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Microbial Genetics
Microbial Genetics: F Factor B Traxler, University of Washington, Seattle, WA, USA
© 2013 Elsevier Inc. All rights reserved.
This article is a revision of the previous edition article by SM Rosenberg and PJ Hastings, volume 2, pp 677–680, © 2001, Elsevier Inc.
Glossary Conjugative pilus An extracellular appendage that mediates the initial contact between a donor and recipient cell during bacterial conjugation. Conjugative plasmid An extrachromosomal DNA element that promotes its DNA transfer from a donor to a recipient cell. Donor A bacterial cell that contains a conjugative plasmid such as the F plasmid. Episome A plasmid that can integrate into and replicate with the chromosome or can replicate
Introduction and Early History The F factor (or F plasmid) has played a critical role in the development of Escherichia coli as a laboratory model organism and has provided general important insights in the genetics, evolution, and physiology of bacteria. This article summarizes certain aspects of the scientific literature surrounding F, with a consideration of how this plasmid continues to contribute to our understanding of bacterial physiology. The first indications of the existence of the F plasmid in laboratory strains of E. coli came from the work Joshua Lederberg and Edward Tatum, starting in the mid-1940s. These investigators showed that mixtures of two E. coli strains with distinct genetic markers could give rise to recombinant progeny, with two or three simultaneously acquired markers. Such recombinants were never identified from either parent alone or from mixtures of living and dead cells. These investi gators recognized that simultaneous transfer of multiple markers was inconsistent with reversion of mutations and that the immediate appearance of the recombinants after the crosses showed that E. coli is a haploid organism. These results differentiated this type of gene exchange from the process of transformation (as described in the Pneumococci) and high lighted a fundamental characteristic of bacteria, in contrast to fungi or other genetic model systems. From this foundation, several other researchers made important observations to the analysis of ‘sexual exchange’ of genetic information among strains of E. coli. Esther Lederberg, Joshua Lederberg, and Luca Cavalli defined this ‘fertility factor’ (F factor) as an extrachromosomal element, or a plasmid, with independent replication and partitioning functions from the chromosome. William Hayes noticed the unidirectional aspect of conjugative DNA transfer from a donor to a recipient bacterium and demonstrated that ‘high frequency of recombination’ (Hfr) donors transfer chromoso mal genes due to the integration of the F plasmid into the E. coli chromosome. Because the F plasmid can replicate independently or can integrate into the chromosome, it is considered an episome.
396
independently from the chromosome. F′ An F plasmid that has acquired a segment of Escherichia coli chromosomal DNA. Mating pair A donor and recipient bacterial cell that are in close stable contact during conjugation. Plasmid An extrachromosomal DNA molecule that carries genes for a variety of functions not essential for cell growth. Recipient A bacterial cell that does not contain a conjugative plasmid.
The capacity of F to integrate at different locations and in either orientation on the chromosome allowed the determina tion that E. coli has a single circular chromosome and the map order of its genes. Researchers determined that different Hfr donors transfer different chromosomal genes at different times in Hfr ‘matings’, showing that there was an defined orientation in the DNA transfer process. For each Hfr, distinct genes were transferred early (and efficiently, usually 5–10 min after mixing donors and recipients) while others were transferred later (and very inefficiently, up to 100 min after mixing). For some time, F was the only known conjugative plasmid. Now researchers generally appreciate that conjugative plasmids are common, and represent a successful strategy for bacteria to exploit varied environmental niches. Plasmids can move through a variety of bacterial hosts, can acquire diverse sets of genes, and can endow upon their host useful traits that provide a fitness advantage. These characteristics reflect aspects of plasmid biology inherent in the F plasmid.
General Features of the F Plasmid F is a 99.2 kb plasmid, with several distinctive genetic loci. While the plasmid backbone contains several genes, conferring a vari ety of traits to F-containing strains, the only features described here include genes specifying replication and partitioning func tions, transposable elements (insertion sequences and a transposon), and conjugative transfer functions (tra genes). All plasmids specify replication functions, which determine the plasmid copy number and host range. F is normally found only in strains of E. coli, although closely related plasmids are found in other enteric bacteria. There are two independent loci that can support the plasmid’s replication. The primary locus is the RepFIA region (containing the plasmid origin of replication, oriV), and deletion derivatives of F that contain only RepFIA have the same copy number (1–2 plasmids/chromosome) and partitioning behavior as the parental F plasmid. The secondary RepFIB region can function in the absence of RepFIA, but is dispensable for the plasmid’s stability under normal conditions.
