You may be wondering...

You may be wondering why I've not been writing. Well, I have. Family and friends, here is my first thesis essay draft of my undergrad. It has nothing to do with bike racing, riding, or otherwise pedaling. But, if you're interested, I've attached everything I know about horizontal gene transfer below. 

Abstract

Horizontal gene transfer (HGT) is the term used to describe a modification of genetic material in an organism that occurs during the lifespan of a single generation. This process interrupts the continuity of normal DNA conservation (heredity) within a population causing changes to the chromosomal complexes of the organisms via homologous recombination, additive integration, or by adding autonomous genetic elements (most often plasmids). These alterations occur through three pathways defined by the terms Transformation, transduction, and conjugation. HGT can occur in plant cells and there is limited evidence for HGT between animal cells. However, it is a process most prevalent among bacteria. The impact of HGT is particularly pertinent to microbiologists and health professionals struggling to contain a growing epidemic of antibiotic resistance that has been partially facilitated through HGT. Enterococcal bacteria, though previously considered commensalistic and harmless, have become a growing concern because of their propensity to disseminate antibiotic resistance genes among populations of diverse bacteria.

                                                      

Introduction to bacteria and their relevance

Imagine an almost invisible alien lifeform running rampant through our world. Unobservable and lethal, they are bombarding us with attacks while learning perfect strategies to evade our defenses. Every effort put forth to combat the invasion is met with aggressive and effective resistance. We are wearing thin, left nearly naked against an ever-growing threat. Unfortunately, this is not science fiction. This is the reality of our war against antibiotic resistant bacteria. Bacteria are individually miniscule, but they have an enormous impact on our lives. Though relatively simplistic, they contribute to an almost unfathomably complex microscopic world. Many bacteria are necessary for human life, however, there is a growing population of antibiotic resistant bacteria that loom over our livelihood and require our attention.

Bacteria are small, unicellular, microscopic organisms with a non-nucleated prokaryotic structure and without membrane bound organelles. Typically, bacteria reproduce via binary fission (one cell enlarging then dividing into two identical cells). The genetic material required for reproduction is contained within a semi-structure known as a nucleoid (Salm et al. 2016). Within the nucleoid, deoxyribonucleic acid (DNA) contains the genetic information unique to each bacterium.

It is paramount to understand genetic material as a physical entity – equal parts information and substance – materially transferable. DNA is made from a dual helix structure of paired bases surrounded by a sugar phosphate backbone. Its replication is a highly-regulated process by which exact copies of the original DNA are separated and established in each resulting cell that arises. In bacteria, this replication happens during a reproductive process of binary fission.

Precise replication of DNA is fundamental to the continuity of life. The accurate copying of genes is responsible for what is known as heredity. Genes are passed from the parental organism to all its resulting progeny. But, this process can be usurped by a mechanism known as horizontal gene transfer (Brooker et al. 2017).

Traditional models of heredity operate vertically. The genes from a parent organism are “passed down” to the next generation. The genes of each specific lifeform are given to resulting related posterity. Horizontal gene transfer is the mechanism by which one organism acquires new genetic material within the same generation and or from a non-related organism. This is a process most prevalent within the phylogenetic kingdom of bacteria (Heurer and Smalla. 2007).

