This study aimed to determine if DNA transfer during bacterial conjugation requires extended F pili or can occur through direct cell surface contact. The researchers used a temperature-sensitive F plasmid traD mutant that accumulates stable mating pairs at the nonpermissive temperature. Treatment with SDS removed F pili from these pairs, yet DNA transfer still occurred upon shifting to the permissive temperature, demonstrating that the traD product acts after F pili are no longer required. Electron microscopy showed donor and recipient cells in direct wall-to-wall contact in accumulated mating pairs. Therefore, DNA transfer occurs through direct cell surface contact and does not necessarily require extended F pili.
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This document discusses various mechanisms of horizontal gene transfer in bacteria, including conjugation, transformation, and transduction. It provides details on the genes and proteins involved in these processes, such as the tra genes for conjugation and the com genes for competence in transformation. Examples are given for using these mechanisms to map genes and determine genetic distances. Order of events in phage lifecycles and the difference between lytic and lysogenic cycles are also covered.
Here are the key steps to open the plasmid polylinker using restriction enzymes:
1. Digest the plasmid with EcoRI and HindIII restriction enzymes and their appropriate buffer.
2. This will cut the plasmid at the EcoRI and HindIII sites, linearizing the plasmid and removing a 51 bp fragment from the polylinker region.
3. Run the digested plasmid on an agarose gel to separate the linearized plasmid from the excised 51 bp fragment.
4. Isolate the linearized plasmid from the gel using a gel extraction kit. This prepares the plasmid with overhangs compatible for ligation of the insert.
The restriction digestion opens up the polylinker region, making room for the insert DNA to
This document characterizes a diversity-generating retroelement (DGR) in Legionella pneumophila that confers adaptive advantages by diversifying a target protein. The DGR contains all the core components found in other DGR systems and is capable of transferring DNA sequence from a template repeat to a variable repeat of the target gene ldtA, accompanied by adenine-specific mutagenesis. The target protein LdtA is anchored in the bacterial outer membrane with its variable C-terminal domain exposed on the surface, allowing it to generate massive protein diversity on the bacterial surface. Analysis of related DGRs in other L. pneumophila strains showed they target different carrier proteins but maintain similar variable domains, demonstrating the modularity and adapt
Genetic Analysis and Mapping in Bacteria and Bacteriophages mpattani
This document discusses various mechanisms of genetic transfer in bacteria, including conjugation, transduction, and transformation. Conjugation involves the physical transfer of genetic material between bacteria via cell-to-cell contact and an apparatus called a sex pilus. Transduction is virus-mediated and occurs when bacteriophages inadvertently package and transfer bacterial DNA. Transformation involves the natural uptake and incorporation of extracellular DNA into bacterial cells. These mechanisms allow for horizontal gene transfer and were important for mapping the bacterial chromosome and determining gene order.
Thomas Morgan observed that genes on the same chromosome can be linked during meiosis. He conducted test crosses on Drosophila with traits for eye color and wing type. The results showed that some offspring received combinations of traits that were not expected if the genes assorted independently, demonstrating genetic linkage. Genetic linkage occurs because homologous chromosomes occasionally exchange sections during meiosis through the process of crossover. Crossover results in new combinations of genes that can become separated from their original linked combinations on the chromosome. The frequency of crossover between genes determines how far apart they will be on the genetic map of the chromosome.
Horizontal gene transfer with gene mappingSalman Khan
This document discusses horizontal gene transfer and gene mapping in prokaryotes. It begins by defining horizontal gene transfer as the movement of genetic information between independent organisms, as opposed to vertical gene transfer between parents and offspring. The three main types of horizontal gene transfer in prokaryotes are transformation, transduction, and conjugation. Gene mapping can be performed using these horizontal gene transfer mechanisms to determine the relative locations of genes on the bacterial chromosome.