Brenner’s Encyclopedia of Genetics, 2nd Edition, Volume 4
doi:10.1016/B978-0-12-374984-0.00512-X
Microbial Genetics: F Factor F carries a single copy of Tn1000 (also known as χ δ) and of IS2, and two copies of IS3. One IS3 inserted into the finO gene, inactivating the FinOP transcriptional repressor, which controls tra gene expression. Because of this IS3 insertion, the F con jugation functions are constitutively expressed, which allowed the original detection of the conjugative activity. The other important consequence of the presence of transposable elements on the F plasmid is that these sequences enable the cointegration of F with the E. coli chromosome at shared trans posable element sequences to form Hfrs. The excision of F from the chromosome usually occurs via a precise reversion of the original cointegration event. However, on occasion, the rever sion of the cointegration goes awry and leads to the formation of F′ (F prime) plasmids, which carry chromosomal sequences that were adjacent to the site of plasmid integration in the parental Hfr. Examples of F′ plasmids that have been widely used include different F′lac derivatives, such as F′42 and F′128. The availability of F′ plasmids, carrying different regions of the chromosome, was critical for E. coli genetic analysis in 1960s and 1970s, before the era of recombinant DNA. Approximately one-third of the F plasmid encodes the conjugative transfer proteins, with ~20 genes expressing pro teins with direct roles in the transfer machinery. For F, the tra genes are contained in a single locus, and the majority of these genes are transcribed from a single promoter as part of a large operon. The process of conjugative DNA transfer and the func tion of the genes are summarized below.
Conjugative F Plasmid Transfer A distinctive feature of F factor transfer (and some other con jugative plasmids) is the ability of recipients and F-containing donors to conjugate in liquid medium or on solid surfaces. The stages in F conjugation were originally characterized, based on the analysis of a large collection of transfer-deficient (Tra−) mutants; many stages in this process are thought to be com mon in all conjugation systems, but the early steps may be characteristic for F and F-like transfer systems. Fundamentally, the stages in F conjugation are as follows: Initiation of contact between an F-containing donor and an F plasmid-free recipient cell, mediated by the F conjugative pilus. Donor cells containing the F plasmid elaborate 1–3 thin, flexible conjugative pili, which are hollow helical filaments, composed entirely of processed TraA pilin protein, usually 1–3 μm long and 8.5 nm in diameter. The presence of the F pilus is correlated with sensitivity of the F donor to a variety of bacteriophages that use the pilus as their primary cell-surface receptor (including the single-strand RNA (ssRNA) phages like MS2 and Qβ and filamentous single-strand DNA (ssDNA) phages like M13). While F pili are composed of only a single subunit, a majority of F knockout mutations in different tra genes result in cells without F pili (and are phage resistant). Retraction of the F pilus, bringing the donor and recipient into close contact. The F pilus will cycle through stages of pilus elonga tion and retraction in the absence of recipients, via subunit depolymerization from the base of the pilus. Contact with an F-deficient recipient cell also causes pilus retraction, with sufficient force to bring two cells into close contact. After pilus retraction, a donor and recipient are somewhat loosely
397
associated in an ‘unstable mating pair’. In all, the process of pilus assembly and retraction requires the proteins expressed by 15 different tra genes. Stabilization of the donor and recipient into a ‘stable mating pair’. After the donor and recipient cells are in close contact, the traG and traN gene products act to make shear-resistant stable mating pairs. This stage may result in transduction of a ‘mating signal’, leading to the DNA processing stage of conjugation. Single-stranded cleavage of the F plasmid at the origin of transfer. Processing of the double-strand DNA (dsDNA) plasmid occurs in a site- and strand-specific manner at the origin of transfer (oriT) sequence, found at one end of the tra locus. The relaxase protein, TraI, carries out the endonucleolytic cleavage in conjunction with two additional F proteins, TraM and TraY (which participate in the cleavage reaction in an incompletely understood manner). Unwinding