Mechanisms of horizontal gene transfer

Three primary processes are responsible for the horizontal dissemination of DNA: transformation, transduction, and conjugation. Each involves a unique pathway of conferring genetic material into cells. Further, each is unique in its role within the natural world (Salm et al. 2016). Transformation is the term used to characterize the general process by which free DNA, that which is suspended in the environment, is absorbed by competent bacteria. Cells may use the absorbed DNA as a source of nutrients, or, integrate the DNA into its genome. DNA may be integrated into the cell’s genome by means of recombination or by becoming an autonomously replicating genetic element (Figure. 1). Those genes integrated by the cell by means of transformation add to cellular genetic diversity and innovation. This genetic variability is especially important to those cells, such as bacteria, that lack a recombination step within their reproductive process and therefore rely on such processes for genetic variation (Croucher et al. 2016). A critical consideration within transformation is the ability of DNA to exist extracellularly. Environmental factors greatly impact the stability of DNA structurally and its ability to enter a cell. Heat and unfavorable pH may cause the DNA to denature. Additionally, the presence of electron charged compounds, such as minerals, may affect the charge of the cell’s environment and push or pull the DNA because DNA itself has a negative charge. Because transformation is defined as the absorption of DNA from the environment, the dominant influence of environmental factors on transformation is a predictable correlation. Environmental factors directly impact the stability of extracellular DNA, the persistence of which is the determining factor in bacterial exposure time, which is the determining factor for transformation frequency (Thomas and Nielson. 2005).  The exact role of transformation within evolutionary history is still debated. It is theorized that transformation may have been significant during the evolution of early lifeforms (Davies and De La Cruz. 2000).

Transduction is the process by which genes are horizontally transferred by bacteriophages known as “specialized transducing phages” via non-infectious virus particles. Bacteriophages are a type of virus that infect prokaryotic cells. Though they normally transport only their own genome and release it into bacterial cells for replication, they may accidently uptake host DNA along with their own replicated genome. The unintentional inclusion of host bacterial DNA occurs during the transitionary period of a temperate phage’s conversion from lysogenic to lytic cycle. An incorrect excision of the temperate phage DNA from the host bacteria’s genome results in the phage DNA being accompanied by a short section of adjacent host DNA and subsequently packaged into the resulting phage head. The subsequent bacteriophage then continues to infect new bacterial cells with its own DNA alongside the DNA from its previous host. By this process, genes are horizontally transferred. Research has shown that most of the genes transferred through transduction are those that contribute to general cellular flexibility and fitness such as Fe uptake systems, proteases, and adhesins. Though, these bacteriophages do not normally transduce virulence factors (Allen et al. 2016).

            Conjugation is a distinct mechanism of HGT because, unlike transformation and transduction, it entails the physical contact of donor and recipient bacteria cells which attach directly to one another by a conjugative pili. Among conjugating cells, the transfer of smaller mobile genetics elements (MGE’s) are prevalent due to the short amount of time needed to transfer these less complex genetic elements. Because conjugation is reliant upon a physical connection between cells, and because the conjugative pili is a relatively delicate structure, the transfer of complex or complete sequences of genetic material is rare because the pilus being too easily broken or otherwise interrupted during the transfer process. Cells select each other for conjugation primarily through pilus specificity mediated by lipopolysaccharides and outer-membrane proteins that respond to hydrophobic peptide pheromones. The release of pheromones is a crucial mechanism for cell-to-cell communication and, in the case of bacteria, is responsible for the activation and deactivation of a cell’s conjugative abilities. As an example, the conjugative ability of Enterococcus faecalis is mediated by the activating pheromone cPD1 and deactivated by the antagonist pheromone iPD1 (Clewell. 2011). This system functions to produce a highly regulated, extremely specific coordination of conjugation between select cells. The genetic element most commonly associated with conjugation is the plasmid, a relatively small, circular, stable genetic structure. Plasmids are an independent self-replicating genetic element that can, if transferred to a new host cell as a complete double stranded structure, negate the requirement of recipient DNA chromosomal integration. Functionally, this means that plasmids are a potentially potent mobile genetic element because their operation within a new cell is not dependent upon the recipient cell’s chromosome being predisposed to the incorporation of the transferred genetic material. Additionally, the transfer of plasmids is made more rampant by the ability of non-self-transmissible plasmids (those plasmids which cannot cross the conjugative pili independently) to become communicable in the presence of a self-transmissible plasmid. In addition to plasmids, other mobile genetic elements, such as insertion sequences and chromosomal DNA, may be transferred via conjugation (Heurer and Smalla. 2007).