The genetic material of bacteria is stored in a chromosome and small extrachromosomal elements like plasmids and episomes. Bacteria reproduce asexually through binary fission. Mutations can arise spontaneously and change bacterial traits like colony color and nutrient usage. Bacteria can exchange genetic material through three main mechanisms - transformation, conjugation, and transduction. Transformation involves uptake of naked DNA from the environment. Conjugation requires cell-to-cell contact and occurs through plasmids. Transduction moves bacterial DNA between cells within bacteriophage virions.
Bacterial genetics /certified fixed orthodontic courses by Indian dental acad...Indian dental academy
The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and offering a wide range of dental certified courses in different formats.
Indian dental academy provides dental crown & Bridge,rotary endodontics,fixed orthodontics,
Dental implants courses.for details pls visit www.indiandentalacademy.com ,or call
0091-9248678078
This document discusses various mechanisms of horizontal gene transfer in bacteria, including conjugation, transformation, and transduction. It provides details on the genes and proteins involved in these processes, such as the tra genes for conjugation and the com genes for competence in transformation. Examples are given for using these mechanisms to map genes and determine genetic distances. Order of events in phage lifecycles and the difference between lytic and lysogenic cycles are also covered.
Here are the key steps to open the plasmid polylinker using restriction enzymes:
1. Digest the plasmid with EcoRI and HindIII restriction enzymes and their appropriate buffer.
2. This will cut the plasmid at the EcoRI and HindIII sites, linearizing the plasmid and removing a 51 bp fragment from the polylinker region.
3. Run the digested plasmid on an agarose gel to separate the linearized plasmid from the excised 51 bp fragment.
4. Isolate the linearized plasmid from the gel using a gel extraction kit. This prepares the plasmid with overhangs compatible for ligation of the insert.
The restriction digestion opens up the polylinker region, making room for the insert DNA to
This document characterizes a diversity-generating retroelement (DGR) in Legionella pneumophila that confers adaptive advantages by diversifying a target protein. The DGR contains all the core components found in other DGR systems and is capable of transferring DNA sequence from a template repeat to a variable repeat of the target gene ldtA, accompanied by adenine-specific mutagenesis. The target protein LdtA is anchored in the bacterial outer membrane with its variable C-terminal domain exposed on the surface, allowing it to generate massive protein diversity on the bacterial surface. Analysis of related DGRs in other L. pneumophila strains showed they target different carrier proteins but maintain similar variable domains, demonstrating the modularity and adapt
Genetic Analysis and Mapping in Bacteria and Bacteriophages mpattani
This document discusses various mechanisms of genetic transfer in bacteria, including conjugation, transduction, and transformation. Conjugation involves the physical transfer of genetic material between bacteria via cell-to-cell contact and an apparatus called a sex pilus. Transduction is virus-mediated and occurs when bacteriophages inadvertently package and transfer bacterial DNA. Transformation involves the natural uptake and incorporation of extracellular DNA into bacterial cells. These mechanisms allow for horizontal gene transfer and were important for mapping the bacterial chromosome and determining gene order.
Thomas Morgan observed that genes on the same chromosome can be linked during meiosis. He conducted test crosses on Drosophila with traits for eye color and wing type. The results showed that some offspring received combinations of traits that were not expected if the genes assorted independently, demonstrating genetic linkage. Genetic linkage occurs because homologous chromosomes occasionally exchange sections during meiosis through the process of crossover. Crossover results in new combinations of genes that can become separated from their original linked combinations on the chromosome. The frequency of crossover between genes determines how far apart they will be on the genetic map of the chromosome.
Horizontal gene transfer with gene mappingSalman Khan
This document discusses horizontal gene transfer and gene mapping in prokaryotes. It begins by defining horizontal gene transfer as the movement of genetic information between independent organisms, as opposed to vertical gene transfer between parents and offspring. The three main types of horizontal gene transfer in prokaryotes are transformation, transduction, and conjugation. Gene mapping can be performed using these horizontal gene transfer mechanisms to determine the relative locations of genes on the bacterial chromosome.