and transport of ssDNA through the F transfer apparatus into the cytoplasm of the recipient. After the processing of oriT, the dsDNA is unwound (probably while transfer occurs) by the bifunctional TraI protein, which also is a DNA helicase. This creates the ssDNA molecule that is transferred with the 5′-end leading into the recipient. F DNA (and that of other plasmids) is transferred to the recipient with the relaxase attached to the ssDNA 5′-end. DNA sequence analysis shows that the conjugative DNA transfer apparatus (of F and other plasmids) is related to a broader set of prokaryotic secretion apparatuses, known as the type IV secretion systems (T4SSs) and that there is likely significant overlap between the proteins involved in pilus assembly/retraction (or pilin secretion) and those directly involved in the DNA export pathway. While the usual mode for F conjugation involves DNA transport in the absence of extended pili (when the cells are in intimate contact as stable mating pairs), ssDNA transport through an extended pilus possibly is a rare event. Based on overlap in the conserved components among the T4SSs and a variety of functional ana lyses (done with F and other conjugative DNA transfer systems), the core DNA transport machinery likely includes the F TraD, TraB, TraV, TraK proteins, and perhaps includes other components (including the TraA pilin). Conjugative DNA transfer likely requires energy in the form of ATP hydrolysis, which could potentially come from TraD (a likely DNA-dependent ATPase). DNA replication is not essential for DNA transfer in either the donor or recipient, so DNA synthesis is unlikely to energize secretion through the membrane. Re-ligation of the cleaved ssDNA and replacement strand DNA synthesis. After complete transfer of the conjugative nucleo protein complex to the recipient cell, the TraI relaxase re-ligates the transferred strand of plasmid DNA. Although replacement strand DNA synthesis is not required in either the donor or recipient cell, DNA replication probably occurs concomitant with conjugative transfer in both cells. After F plasmid conjugation, both cells involved in the transfer contain complete copies of the plasmid and are proficient for subsequent rounds of conjugation. In contrast, the trans fer of DNA during Hfr matings rarely results in transfer of the F plasmid tra sequences, which are transferred last due to the orientation of oriT, so the recipients in Hfr matings rarely are competent donors.
398
Microbial Genetics: F Factor
Concluding Remarks
Further Reading
The discovery of F plasmid-mediated conjugative transfer contributed significantly to the importance of E. coli as a genetic model system. The continuing importance of F-mediated conjugation to the analysis of bacterial physiol ogy has recently been shown by screens for synthetic interactions between known and unknown genes, as demon strated by studies led by the groups of Carol Gross and Andrew Emili. This genetic workhorse will likely continue to allow investigators to attack diverse problems with power ful genetic tools.
Butland G, Babu M, Díaz-Mejía JJ, et al. (2008) eSGA: E. coli synthetic genetic array analysis. Nature Methods 5: 789–795. Firth N, Ippen-Ihler K, and Skurray R (1996) Structure and function of the F factor and mechanism of conjugation. In: Neidhardt FC, Curtiss III R, Ingraham JL, et al. (eds.) Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn., pp. 2377–2401. Washington, DC: ASM Press. Hayes W (1968) The Genetics of Bacteria and Their Viruses. Oxford: Blackwell Scientific Publications. Jacob F and Wollman EL (1961) Sexuality and the Genetics of Bacteria. New York: Academic Press. Lawley TD, Klimke WA, Gubbins MJ, and Frost LS (2003) F factor conjugation is a true type IV secretion system. FEMS Microbiology Letters 224: 1–15. Lawley T, Wilkins BM, and Frost LS (2004) Bacterial conjugation in Gram-negative bacteria. In: Funnell BF and Phillips GJ (eds.) Plasmid Biology, pp. 203–226. Washington, DC: ASM Press. Silverman PM and Clarke MB (2010) New insights into F-pilus structure, dynamics, and function. Integrative Biology 2: 25–31. Typas A, Nichols RJ, Siegele D, et al. (2008) High-throughput, quantitative analyses of genetic interactions in E. coli. Nature Methods 5: 781–787.
See also: BACs (Bacterial Artificial Chromosomes); Bacterial Transformation; Conjugation; Conjugative Transposons and Integrative and Conjugative Elements; Horizontal Gene Transfer; Plasmids; Sex Plasmid; Single-Copy Plasmids.
