1 is essential for replication of pSA564a. This hypothesis was further strengthened by cloning the region encoding RNA1 on a multi-copy plasmid, which led to rapid elimination of pSA564a. RNase J1 is a major player in RNA decay of Gram-positive bacteria, but has no ortholog in E. coli, perhaps explaining why the ßlactamase/heavy-metal resistance plasmids have such a narrow host range.
The DEAD-box helicase CshA and the 3' to 5' exonuclease PNPase are also factors in RNA decay, and it was therefore surprising that a RNase J1, CshA, PNPase triple mutant, although extremely sick, nevertheless maintains pSA564a replication.
Full-genome sequencing was used to verify that pSA564a was not integrated into the chromosome of the triple mutant, but is indeed kept as an episome, which should be reliant on its own origin of replication. Complementing the triple mutant with wild-type CshA led to immediate loss of pSA564a, whereas a CshAK52A active-site mutant did not, showing that the helicase activity of CshA is an important factor for plasmid replication.
Current work is focused on confirming that RNase J1 is needed as host-factor for other members of the pSA564a plasmid family, and to determine the RNase J1 mediated decay-path of the small regulatory RNA1. Furthermore, we are examining the molecular mechanism by which the deletion of CshA counteracts the effects of the RNase J1 deletion, and whether CshA directly modifies the formation of secondary structures within the RNA1, the 5'-UTR target, or the interaction between the two.
Masakazu Kataoka
Shinshu Universiry, Nagano, Japan
Conjugative transfer of bacterial plasmids has been studied from the viewpoint of drug- resistance spreading in the hospital and bacterial genome evolution by the horizontal gene transfer. In addition, the F factor has contributed largely in genetic of Eschericia coli, and Ti plasmid has opened the door of the current plant biotechnology. We believe the horizontal gene transfer using three main mechanisms, transformation, transfection, and conjugation has increased the bacterial genetic diversity. The conjugal transfer has suitable characteristics for synthetic biology since free from the capability to transfer the large DNA without exposing it in water. The huge genome information of various organisms has been accumulated by appearance of the next-generation sequencer and high performance computers. The synthetic biology of designed genome using the huge information must be one of the targets in next generation biology. However, the handling and cloning method of a large DNA fragment are still difficult way, and limited bacterial species can be used for artificial transformation. We have been trying to solve these difficulties by using the conjugal transfer, relatively old knowledge. In this CSHL meeting, I would like to present and discuss our recent three challenges for handling Streptomyces and Bacillus bacteria.
The conjugal transfer for the handling of Streptomyces secondary metabolism.
We use three techniques, the conjugal transfer between the E. coli - Streptomyces using RP4 system,
the insertion of the large DNA fragment onto linear plasmid by actinophage system,
the conjugal transfer between Streptomyces by the conjugation system of linear Streptomyces plasmid. The established system can be used to improve various biosynthesis of many Streptomyces strains without developing protoplast operation and transformation.
High-frequency transmission of the Streptomyces genome.
We developed the high frequency genome mobilization system in Streptomyces. The system was accomplished by insertion of 4kb fragment coding transfer genes of Streptomyces RCR type small size plasmid named pSN22.
Optimization by the conjugal transfer to Bacillus.
We optimized the inter-genus conjugal transfer between E. coli and B. subtilis using RP4 system.
We expect this technique can be applied to strains being closely related to B. subtilis with difficulties of normal transformation including B. natto.