The genetic material of bacteria is stored in a chromosome and small extrachromosomal elements like plasmids and episomes. Bacteria reproduce asexually through binary fission. Mutations can arise spontaneously and change bacterial traits like colony color and nutrient usage. Bacteria can exchange genetic material through three main mechanisms - transformation, conjugation, and transduction. Transformation involves uptake of naked DNA from the environment. Conjugation requires cell-to-cell contact and occurs through plasmids. Transduction moves bacterial DNA between cells within bacteriophage virions.
1. The study aimed to investigate why clade A cyanopodoviruses are more virulent than clade B by examining whether they encode a thioredoxin (trx) gene, which increases replication rate.
2. PCR and sequencing were performed on genomic regions of interest from clade A and B cyanopodoviruses. Results showed that clade A viruses TIP28 and TIP33 contained homologs of trx and other genes, while clade B virus TIP41 contained a hypothetical protein gene.
3. The findings support the hypothesis that encoding of trx by clade A but not clade B viruses contributes to their higher virulence, through increasing replication
Vectors are carriers used to transfer genes during cloning. Plasmids are commonly used vectors that contain marker genes to identify successful insertion into a host organism. The DNA cloning process using plasmids involves isolating a gene of interest, cutting it and the plasmid with the same restriction enzyme, ligating the gene into the plasmid, transforming bacteria by adding the recombinant plasmid, and allowing the bacteria to replicate and express the cloned gene. Colonies containing the recombinant plasmid can be identified by their antibiotic resistance.
Genetic transformation is the incorporation of naked DNA from the extracellular environment into bacterial cells. There are two types of transformation: natural transformation which occurs naturally in some bacteria, and artificial transformation which is done through chemical, physical, or enzymatic treatment in the laboratory. The basic procedure of transformation involves isolating naked donor DNA, mixing it with competent recipient bacterial cells, and allowing the donor DNA to enter the recipient cells and recombine with the recipient genome.
This document discusses several key concepts related to gene structure:
1. Genes in eukaryotes contain both coding (exon) and non-coding (intron) sequences. Introns are removed during RNA processing to form mRNA.
2. Benzer performed fine structure analysis of phage T4 genes which demonstrated that genes can undergo intragenic recombination, or crossing over within the gene. This established that genes have an internal structure smaller than previously believed.
3. Split genes were discovered, which have interrupted sequences (introns) between coding sequences (exons). RNA splicing removes introns to form mature mRNA from split genes.
Benzer conducted experiments to study intragenic recombination within the rII gene of bacteriophage T4. He isolated rare recombinants that arose from exchanges between DNA of co-infecting phages. This allowed him to map mutations within the rII gene at high resolution. He determined that rII mutations occurred in two complementation groups, rIIA and rIIB, representing two different genes. Through deletion mapping, Benzer was able to localize many rII mutations to a short region within gene A or B. His work established the concept of the cistron as the smallest genetic unit.
Recombination model and cytological basis of crossing overAlex Harley
This study evaluated the effect of expressing multiple heterologous recombinases on increasing homologous recombination in tobacco plants. The recombinases RecA, RecG, RuvC, Rad51, Rad52 and DMC1 were expressed individually and in combinations in tobacco plants containing a recombination substrate. Expression of DMC1 alone produced the greatest stimulation of homologous recombination, increasing recombination frequency up to 1000-fold. Expression of other recombinases also increased recombination 2 to 380-fold. Increasing homologous recombination could improve the efficiency of gene targeting for plant biotechnology applications using CRISPR/Cas.
- Linkage refers to the tendency of genes located near each other on the same chromosome to be inherited together during meiosis. This is because genes located close together on a chromosome move together to the same pole during cell division.
- There are different types of linkage based on whether crossing over occurs, the genes involved, and the chromosomes. Linkage can be complete or incomplete depending on the presence or absence of crossing over. It can involve dominant or recessive alleles.
- Linkage is detected through test crosses, where deviations from expected Mendelian ratios indicate genes are linked. The strength of linkage depends on distance between genes, with closer genes showing stronger linkage.