The examples and mechanisms of HGT thus far have been exclusively related to bacterial cells because most HGT happens within the context of bacterial interaction. However, HGT is not isolated to the bacterial kingdom alone. In the 2017 Annual Review of Genetics, Ralph Bock published an article documenting the recent progress in reconstructing the horizontal transfer of genes between plants where HGT was established as a viable process (Bock. 2017). The subject of HGT between eukaryotic animal cells, however, remains a subject of intense debate. Recently, in a study published in the Proceedings of the National Academy of Sciences by Boothby et al., the possibility of extensive HGT (Figure. 2) within the tardigrade genome was suggested (2015). Shortly thereafter, researcher Kazuharu Arakawa responded to this claim by suggesting the evidence of HGT within the draft genome of the tardigrade may have been the result of bacterial contamination within the data (Arakawa. 2016). The result of this debate is still forthcoming.

Integration of horizontally transferred genetic material

After a genetic element has been accepted into a competent cell, there exist three mechanisms by which it may be integrated by the recipient. The most simplistic genetic element that can be transferred is known as an insertion sequence element (IS). The introduction of an insertion sequence can only result in genome modification if the conditions are conducive to homologous recombination, the first of the three integration options. Homologous recombination is a type of gene modification that depends on base sequence similarity between the insertion sequence and the host chromosomal DNA. At a minimum, 25 base pairs of similarity must exist between the donor DNA sequence and that of the recipient if there is to be any amount of recombination. The amount of recombination induced by an insertion sequence is dependent on the type of bacteria which receives the insertion sequence. Acinetobacter baylyi shows recombination rates as low as 0.1% where Bacillus subtilis and Streptococcus pneumonia have recombination rates as high as 25-50%. These differences in recombination frequency are dependent on the level of similarity between the genome of the donor insertion sequence and the recipient’s own chromosome. The term used to define this level of similarity is “divergence”. The more dissimilar a DNA sequence is after recombination, the higher percentage of divergence. A log-linear relationship has been established between a decrease of insertion sequence equivalence to the host’s chromosome and a prevalence in resulting divergence – the more dissimilar resultant chromosome, the less likely a recombination will occur (Thomas and Nielsen. 2005).

The second type of DNA incorporation that may take place within a recipient chromosome is known as additive integration. The previous process of homologous recombination replaced sections of chromosomal DNA without adding to the DNA’s length. Conversely, additive integration adds genetic information to the recipient cell’s DNA by physically increasing DNA sequence length. This process requires only a small region of DNA similarity to initiate, while the subsequently added DNA may have little to no similarity to that which is being pushed or replaced. Additive integration is far less likely to occur than its homologous recombination counterpart. Neither homologous recombination or additive integration are limited by cell type (Thomas and Nielsen. 2005).

            Finally, transferred genes may, as is often the case for plasmids, integrate into the recipient cells gene schematic as an autonomous element. Autonomous genetic elements are not reliant on integration within the chromosomal complex of the cell for their expression (unlike homologous recombination and additive integration). Further, they are self-replicating and structurally stable (Thomas and Nielsen. 2005).

Competence and other factors impacting horizontal gene transfer

 Genes cannot be transferred between bacterial cells unless they first develop a state of competence. Competence is a physiological state effecting the bacteria’s membrane that is most often induced as the result of environmental stimulants, and is dictated further by genetic individual bacteria’s predisposition. “The ability of bacteria to take up extracellular DNA” is the succinct definition of competence given by Christopher M. Thomas and Kaare M. Nielson in their review “Mechanisms of, and barriers to, horizontal gene transfer between bacteria” (Thomas and Nielsen. 2005). Competence is not a static state. Rather it must be both induced and maintained. Most competent bacterial cells are “time limited” in their ability to sustain a competent state. Cell’s time limited competence can often be linked to variable external factors such as fluctuations in growth conditions, nutrient competition and or starvation, along with the relative density of other cells within the environment. Cells differ in their level of competence based on their exposure to environmental factors and their individual levels of genetic predisposition to a competent state which predetermines the level and type of gene expression that impact the structural integrity of the cell membrane. But, a state of competence alone is often not enough to induce HGT. Therefore, competence can be understood as a membrane permeable state required for HGT, but not a sufficient condition for HGT in and of itself (Thomas and Nielsen. 2005).