© 2013 Elsevier Inc. All rights reserved.
This article is a revision of the previous edition article by SM Rosenberg and PJ Hastings, volume 2, pp 677–680, © 2001, Elsevier Inc.
Glossary Conjugative pilus An extracellular appendage that mediates the initial contact between a donor and recipient cell during bacterial conjugation. Conjugative plasmid An extrachromosomal DNA element that promotes its DNA transfer from a donor to a recipient cell. Donor A bacterial cell that contains a conjugative plasmid such as the F plasmid. Episome A plasmid that can integrate into and replicate with the chromosome or can replicate
Introduction and Early History The F factor (or F plasmid) has played a critical role in the development of Escherichia coli as a laboratory model organism and has provided general important insights in the genetics, evolution, and physiology of bacteria. This article summarizes certain aspects of the scientific literature surrounding F, with a consideration of how this plasmid continues to contribute to our understanding of bacterial physiology. The first indications of the existence of the F plasmid in laboratory strains of E. coli came from the work Joshua Lederberg and Edward Tatum, starting in the mid-1940s. These investigators showed that mixtures of two E. coli strains with distinct genetic markers could give rise to recombinant progeny, with two or three simultaneously acquired markers. Such recombinants were never identified from either parent alone or from mixtures of living and dead cells. These investi gators recognized that simultaneous transfer of multiple markers was inconsistent with reversion of mutations and that the immediate appearance of the recombinants after the crosses showed that E. coli is a haploid organism. These results differentiated this type of gene exchange from the process of transformation (as described in the Pneumococci) and high lighted a fundamental characteristic of bacteria, in contrast to fungi or other genetic model systems. From this foundation, several other researchers made important observations to the analysis of ‘sexual exchange’ of genetic information among strains of E. coli. Esther Lederberg, Joshua Lederberg, and Luca Cavalli defined this ‘fertility factor’ (F factor) as an extrachromosomal element, or a plasmid, with independent replication and partitioning functions from the chromosome. William Hayes noticed the unidirectional aspect of conjugative DNA transfer from a donor to a recipient bacterium and demonstrated that ‘high frequency of recombination’ (Hfr) donors transfer chromoso mal genes due to the integration of the F plasmid into the E. coli chromosome. Because the F plasmid can replicate independently or can integrate into the chromosome, it is considered an episome.
396
independently from the chromosome. F′ An F plasmid that has acquired a segment of Escherichia coli chromosomal DNA. Mating pair A donor and recipient bacterial cell that are in close stable contact during conjugation. Plasmid An extrachromosomal DNA molecule that carries genes for a variety of functions not essential for cell growth. Recipient A bacterial cell that does not contain a conjugative plasmid.
The capacity of F to integrate at different locations and in either orientation on the chromosome allowed the determina tion that E. coli has a single circular chromosome and the map order of its genes. Researchers determined that different Hfr donors transfer different chromosomal genes at different times in Hfr ‘matings’, showing that there was an defined orientation in the DNA transfer process. For each Hfr, distinct genes were transferred early (and efficiently, usually 5–10 min after mixing donors and recipients) while others were transferred later (and very inefficiently, up to 100 min after mixing). For some time, F was the only known conjugative plasmid. Now researchers generally appreciate that conjugative plasmids are common, and represent a successful strategy for bacteria to exploit varied environmental niches. Plasmids can move through a variety of bacterial hosts, can acquire diverse sets of genes, and can endow upon their host useful traits that provide a fitness advantage. These characteristics reflect aspects of plasmid biology inherent in the F plasmid.
General Features of the F Plasmid F is a 99.2 kb plasmid, with several distinctive genetic loci. While the plasmid backbone contains several genes, conferring a vari ety of traits to F-containing strains, the only features described here include genes specifying replication and partitioning func tions, transposable elements (insertion sequences and a transposon), and conjugative transfer functions (tra genes). All plasmids specify replication functions, which determine the plasmid copy number and host range. F is normally found only in strains of E. coli, although closely related plasmids are found in other enteric bacteria. There are two independent loci that can support the plasmid’s replication. The primary locus is the RepFIA region (containing the plasmid origin of replication, oriV), and deletion derivatives of F that contain only RepFIA have the same copy number (1–2 plasmids/chromosome) and partitioning behavior as the parental F plasmid. The secondary RepFIB region can function in the absence of RepFIA, but is dispensable for the plasmid’s stability under normal conditions.