Ersi Emmanouilidou1, NikosTaliouras1, Valia Tampakopoulou1, Karen Davenport2, David Bruce3, Chris Detter2, Roxanne Tapia2, Cliff Han2, Miriam L. Land4, Loren Hauser4, Yun-Juan Chang4, Chrongle Pan4, Vassili N. Kouvelis1, Lynne A. Goodwin2, Tanja Woyke2, Nikos C. Kyrpides3, Milton A. Typas1, and Katherine M. Pappas1*
1Department of Genetics & Biotechnology, Faculty of Biology, University of Athens, Panepistimiopolis, Athens 15701, Greece
2DOE Joint Genome Institute, Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
3DOE Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, California 94598
4Oak Ridge National Laboratory, Bioscience Division, Oak Ridge, Tennessee 37831
*corresponding: kmpappas@biol.uoa.gr
Zymomonas mobilis is a candidate organism for large scale bioethanol production due to its ability to ferment sugars to ethanol faster than yeasts and to higher final yields. In order to understand the biology of Z. mobilis, six different strains belonging to its major subspecies, isolated from various parts of the globe, are being sequenced at the US Department of Energy Joint Genome Institute in collaboration with the University of Athens (CSP_788284 (DNAseq); CSP_52 (RNAseq); http://jgi.doe.gov/why-sequence-zymomonas-mobilis-strains/). Plasmids of the examined strains have received special attention and have been sequenced twice for each strain: once in terms of WGS sequencing and also from plasmid material provided in separate. All Z. mobilis strains harbor plasmids that range in numbers from two to eight per strain, in sizes from 1.6 kb to 53 kb, and often sum-up to 8% of a strains genome. The smallest plasmids are high-copy rolling-circle-replicating and some are mobilizable; they are suitable for the generation of species-specific cloning and expression vectors. The larger plasmids are theta-replicating, and are also important for the creation of low-copy vehicles for gene or library introduction. Of the over 600 genes annotated on the sequenced Z. mobilis plasmids, genes of interest contain genes involved in basic metabolism, structure formation, regulation, transposition, immunity (CRISPR) and tolerance to adverse agents, as well as housekeeping genes. Among these last, well discerned are replication, active partitioning and toxin-antitoxin genes. Plenty of the Z. mobilis plasmid genes are strain-specific, while others have homologs in different strains. Apart from instances where distinct syntenic regions are found on plasmids of more than one strain, no particular plasmid seems to have been horizontally dispersed and retained between strains in its entirety. Additionally, and despite the plentiful IS elements harbored in most strains, strain chromosomes do not appear to host traces of plasmid material. In fact, in one strain (NCIMB 11163) an entire conjugative region is met on the chromosome and not on any of its plasmids (or any other strain's plasmids), as a whole or part of. This gives the impression that the plasmidome of Z. mobilis is highly conserved, which is reinforced by the fact that plasmids of different Z. mobilis strains have been used consistently throughout the years for strain-profiling purposes.
Xavier Bellanger, Hélène Guilloteau, Christophe Merlin*
Laboratoire de Chimie Physique et Microbiologie pour l'Environnement,
UMR 7564 Université Lorraine - CNRS,
15 avenue du Charmois, 54500 Vandoeuvre-lès-Nancy, Nancy, France
*corresponding: christophe.merlin@univ-lorraine.fr
Antibiotic resistance gene transfer mediated by plasmids is a matter of concern for public health, but permissive environments/conditions supporting plasmid dissemination are still quite difficult to identify. Lately, we have developed a molecular approach based on quantitative PCR to monitor the fate of known plasmids in natural microbial communities maintained in microcosms. Practically, it consists in inoculating microcosms with a donor bacterium and monitoring the evolution of both the plasmid and the initial host DNAs over time. Because conjugative transfer is an intercellular form of DNA replication, the plasmid to donor DNA ratio increases in community DNA when the plasmid disseminates by conjugation into the indigenous population. As far as very specific sets of primers and probes are available for the non-ambiguous quantification of donor and plasmid DNAs, this method provides the advantage of considering plasmid transfer in a wide range of possible indigenous recipient bacteria, culturable or not, and it is sensitive enough to detect rare transfer events under low (but realistic) donor inoculum size. Using the broad host range IncP-1β plasmid pB10 as model, such transfer experiments were carried out in various environmental matrices from river sediments, to activated sludge, and manure. These experiments demonstrated that the transfer of the conjugative-proficient plasmid pB10 in complex environments is relatively rare and is strongly matrix dependent. The detection of successful transfer events in a given environmental matrix seemed to be linked to the initial stability of the donor inoculum. Depending on the matrix considered, eukaryotic predation plays a significant role in either limiting or promoting the plasmid transfer events. An attempt to link the microbial community structure and the matrix permissiveness showed that TTGE analysis is not resolutive enough to point out common features among comparable communities supporting pB10 transfer. However, an estimation of the IncP-1α/β plasmids abundance by quantitative PCR demonstrated that pB10 transfer tends to be supported by environmental matrices exhibiting a higher content of IncP-1α/β plasmids. We suggest that the relative abundance of IncP-1 plasmids in a given microbial community reflects its permissiveness to the transfer of plasmids belonging to the same incompatibility group, which prevails over transfer limitation due to the phenomenon known as superinfection immunity.
Porse A, Munck C, Sommer MO
Technical University of Denmark
It is not well understood how plasmids persist under non-selective conditions in nature1. The first efforts to address this question were carried out in the 1970s through mathematical modelling of plasmid bearing populations2,3,4. These first models predicted a broad range of conditions under which conjugative plasmids will persist by means of infectious transfer alone. However, they were often based on parameters obtained in vitro using laboratory strains and plasmids4,5. While some suggest high transfer rates to be vital for parasitic plasmid survival, others argue that the transfer rates predicted to be necessary are not attainable, nor necessary, for plasmid persistence in natue6,7,8,9. Consequently, the importance of conjugation in the persistence of plasmids has been heavily debated in the plasmid community6,8,10.