Researchers John W. Beaber, Bianca Hochhut, and Matthew K. Waldor investigated the correlation between the DNA damage SOS response in cells and the spread of antibiotic resistance. Their findings revealed a positive relationship between the “DNA damage SOS response”, which is a ubiquitous DNA repair response that causes the alleviation of repression genes that otherwise prevent the integrating conjugative element STX (a small kilobase resulting from Vibrio cholerae that is known to confer antibiotic resistance) from horizontally transferring into a new cell, and elicitation of a HGT reaction. A cellular SOS response can be stimulated through several mechanisms including environmental stimulus such as antibiotic pressure. Sub-lethal dosing of mitomycin C, a known DNA-damaging treatment used by researchers to induce an SOS response, increased the transfer frequency rate of the investigated integrating conjugative element STX by over 300-fold. Ciprofloxacin incubated donor cells also showed an increased rate of STX transfer. Ciprofloxacin, a fluoroquinolone antibiotic, was also shown to successfully activate the SOS response. Positive induction of an SOS response to promote the HGT of STX was shown in Escherichia coli and Vibrio cholerae with either mitomycin or ciprofloxacin treatment. This result suggests a positive correlation between the cellular SOS DNA damage response and HGT. The mechanisms responsible for generating this reaction is the mitigation of SetR, an STX encoded repressor. This results in amplified expression of genes essential to HGT, and therefore increases the frequency of STX transfer (Beaber et al. 2004).

One genetic element experimentally shown to contribute to the regulation of horizontally transferred genes are CRISPR (clustered regularly interspace short palindromic repeats) sequences. These relatively short genetic sequences facilitate bacteria in defense against foreign DNA, especially that which is injected by bacteriophages. Luciano A. Marrafini and Erik J. Sontheimer conducted research that found “strong functional evidence” that CRISPR sequences play a general role in preventing HGT, in addition to establishing immunity against bacteriophages. Specifically, it was concluded that the presence of CRISPR loci existent on CRISPR-positive RP62a strains of Staphylococci made experimental cells “refractory” to the conjugation of the pG0400 plasmid. Experimentally, this result was inferred by the presence of genome spacer spc1 in S. epidermis RP62a. This base sequence contains segments of homologous similarity to the nickase gene present in ubiquitous MRSA and VRSA plasmids such as pG0400. Thus, it could be expected that S. epidermis RP62a would be enabled to mount a CRISPR defense response against the introduction of the pG0400 plasmid. As anticipated, only experimentally muted versions of pG0400 (pG0mut) were horizontally transferred into the recipient cell. A similarly exclusive result was demonstrated when researchers attempted to transfer plasmid pLM6 into recipient strain RP62a (Figure. 4) Here, again, only the muted version of the plasmid was transferable. The wild-type plasmid had a transformation efficiency of 0 while the muted plasmid was transmitted with an efficiency of 2.3x10^-4 (conjugation efficiency was calculated as the recipient/transconjugants ratio). It was thus shown that CRISPR regulation of HGT is not limited to cellular defense against bacteriophages. Rather, it is capable of fulfilling a more general regulatory role in preventing HGT (Marraffini and Sontheimer. 2008).   

Horizontal gene transfer and antibiotic resistance

Genes that are horizontally transferred fall into three categories. First, it is possible for transferred genes to be deleterious. This practically translates into the loss of relevant information with the recipient cell. Loss of useful genetic information is disadvantageous and results in lower rates of propagation and or survival and are therefore invariably terminated by process of natural selection. Second, horizontally transferred genes may be neutral and confer no consequential changes in genetic composition. Neutral genetic variations represent no lasting significant genomic change within bacterial populations because they are perpetually outpaced by the final category of HGT, that which is advantageous. Advantageous genes benefit the recipient cell in some manner, effecting their general adaptability and fitness (Thomas and Nielson. 2005; Gogarten and Townsend 2005). Genes conferring antibiotic resistance are advantageous to bacterial populations are therefore a subject of tremendous relevance because of their effect on our cost and quality of healthcare options.