Brenner’s Encyclopedia of Genetics, 2nd Edition, Volume 4
doi:10.1016/B978-0-12-374984-0.00512-X
Microbial Genetics: F Factor F carries a single copy of Tn1000 (also known as χ δ) and of IS2, and two copies of IS3. One IS3 inserted into the finO gene, inactivating the FinOP transcriptional repressor, which controls tra gene expression. Because of this IS3 insertion, the F con jugation functions are constitutively expressed, which allowed the original detection of the conjugative activity. The other important consequence of the presence of transposable elements on the F plasmid is that these sequences enable the cointegration of F with the E. coli chromosome at shared trans posable element sequences to form Hfrs. The excision of F from the chromosome usually occurs via a precise reversion of the original cointegration event. However, on occasion, the rever sion of the cointegration goes awry and leads to the formation of F′ (F prime) plasmids, which carry chromosomal sequences that were adjacent to the site of plasmid integration in the parental Hfr. Examples of F′ plasmids that have been widely used include different F′lac derivatives, such as F′42 and F′128. The availability of F′ plasmids, carrying different regions of the chromosome, was critical for E. coli genetic analysis in 1960s and 1970s, before the era of recombinant DNA. Approximately one-third of the F plasmid encodes the conjugative transfer proteins, with ~20 genes expressing pro teins with direct roles in the transfer machinery. For F, the tra genes are contained in a single locus, and the majority of these genes are transcribed from a single promoter as part of a large operon. The process of conjugative DNA transfer and the func tion of the genes are summarized below.
Conjugative F Plasmid Transfer A distinctive feature of F factor transfer (and some other con jugative plasmids) is the ability of recipients and F-containing donors to conjugate in liquid medium or on solid surfaces. The stages in F conjugation were originally characterized, based on the analysis of a large collection of transfer-deficient (Tra−) mutants; many stages in this process are thought to be com mon in all conjugation systems, but the early steps may be characteristic for F and F-like transfer systems. Fundamentally, the stages in F conjugation are as follows: Initiation of contact between an F-containing donor and an F plasmid-free recipient cell, mediated by the F conjugative pilus. Donor cells containing the F plasmid elaborate 1–3 thin, flexible conjugative pili, which are hollow helical filaments, composed entirely of processed TraA pilin protein, usually 1–3 μm long and 8.5 nm in diameter. The presence of the F pilus is correlated with sensitivity of the F donor to a variety of bacteriophages that use the pilus as their primary cell-surface receptor (including the single-strand RNA (ssRNA) phages like MS2 and Qβ and filamentous single-strand DNA (ssDNA) phages like M13). While F pili are composed of only a single subunit, a majority of F knockout mutations in different tra genes result in cells without F pili (and are phage resistant). Retraction of the F pilus, bringing the donor and recipient into close contact. The F pilus will cycle through stages of pilus elonga tion and retraction in the absence of recipients, via subunit depolymerization from the base of the pilus. Contact with an F-deficient recipient cell also causes pilus retraction, with sufficient force to bring two cells into close contact. After pilus retraction, a donor and recipient are somewhat loosely
397
associated in an ‘unstable mating pair’. In all, the process of pilus assembly and retraction requires the proteins expressed by 15 different tra genes. Stabilization of the donor and recipient into a ‘stable mating pair’. After the donor and recipient cells are in close contact, the traG and traN gene products act to make shear-resistant stable mating pairs. This stage may result in transduction of a ‘mating signal’, leading to the DNA processing stage of conjugation. Single-stranded cleavage of the F plasmid at the origin of transfer. Processing of the double-strand DNA (dsDNA) plasmid occurs in a site- and strand-specific manner at the origin of transfer (oriT) sequence, found at one end of the tra locus. The relaxase protein, TraI, carries out the endonucleolytic cleavage in conjunction with two additional F proteins, TraM and TraY (which participate in the cleavage reaction in an incompletely understood manner). Unwinding