Cytogenetic techniques for gene location and transferPratik Satasiya
This document discusses various cytogenetic techniques for gene location and transfer. It describes techniques for locating genes such as using structural and numerical chromosomal aberrations, chromosome banding, and in situ hybridization. Structural aberrations discussed include deficiencies, inversions, and translocations. Numerical aberrations discussed include aneuploids like trisomics, monosomics, and nullisomics. The document also describes techniques for transferring genes between species such as transferring whole genomes, whole chromosomes, chromosome arms, and through various types of interchanges. Specific examples of using these techniques in plants are provided.
Linkage and crossing over involve the exchange and recombination of genetic material between homologous chromosomes. Crossing over occurs during prophase I of meiosis through the breakage and rejoining of non-sister chromatids, producing new combinations of genes. It increases genetic variation and is important for evolution and the development of new species through natural selection. The frequency of crossing over is used to map the linear positions of genes on chromosomes.
1. The document discusses selection experiments in chickens for early growth rate (EGR) and correlated responses, as well as QTL analysis using F2 segregating generations.
2. It describes different types of molecular markers like RFLP, RAPD, and microsatellites that can be used in QTL detection experiments.
3. Experimental designs for QTL detection are discussed, including backcross, test cross, recombinant inbred lines, and grand-daughter designs.
The document summarizes experiments conducted to isolate and analyze a gene called mrfA involved in aerial hyphae development in Monascus ruber. Key findings include:
1. M. ruber mutants with disrupted mrfA showed abnormal hyphae growth compared to the original strain.
2. TAIL-PCR and other techniques were used to isolate the mrfA gene sequence.
3. A knock-out vector was constructed and transformed into M. ruber, resulting in transformants that exhibited autolytic aerial hyphae.
F plasmid is a conjugative plasmid found in Escherichia coli that was the first plasmid discovered. It plays an important role in bacterial reproduction by containing genes that code for the production of sex pili and enzymes required for conjugation. The F plasmid replicates through a rolling circle mechanism and transfers between bacteria via conjugation using an F pilus. During conjugation, the F plasmid unwinds and one strand is transferred to the recipient cell where it is replicated to form a double-stranded circular plasmid, converting the recipient into an F+ cell capable of plasmid transfer.
One of the first plasmids to be used in recombinant genetics was called pBR322. It is approximately 4300 bp in length and has two antibiotic resistance genes: Ap (Ampicillin) and Tc (Tetracycline). Bacteria cells that are successfully transformed with this plasmid are able to grow in the presence of both ampicillin and tetracycline antibiotics
chloroplast being the second semi-autonomous organelle of the plant cell also harbours its genome. the presentation includes various characteristic features of this organelle genome along with its functional pecularities and significance
This document discusses high and low copy number plasmids. It defines plasmids as small, circular DNA molecules that can replicate independently of bacterial chromosomal DNA. Plasmids are classified based on their copy number per cell as either low (1-4 copies), moderate, or high (>100 copies). Examples are given of common plasmids and their typical copy numbers. The advantages of plasmids include being easily isolated, replicated, and having multiple cloning sites, while disadvantages include smaller size limits and replication rate decreasing with larger insert size.
This study developed a new genetic assay to detect transcription errors in vivo using Saccharomyces cerevisiae. The assay uses a mutant form of the Cre recombinase gene with a missense mutation in the active site tyrosine. Rare transcription errors that restore the wild-type tyrosine codon can be detected by Cre-dependent rearrangement of reporter genes. Using this assay, the researchers screened for mutations in the largest subunit of RNA polymerase II, Rpb1, that increase the rate of transcription errors. They identified mutations in three domains of Rpb1 - the trigger loop, bridge helix, and TFIIS binding sites - that lead to higher misincorporation rates or defects in error correction. Biochemical characterization confirmed
pUC vectors are plasmids derived from pBR322 that have a higher copy number of 500-600 copies per cell. They contain an ampicillin resistance gene for selection, as well as the lacZ' gene containing multiple cloning sites. When a gene of interest is inserted, it disrupts the lacZ' gene, allowing for blue-white screening on media containing IPTG and X-gal to identify recombinant colonies that appear white instead of blue. pUC vectors offer advantages over pBR322 such as high copy number and easy selection, though they cannot accommodate inserts larger than 15kb.