An example of an advantageous horizontally transferred gene would be one that causes an alteration of multidrug efflux pump expression in the bacteria. The expression of these pumps is controlled by transcription regulators (repressors or activators) which can be present on MGEs such as plasmids. If genes promoting the expression of these pumps exist on a plasmid transferred into a previously antibiotic susceptible bacterium, then the recipient cell will be advantageously altered towards being able to survive in a greater spectrum of environments. Additionally, plasmids may carry genes that allow bacterium to express enzymes that catabolize antibiotics (Lin et al 2015).

In the highly relevant article, “Horizontal gene transfer-emerging multidrug resistance in hospital bacteria” this reality is explored in some detail. Dissemination of resistance was found as least common through the mechanisms of transformation. This because transformation requires a competent state, and because achieving bacterial competence is predicated upon dissimilar conditions for different bacterial cells. Consequently, the movement of resistance genes by transformation between bacterial types is extremely limited. As a result, integration of resistance genes through transformation is regarded as possible but not prevalent (Dzidic and Bedekovic. 2003).

Transduction of resistance genes is more viable than transformation because bacteriophages provide stability for the resistance genes by engulfing them with their protein coat thereby protecting resistance gene DNA from possible unfavorable extracellular conditions. Additionally, the pervasiveness of bacteriophages within the bacterial ecosystem provides abundant opportunity for resistance genes to be enveloped by a bacteriophage and transferred. However, conjugation is the dominant form of HGT in the environment. Resistance genes conferred into plasmids, a highly versatile and stable mobile genetic element, can be transferred between identical populations of bacteria in addition to being suited for transfers across bacterial genera. This ability of bacteria to transfer resistance genes across species (including across the Gram-negative Gram positive line) has resulted in the presence of almost identical antibiotic resistance genes across phylogenetically diverse bacterial populations (Dzidic and Bedekovic. 2003). HGT has been demonstrated as effective by the steadily increasing resistance to vancomycin based drugs among Enterococcal bacteria. Amongst other resistances, HGT has been directly linked to the emergence of multidrug-resistant Enterobacteriaceae, an extremely widespread, potentially harmful family of Gram-negative bacteria. This type of bacteria is particularly rampant in immunologically susceptible or otherwise homeostatically compromised individuals such as those in intensive care units. Because the effects of HGT are so clearly present in healthcare settings, burdening healthcare providers with both “escalating costs and morbidity”, research involving HGT has been, and continues to be, an area of intense interest (Palmer et al. 2010).   

Enterococcal bacteria and the Horizontal transfer of antibiotic resistance

The National Institute of Health, in an article with joint contributions from Harvard Medical School and the Harvard Microbial Initiative, published a specific review of “Horizontal Gene Transfer and the Genomics of Enterococcal Antibiotic Resistance” (Palmer et al. 2010). Enterococci were predominantly considered harmless, commensalistic, gastrointestinal bacteria. Now, because of the immense number of resistance genes that have become commonplace within the Enterococcal population, they are considered a potentially dangerous pathogen and are frequently found in hospital-acquired infections. Plasmids are the primary movers in the spread of resistance within enterococci and beyond; vancomycin resistance has been transferred from enterococci to methicillin-resistant Staphylococcus aureus (MRSA).     

Those plasmids most highly adapted for efficient and swift dissemination of antibiotic resistance are those which are pheromone responsive. Plasmids of this class appear to be most common among the species E. faecalis. Pheromone-responsive class plasmids promote cell-specific donor-receptor adhesins. These adhesins sense the presence of pheromone-producing cells in close vicinity so that the cells, once activated via the pheromones released, may initiate conjugation (Palmer et al. 2010). Though pheromone specific HGT is generally cell specific, some instances of inter-genus plasmid transfer have been documented. A pheromone similar to cAM373 produced by Streptococcus gordonii elicited a response form E. faecalis in the form of pAM373 (plasmid) mobilization. The result was the transfer of the pAM373 erythromycin resistance encoding plasmid from E. faecalis to S. gordonii in Todd-Hewitt broth prepared by researchers in 1.5% solid agar incubated at 37º C (Vickerman et al. 2010).