and transport of ssDNA through the F transfer apparatus into the cytoplasm of the recipient. After the processing of oriT, the dsDNA is unwound (probably while transfer occurs) by the bifunctional TraI protein, which also is a DNA helicase. This creates the ssDNA molecule that is transferred with the 5′-end leading into the recipient. F DNA (and that of other plasmids) is transferred to the recipient with the relaxase attached to the ssDNA 5′-end. DNA sequence analysis shows that the conjugative DNA transfer apparatus (of F and other plasmids) is related to a broader set of prokaryotic secretion apparatuses, known as the type IV secretion systems (T4SSs) and that there is likely significant overlap between the proteins involved in pilus assembly/retraction (or pilin secretion) and those directly involved in the DNA export pathway. While the usual mode for F conjugation involves DNA transport in the absence of extended pili (when the cells are in intimate contact as stable mating pairs), ssDNA transport through an extended pilus possibly is a rare event. Based on overlap in the conserved components among the T4SSs and a variety of functional ana lyses (done with F and other conjugative DNA transfer systems), the core DNA transport machinery likely includes the F TraD, TraB, TraV, TraK proteins, and perhaps includes other components (including the TraA pilin). Conjugative DNA transfer likely requires energy in the form of ATP hydrolysis, which could potentially come from TraD (a likely DNA-dependent ATPase). DNA replication is not essential for DNA transfer in either the donor or recipient, so DNA synthesis is unlikely to energize secretion through the membrane. Re-ligation of the cleaved ssDNA and replacement strand DNA synthesis. After complete transfer of the conjugative nucleo protein complex to the recipient cell, the TraI relaxase re-ligates the transferred strand of plasmid DNA. Although replacement strand DNA synthesis is not required in either the donor or recipient cell, DNA replication probably occurs concomitant with conjugative transfer in both cells. After F plasmid conjugation, both cells involved in the transfer contain complete copies of the plasmid and are proficient for subsequent rounds of conjugation. In contrast, the trans fer of DNA during Hfr matings rarely results in transfer of the F plasmid tra sequences, which are transferred last due to the orientation of oriT, so the recipients in Hfr matings rarely are competent donors.
398
Microbial Genetics: F Factor
Concluding Remarks
Further Reading
The discovery of F plasmid-mediated conjugative transfer contributed significantly to the importance of E. coli as a genetic model system. The continuing importance of F-mediated conjugation to the analysis of bacterial physiol ogy has recently been shown by screens for synthetic interactions between known and unknown genes, as demon strated by studies led by the groups of Carol Gross and Andrew Emili. This genetic workhorse will likely continue to allow investigators to attack diverse problems with power ful genetic tools.
Butland G, Babu M, Díaz-Mejía JJ, et al. (2008) eSGA: E. coli synthetic genetic array analysis. Nature Methods 5: 789–795. Firth N, Ippen-Ihler K, and Skurray R (1996) Structure and function of the F factor and mechanism of conjugation. In: Neidhardt FC, Curtiss III R, Ingraham JL, et al. (eds.) Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn., pp. 2377–2401. Washington, DC: ASM Press. Hayes W (1968) The Genetics of Bacteria and Their Viruses. Oxford: Blackwell Scientific Publications. Jacob F and Wollman EL (1961) Sexuality and the Genetics of Bacteria. New York: Academic Press. Lawley TD, Klimke WA, Gubbins MJ, and Frost LS (2003) F factor conjugation is a true type IV secretion system. FEMS Microbiology Letters 224: 1–15. Lawley T, Wilkins BM, and Frost LS (2004) Bacterial conjugation in Gram-negative bacteria. In: Funnell BF and Phillips GJ (eds.) Plasmid Biology, pp. 203–226. Washington, DC: ASM Press. Silverman PM and Clarke MB (2010) New insights into F-pilus structure, dynamics, and function. Integrative Biology 2: 25–31. Typas A, Nichols RJ, Siegele D, et al. (2008) High-throughput, quantitative analyses of genetic interactions in E. coli. Nature Methods 5: 781–787.
See also: BACs (Bacterial Artificial Chromosomes); Bacterial Transformation; Conjugation; Conjugative Transposons and Integrative and Conjugative Elements; Horizontal Gene Transfer; Plasmids; Sex Plasmid; Single-Copy Plasmids.