This document discusses genetic linkage and crossing over during meiosis. It defines linkage as genes located near each other on the same chromosome tending to be inherited together. Crossing over occurs when non-sister chromatids exchange genetic material, producing new allele combinations and reducing linkage. The arrangement of alleles on homologous chromosomes, whether in coupling or repulsion phase, determines the outcome of crosses between linked genes. Recombination frequency is used to measure linkage and construct genetic maps showing gene order on chromosomes.
Vinay ju 5076 Virulence in plant pathogenic bacteriavinay ju
Mutations and three mechanisms of genetic exchange allow bacteria to gain new genetic material: transformation, conjugation, and transduction. Conjugation involves the transfer of DNA between bacteria via a plasmid. Transduction involves the transfer of DNA between bacteria through bacteriophages. Studies have shown differences in virulence and presence of effector proteins among strains of Pseudomonas syringae isolated in California, and differences in plasmid profiles and copper resistance among strains of Pseudomonas syringae isolated from mango trees.
1. Conjugation is the transfer of genetic material between bacterial cells through direct contact or a bridge. It is a form of horizontal gene transfer.
2. Some plasmids called self-transmissible or conjugative plasmids can transfer themselves between bacteria. They encode proteins for transfer in a process requiring mating pair formation and DNA transfer systems.
3. The mating pair formation system forms a pilus for bacterial attachment and a channel for DNA passage. The DNA transfer system nicks DNA at the origin of transfer and replicates it unidirectionally for transfer to the recipient cell.
1. There are three main types of gene transfer between bacteria: conjugation, transformation, and transduction.
2. Conjugation involves the direct transfer of genetic material between bacterial cells via cell-to-cell contact through a conjugation tube or pilus. Transformation occurs through the uptake of naked DNA from the environment. Transduction is the transfer of DNA from one bacterium to another via bacteriophages.
3. The mechanisms of conjugation, transformation, and transduction are described, including the roles of F factors, Hfr strains, competence factors, and specialized vs. generalized transduction. Key experiments in the discovery of these processes are also summarized.
Genetics is the science of cell characteristics and how they are passed from generation to generation. The main molecules of genetics are nucleic acids, namely DNA and RNA, which store genetic information as sequences of bases. There are three main types of genetic replication in bacteria: transformation, transduction, and conjugation. Conjugation involves the exchange of DNA between bacteria mediated by plasmids, through which genetic factors are transferred from donor to recipient cells. Transduction transfers genetic material between bacteria through temperate bacteriophages. Transformation transfers genetic material between bacteria of the same species through uptake of free DNA from the environment.
1. The study aimed to investigate why clade A cyanopodoviruses are more virulent than clade B by examining whether they encode a thioredoxin (trx) gene, which increases replication rate.
2. PCR and sequencing were performed on genomic regions of interest from clade A and B cyanopodoviruses. Results showed that clade A viruses TIP28 and TIP33 contained homologs of trx and other genes, while clade B virus TIP41 contained a hypothetical protein gene.
3. The findings support the hypothesis that encoding of trx by clade A but not clade B viruses contributes to their higher virulence, through increasing replication
Vectors are carriers used to transfer genes during cloning. Plasmids are commonly used vectors that contain marker genes to identify successful insertion into a host organism. The DNA cloning process using plasmids involves isolating a gene of interest, cutting it and the plasmid with the same restriction enzyme, ligating the gene into the plasmid, transforming bacteria by adding the recombinant plasmid, and allowing the bacteria to replicate and express the cloned gene. Colonies containing the recombinant plasmid can be identified by their antibiotic resistance.
Genetic transformation is the incorporation of naked DNA from the extracellular environment into bacterial cells. There are two types of transformation: natural transformation which occurs naturally in some bacteria, and artificial transformation which is done through chemical, physical, or enzymatic treatment in the laboratory. The basic procedure of transformation involves isolating naked donor DNA, mixing it with competent recipient bacterial cells, and allowing the donor DNA to enter the recipient cells and recombine with the recipient genome.