We have investigated the genetic changes implicated in host-plasmid adaptation between a clinical ESBL plasmid and different clinically relevant E. coli and K. pneumoniae hosts. Using adaptive evolution and subsequent population sequencing, we show that the burden imposed on naïve cells receiving a conjugative plasmid is mainly caused by the conjugational transfer machinery itself and varies substantially between hosts. We suggest that variations in the ability to regulate genes involved in conjugation is the major cause of the observed differences in plasmid cost, ultimately resulting in deletion of non-regulated genes. These observations highlight the trade-off between horizontal and vertical transfer and indicate that the fitness cost, having to be compensated by the conjugational machinery in order for the plasmid to persist, is highly dependent on the host background and that the conjugational machinery can be strongly disfavoured in certain hosts. Our findings add an extra layer of complexity to the ongoing discussion concerning the importance of conjugational transfer for plasmid persistence in diverse bacterial populations.
References
1Harrison, E. & Brockhurst, M. a. Plasmid-mediated horizontal gene transfer is a coevolutionary process. Trends Microbiol. 20, 2627 (2012).
2Stewart, F. & Levin, B. The population biology of bacterial plasmids: a priori conditions for the existence of conjugationally transmitted factors. Genetics 209228 (1977).
3Levin, B. R. & Stewart, F. M. Probability of establishing chimeric plasmids in natural populations of bacteria. Science 196, 21820 (1977).
4Levin, B. R. TRANSITORY DEREPRESSION AND THE MAINTENANCE. 483497 (1986).
5Slater, F. R., Bailey, M. J., Tett, A. J. & Turner, S. L. Progress towards understanding the fate of plasmids in bacterial communities. FEMS Microbiol. Ecol. 66, 313 (2008).
6Lili, L. N., Britton, N. F. & Feil, E. J. The persistence of parasitic plasmids. Genetics 177, 399405 (2007).
7Bergstrom, C. T., Lipsitch, M. & Levin, B. R. Natural selection, infectious transfer and the existence conditions for bacterial plasmids. Genetics 155, 150519 (2000).
8Freter, R., Freter, R. R. & Brickner, H. Experimental and mathematical models of Escherichia coli plasmid transfer in vitro and in vivo. Infect. Immun. 39, 6084 (1983).
9Imran, M., Jones, D. & Smith, H. Biofilms and the plasmid maintenance question. Math. Biosci. 193, 183204 (2005).
10Tazzyman, S. J. & Bonhoeffer, S. Fixation probability of mobile genetic elements such as plasmids. Theor. Popul. Biol. 90, 4955 (2013).
Piotr Zaleski*, Pawel Wawrzyniak, Agnieszka Sobolewska, Natalia Lukasiewicz, Piotr Baran, Katarzyna Romanczuk, Katarzyna Daniszewska, Piotr Kieryl, Grazyna Plucienniczak, Andrzej Plucienniczak
Department of Bioengineering, Institute of Biotechnology and Antibiotics
Staroscinska 5, 02-516, Warsaw Poland
*zaleskip@iba.waw.pl
Plasmids are the most abundant extrachromosal mobile genetic elements (MGEs). They are very widespread and mostly found in bacteria. The ever growing number of completed sequences of different mobile genetic elements (including plasmids) showed that they are built from smaller elements modules, which group genes responsible for individual function (e.g. REP module plasmid replication; MOB module conjugative transfer and/or mobilization). Our recent research showed that it is possible to separate plasmid modules and that they might possess different characteristic (Smorawinska et al., 2012) hence they might evolve separately. pIGWZ12 is a cryptic plasmid described in 2006 as a completely new molecule with only two small regions of nucleotide homology to already known plasmids (Zaleski et al.,2006). One almost identical plasmid pSM35_4 was reported later (Fricke et al., 2008). Our results presented here show that pIGWZ12 consist of REP module from IncF family and MOB module characteristic for MOBQ family. Such results indicate that both modules originate from a distinct plasmid families. IncF replicone is characteristic for low copy number, narrow-host-range plasmids. MOBQ usually is a part of broad-host-range plasmids from RSF1010/R1162 family. Furthermore we were able to separate REP and MOB regions showing that both modules might function independently. REP module by means of iteron region is responsible for binding of RepA (replication initation protein), repA expression and incompatibility. MOB module (responsible for plasmid mobilization) consists of two genes (mobA and mobC) and origin of transfer (oriT).