To address the question of Enterococcal plasmid’s effect the plasticity of recipient genomes, research performed in 2010 by Manson et al. was referenced extensively by Palmer et al (2010). The key finding of this work was the existence of large post transfer amounts (up to 25%) of highly conserved donor genome sequences from E. faecalis V583 in recipient strain of OG1RF when plasmids, pTEF1 or pTEF2 were present. This result was confirmed by the traceable presence of selectable tetracycline resistance markers that were followed from donor to recipient (Figure. 4).  This result suggests a relationship between the presence of plasmids and the mobilization of genomic elements though the mechanisms of this relationship are yet unknown (Palmer et al. 2010). pAMb1 promotes resistance to macrolide class drugs such as erythromycin, lincosamides, along with streptogramin B and is transmissible among other Graham-positive bacteria. pIP501 bolsters resistance to chloramphenicol, and is transferable to both Graham-positive and Gram-negative bacteria groups. Other plasmids inside this category include pRE25 which provides resistance to 12 unique antibiotics, and pRUM which is attributed with resistance to erythromycin, chloramphenicol, streptomycin, and streptothricin. These genes are found in a broad host range of Graham-positive and Gram-negative bacteria suggesting a flexible mechanism of HGT. Factors that induce Inc 18 plasmid transfer are largely unknown. The transposon Tn1546 is known to reside on many of the Inc 18 plasmids and is an established vancomycin resistance gene. It is also re-established that CRISPR sequences, as examined earlier in Luciano A. Marrafini and Erik J. Sontheimer’s report, play a pivotal role in the ability of genetic material to be transferred (Palmer et al. 2010).

Conclusions

Horizontal gene transfer modifies the genetic complex of an organism during the lifespan of a single generation or across genera. This process disrupts the stability of normal DNA conservation (heredity) producing changes to the chromosome of organisms via homologous recombination, additive integration, or by adding autonomous genetic elements (most often plasmids). These alterations can result from of three distinct pathways; Transformation, transduction, and conjugation. HGT can occur in plant cells, but, debate remains regarding the role of HGT between animal cells. HGT is a process most prevalent among bacteria. The impact of HGT is a subject of particular importance to microbiologists and health professionals struggling to contain a growing epidemic of antibiotic resistance that has been facilitated through HGT. Enterococcal bacteria, though previously considered commensalistic and harmless, have become a growing concern because of their propensity to disseminate antibiotic resistance genes among populations of diverse bacteria. With an emerging understanding of HGT, its mechanisms, applications, and medical ramifications, it is now time to conduct research with the goal of employing relevant knowledge of HGT in combating the spread of antibiotic resistance.

           

Figure 1 DNA is release into the environment and extracellularly stabilized, exposed to bacteria and absorbed, or extracellularly incapacitated. Restricted DNA follows a recombination pathway, while independent genetic elements may re-form into circularized structures. Mutagenic recombination of genetic material is expressed as Negative, neutral, or positive (Thomas and Nielson. 2005)

Figure 2 Percentage of gene sources in the tardigrade H. dujardini genome calculated by HGT index Galaxy tools taxonomy extraction (Boothby et al. 2015).

Figure 3 The volume of conjugation is here shown in terms of colony forming units (CFU) per milliliter allowing for visual comparison of transfer efficacy between wild-type (wt) and muted (mut) plasmids in the presence of CRISPR. The volumetric number of organisms that received the conjugated DNA is represented by the dark grey shaded bars, and the amount of successfully transformed organisms (transconjugants) is shown by the bars shaded white (Marraffini and Sontheimer. 2008).

Figure 4 This bar graph visually represents the prevalence and location of introduced traceable tetracycline resistance markers with the relative position of the selectable marker on the X-axis and the rate of transfer per donor cell on the Y-axis (Palmer et al. 2010).

Annotated Bibliography

Allen DP, Anderson DG, Nester EW, Salm SN. 2016. Bacterial Genetics. In: Nester’s Microbiology: A Human Perspective 8^th ed. New York (NY): McGraw-Hill Education. p 204-31.