This document discusses several key concepts related to gene structure:
1. Genes in eukaryotes contain both coding (exon) and non-coding (intron) sequences. Introns are removed during RNA processing to form mRNA.
2. Benzer performed fine structure analysis of phage T4 genes which demonstrated that genes can undergo intragenic recombination, or crossing over within the gene. This established that genes have an internal structure smaller than previously believed.
3. Split genes were discovered, which have interrupted sequences (introns) between coding sequences (exons). RNA splicing removes introns to form mature mRNA from split genes.
Benzer conducted experiments to study intragenic recombination within the rII gene of bacteriophage T4. He isolated rare recombinants that arose from exchanges between DNA of co-infecting phages. This allowed him to map mutations within the rII gene at high resolution. He determined that rII mutations occurred in two complementation groups, rIIA and rIIB, representing two different genes. Through deletion mapping, Benzer was able to localize many rII mutations to a short region within gene A or B. His work established the concept of the cistron as the smallest genetic unit.
Recombination model and cytological basis of crossing overAlex Harley
This study evaluated the effect of expressing multiple heterologous recombinases on increasing homologous recombination in tobacco plants. The recombinases RecA, RecG, RuvC, Rad51, Rad52 and DMC1 were expressed individually and in combinations in tobacco plants containing a recombination substrate. Expression of DMC1 alone produced the greatest stimulation of homologous recombination, increasing recombination frequency up to 1000-fold. Expression of other recombinases also increased recombination 2 to 380-fold. Increasing homologous recombination could improve the efficiency of gene targeting for plant biotechnology applications using CRISPR/Cas.
- Linkage refers to the tendency of genes located near each other on the same chromosome to be inherited together during meiosis. This is because genes located close together on a chromosome move together to the same pole during cell division.
- There are different types of linkage based on whether crossing over occurs, the genes involved, and the chromosomes. Linkage can be complete or incomplete depending on the presence or absence of crossing over. It can involve dominant or recessive alleles.
- Linkage is detected through test crosses, where deviations from expected Mendelian ratios indicate genes are linked. The strength of linkage depends on distance between genes, with closer genes showing stronger linkage.
Cytogenetic techniques for gene location and transferPratik Satasiya
This document discusses various cytogenetic techniques for gene location and transfer. It describes techniques for locating genes such as using structural and numerical chromosomal aberrations, chromosome banding, and in situ hybridization. Structural aberrations discussed include deficiencies, inversions, and translocations. Numerical aberrations discussed include aneuploids like trisomics, monosomics, and nullisomics. The document also describes techniques for transferring genes between species such as transferring whole genomes, whole chromosomes, chromosome arms, and through various types of interchanges. Specific examples of using these techniques in plants are provided.
Linkage and crossing over involve the exchange and recombination of genetic material between homologous chromosomes. Crossing over occurs during prophase I of meiosis through the breakage and rejoining of non-sister chromatids, producing new combinations of genes. It increases genetic variation and is important for evolution and the development of new species through natural selection. The frequency of crossing over is used to map the linear positions of genes on chromosomes.
1. The document discusses selection experiments in chickens for early growth rate (EGR) and correlated responses, as well as QTL analysis using F2 segregating generations.
2. It describes different types of molecular markers like RFLP, RAPD, and microsatellites that can be used in QTL detection experiments.
3. Experimental designs for QTL detection are discussed, including backcross, test cross, recombinant inbred lines, and grand-daughter designs.
The document summarizes experiments conducted to isolate and analyze a gene called mrfA involved in aerial hyphae development in Monascus ruber. Key findings include:
1. M. ruber mutants with disrupted mrfA showed abnormal hyphae growth compared to the original strain.
2. TAIL-PCR and other techniques were used to isolate the mrfA gene sequence.
3. A knock-out vector was constructed and transformed into M. ruber, resulting in transformants that exhibited autolytic aerial hyphae.
