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 (Kan^R) 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

What a man can make …….

RESEARCH ARTICLES

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
UofI

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.

PRIMARY ARTICLE:
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.

ADDITIONAL REFERENCES:
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

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.


References:

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

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

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.
2008
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

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