The brief introduction to the principles of horizontal gene transfer contained in this text book served as a spring board for researching horizontal gene transfer as a topic in general.

Beaber JW, Hochhut B, Waldor M. 2004. SOS response promotes horizontal dissemination of antibiotic resistance genes [internet]. Nature [cited 2017 Oct 9]; (427):72-74. Available from: https://search.proquest.com/docview/204560221?pq-origsite=gscholar

            Horizontal gene transfer is effected by a variety of factors, this article specifically investigates the role of mobile genetic elements, particularly that of integrating conjugation elements.

Boothby T, Tenlen J, Smith F, Wang J, Patanella K, Nishimura E, Tintori S, Li Q, Jones C, Yandell M, Messina D, Glasscock J, and Goldstein B. 2015

Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade [internet] PNAS [cited 2017 Oct 7];112 (52) 15976-1598. Available from: http://www.pnas.org/content/112/52/15976.short

This research article proposes the possibility of extensive HGT by siting base sequences discovered in the draft genome of tardigrade.

Bock R. 2017. Witnessing Genome Evolution: Experimental Reconstruction of Endosymbiotic and Horizontal Gene Transfer. [internet] Ann Rev of Genetics [cited 2017 Oct 7]: 51:1-22. Available from: http://www.annualreviews.org/doi/abs/10.1146/annurev-genet-120215-035329

This review frames the first event of DNA containing organelles being phagocytized in primary endosymbiosis as the most extreme example of horizontal gene transfer which was pivotal in establishing the far-reaching importance of horizontal gene transfer in the context of evolutionary.

Brooker RJ, Widmaier EP, Graham LE, Stiling PD. 2017. Taxonomy and Systematics. In: Biology 4^th ed. New York (NY): McGraw-Hill Education. p 540-4.

            This brief section of text proposes the existence of horizontal evolutionary movement in organismal phylogeny due to the presence and prevalence of horizontal gene transfer.

Clewell, DB. 2011. Tales of conjugation and sex pheromones: A plasmid and enterococcal odyssey. [Internet] [cited 2017 Dec 5]; Mobile Genetic Elements 1(1), 38–54. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3190283/

            This detailed account of research regarding the role of conjugation sex pheromones was a critical resource in understanding the pheromone dependent conjugation mechanism. 

Croucher NJ, Mostowy R, Wymant C, Turner P, Bentley SD, and Fraser C. 2016. Horizontal DNA Transfer Mechanisms of Bacteria as Weapons of Intragenomic Conflict. [internet]. [cited 2017 Oct 7]. Available from: http://journals.plos.org/plosbiology/article/file?id=10.1371/journal.pbio.1002394&type=printable

This article briefly explains the variety of conditions and mechanisms that are known to facilitate horizontal gene transfer while simultaneously deconstructing any preconceptions that may lead to an over confidence of our scientific understanding of a still relatively novel genetic phenomenon. 

Davies J, and De La Cruz F. 2000. Horizontal gene transfer and the origin of species: lessons from bacteria. [internet]. Trends in Microbiol [cited 2017 Oct 7]; 8(3): 128-133. Available from: http://www.cell.com/trends/microbiology/fulltext/S0966-842X(00)01703-0?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0966842X00017030%3Fshowall%3Dtrue

The western world’s wide spread use of antibiotics is alluded to in this article as an ‘experiment’ which has resulted in the rapid adaptation of huge populations of bacteria.

Dzidic S, Bedekovic V. 2003. Horizontal gene transfer-emerging multidrug resistance in hospital bacteria. [internet] Acta Pharmacol Sin [cited 2017 Oct 7]; (6): 519-526. Available from: http://www.chinaphar.com/article/view/9150/9815

            Focused on the rapid adaptations and prevalence of hospital microorganisms, a general knowledge of the current bacterial and antibiotic landscape of hospital’s present struggles can be gleaned for this article.

