Monday, September 22, 2008

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.

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.

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

Tuesday, September 16, 2008

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.


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

Thursday, September 4, 2008

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