F plasmid is a conjugative plasmid found in Escherichia coli that was the first plasmid discovered. It plays an important role in bacterial reproduction by containing genes that code for the production of sex pili and enzymes required for conjugation. The F plasmid replicates through a rolling circle mechanism and transfers between bacteria via conjugation using an F pilus. During conjugation, the F plasmid unwinds and one strand is transferred to the recipient cell where it is replicated to form a double-stranded circular plasmid, converting the recipient into an F+ cell capable of plasmid transfer.
One of the first plasmids to be used in recombinant genetics was called pBR322. It is approximately 4300 bp in length and has two antibiotic resistance genes: Ap (Ampicillin) and Tc (Tetracycline). Bacteria cells that are successfully transformed with this plasmid are able to grow in the presence of both ampicillin and tetracycline antibiotics
chloroplast being the second semi-autonomous organelle of the plant cell also harbours its genome. the presentation includes various characteristic features of this organelle genome along with its functional pecularities and significance
This document discusses high and low copy number plasmids. It defines plasmids as small, circular DNA molecules that can replicate independently of bacterial chromosomal DNA. Plasmids are classified based on their copy number per cell as either low (1-4 copies), moderate, or high (>100 copies). Examples are given of common plasmids and their typical copy numbers. The advantages of plasmids include being easily isolated, replicated, and having multiple cloning sites, while disadvantages include smaller size limits and replication rate decreasing with larger insert size.
This study developed a new genetic assay to detect transcription errors in vivo using Saccharomyces cerevisiae. The assay uses a mutant form of the Cre recombinase gene with a missense mutation in the active site tyrosine. Rare transcription errors that restore the wild-type tyrosine codon can be detected by Cre-dependent rearrangement of reporter genes. Using this assay, the researchers screened for mutations in the largest subunit of RNA polymerase II, Rpb1, that increase the rate of transcription errors. They identified mutations in three domains of Rpb1 - the trigger loop, bridge helix, and TFIIS binding sites - that lead to higher misincorporation rates or defects in error correction. Biochemical characterization confirmed
pUC vectors are plasmids derived from pBR322 that have a higher copy number of 500-600 copies per cell. They contain an ampicillin resistance gene for selection, as well as the lacZ' gene containing multiple cloning sites. When a gene of interest is inserted, it disrupts the lacZ' gene, allowing for blue-white screening on media containing IPTG and X-gal to identify recombinant colonies that appear white instead of blue. pUC vectors offer advantages over pBR322 such as high copy number and easy selection, though they cannot accommodate inserts larger than 15kb.
This document discusses genetic linkage and crossing over during meiosis. It defines linkage as genes located near each other on the same chromosome tending to be inherited together. Crossing over occurs when non-sister chromatids exchange genetic material, producing new allele combinations and reducing linkage. The arrangement of alleles on homologous chromosomes, whether in coupling or repulsion phase, determines the outcome of crosses between linked genes. Recombination frequency is used to measure linkage and construct genetic maps showing gene order on chromosomes.
Vinay ju 5076 Virulence in plant pathogenic bacteriavinay ju
Mutations and three mechanisms of genetic exchange allow bacteria to gain new genetic material: transformation, conjugation, and transduction. Conjugation involves the transfer of DNA between bacteria via a plasmid. Transduction involves the transfer of DNA between bacteria through bacteriophages. Studies have shown differences in virulence and presence of effector proteins among strains of Pseudomonas syringae isolated in California, and differences in plasmid profiles and copper resistance among strains of Pseudomonas syringae isolated from mango trees.
1. Conjugation is the transfer of genetic material between bacterial cells through direct contact or a bridge. It is a form of horizontal gene transfer.
2. Some plasmids called self-transmissible or conjugative plasmids can transfer themselves between bacteria. They encode proteins for transfer in a process requiring mating pair formation and DNA transfer systems.
3. The mating pair formation system forms a pilus for bacterial attachment and a channel for DNA passage. The DNA transfer system nicks DNA at the origin of transfer and replicates it unidirectionally for transfer to the recipient cell.