Gogarten JP, Townsend JP. 2005. HORIZONTAL GENE TRANSFER, GENOME INNOVATION AND EVOLUTION. [internet] Nat Rev [cited 2017 Oct 7]; (3): 679-687. Available from: https://search.proquest.com/docview/224638021?pq-origsite=gscholar

            It can be inferred from the latter sections of this research article that many horizontally transferred genes are deleterious or neutral, additionally, its authors suggest that accuracy in testing for horizontally transferred genes is sometimes lacking.

Heurer H, and Smalla K*. 2007. Horizontal gene transfer between bacteria. [internet] Environ. Biosafety Res. [cited 2017 Oct 7]; (6):3-13. Available from: https://www.ebr-journal.org/articles/ebr/pdf/2007/01/ebr0633.pdf

            An in depth yet comprehensive article, the principles and mechanisms of horizontal gene transfer are outlined in exceptional clarity by the author’s review.

Kazuharu Arakawa, 2016. No evidence for extensive horizontal gene transfer from the draft genome of a tardigrade [internet] PNAS [cited 2017 Oct 7]; 113 (22) E3057. Available from: http://www.pnas.org/content/113/22/E3057.extract.html?etoc=&utm_source=TrendMD&utm_medium=cpc&utm_campaign=Proc_Natl_Acad_Sci_U_S_A_TrendMD_0

            This article is a specific rebuttal of the research conducted by Boothby et al. (referenced above) regarding the role of HGT in tardigrade evolution.

Lin J, Nishino K. Roberts MC, Tolmasky M, Aminov RI, and Zhang L. 2015. Mechanisms of antibiotic resistance. [internet] Frontiers in Microbiology [cited 2017 Dec 6] 6:34 Available from: http://doi.org/10.3389/fmicb.2015.00034

            This article provides a brief and insightful look at the mechanisms of antibiotic resistance.

Marraffini LA, Sontheimer EJ*. 2008. CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA. [Internet] Sci. [cited 2017 Oct 7]; 322 (5909), 1843-1845. Available from: http://science.sciencemag.org/content/sci/322/5909/1843.full.pdf

            The research of this article is specific to the role of CRISPR (Clustered Regularly Interspaced Palindromic Repeats) loci and their effect on conjugation (the most prevalent form of horizontal gene transfer). 

Palmer KL*, Kos VN*, and Gilmore MS. 2010. Horizontal Gene Transfer and the Genomics of Enterococcal Antibiotic Resistance [internet]. Curr Opin Microbiol [cited 2017 Oct 7]; 13(5): 632-639. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2955785/

            This article explores adaptation of Enterococcal bacteria to resist antiobiotics and their (enterococcal’s) evolution from harmless commensals to harmful hospital infection. 

Salm S, Allen D, Nester E, Anderson D. 2016. Nester's Microbiology: A Human Perspective. 8th edition | 9780073522593 | VitalSource, McGraw-Hill Higher Education, www.vitalsource.com/products/nester-39-s-microbiology-a-human-perspective-sarah-salm-deborah-allen-v9780077730932.

           This text serves as a general overview of microbiology which is helpful in foundational research.

Thomas CM*, Nielsen KM. 2005. Mechanisms Of, And Barriers To, Horizontal Gene Transfer Between Bacteria [internet]. Nat Rev [cited 2017 Oct 7]; 3:711-721. Available from: https://search.proquest.com/docview/224637488?pq-origsite=gscholar_between_bacteria_Nat_Rev_Micro_3_711-721/links/0046352aafc4a1330d000000.pdf

            This article contains key information regarding the mechanistic operations of bacterial horizontal gene transfers and the limitations to their influence and success. 

Vickerman M, Flannagan SE, Jesionowski AM, Brossard KA, Clewell DB, and Sedgley CM. 2010. A Genetic Determinant in Streptococcus gordonii Challis Encodes a Peptide with Activity Similar to That of Enterococcal Sex Pheromone cAM373, Which Facilitates Intergeneric DNA Transfer [internet] Journal of Bacteriology [cited 2017 Oct 7] 192(10): 2535–2545. Available from: http://doi.org/10.1128/JB.01689-09

            This article was mentioned by Palmer et al. and highly pertinent to the subject of intergenic dissemination of antibiotic resistance.