1. There are three main types of gene transfer between bacteria: conjugation, transformation, and transduction.
2. Conjugation involves the direct transfer of genetic material between bacterial cells via cell-to-cell contact through a conjugation tube or pilus. Transformation occurs through the uptake of naked DNA from the environment. Transduction is the transfer of DNA from one bacterium to another via bacteriophages.
3. The mechanisms of conjugation, transformation, and transduction are described, including the roles of F factors, Hfr strains, competence factors, and specialized vs. generalized transduction. Key experiments in the discovery of these processes are also summarized.
Genetics is the science of cell characteristics and how they are passed from generation to generation. The main molecules of genetics are nucleic acids, namely DNA and RNA, which store genetic information as sequences of bases. There are three main types of genetic replication in bacteria: transformation, transduction, and conjugation. Conjugation involves the exchange of DNA between bacteria mediated by plasmids, through which genetic factors are transferred from donor to recipient cells. Transduction transfers genetic material between bacteria through temperate bacteriophages. Transformation transfers genetic material between bacteria of the same species through uptake of free DNA from the environment.
This document discusses genetic recombination in prokaryotes. It describes three main mechanisms of genetic recombination: transformation, transduction, and conjugation. Conjugation involves the transfer of DNA from one bacterium to another through direct cell-to-cell contact and requires a conjugative plasmid. The document then discusses bacterial conjugation in more detail, covering the history of its discovery, the roles of different plasmids, and the key steps and genes involved in the conjugation process. It also describes three main conjugative processes: F+ conjugation, Hfr conjugation, and resistant plasmid conjugation.
A high copy number screen of the trs23Δ99C mutantGabriel Bendavit
This document summarizes a study investigating the role of the TRAPP I subunit TRS23, a guanine nucleotide exchange factor for the Ypt1/Rab GTPase, in the secretion pathway of yeast. A high copy number screen was performed on a yeast mutant with a C-terminal deletion of TRS23 (trs23Δ99C) to identify suppressors of its thermosensitive phenotype. From screening 36,000 transformed yeast colonies, 37 colonies showed suppression. Sequencing identified the gene CIN5, involved in chromosome instability, as a potential suppressor. Further experiments, such as isolating and transforming CIN5 into the trs23Δ99C mutant, are needed to confirm its role in suppress
This document provides an overview of gene transfer in bacteria through three main methods: conjugation, transformation, and transduction. Conjugation involves the transfer of genetic material between bacteria via cell-to-cell contact through sex pili. Transformation refers to the uptake of naked DNA by competent bacterial cells. Transduction is the transfer of DNA from one bacterium to another via bacteriophage. Each method is described in 1-2 paragraphs detailing its history of discovery and basic mechanisms.
This document provides an overview of gene transfer in bacteria through three main methods: conjugation, transformation, and transduction. Conjugation involves the transfer of genetic material between bacteria via cell-to-cell contact through sex pili. Transformation refers to the uptake of naked DNA by competent bacterial cells. Transduction is the transfer of DNA from one bacterium to another via bacteriophage. Each method is described in 1-2 paragraphs detailing its history of discovery and basic mechanisms.
The study examines DNA double-strand break repair in Drosophila melanogaster mutants lacking both the Pif1 and Pol32 genes. PCR analysis confirms the generation of homozygous pif1 pol32 double mutant fly stocks, unlike in yeast where such double mutants are lethal. The pif1 pol32 double mutants are viable and fertile. Using a P-element excision assay to assess DNA repair, the study finds the double mutants exhibit significant defects in somatic repair of excised P-elements, compared to single mutants and wildtype flies.
Tryptophan scanning mutagenesis was used to identify sites of interaction between the transmembrane domains of connexin32 (Cx32), a gap junction protein. Tryptophan was substituted for residues in all four transmembrane domains of Cx32. Function was then assayed in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, especially TM1 and TM4, indicating tight packing. A region midway through the membrane appeared highly sensitive to substitution. Pore-facing regions were also highly sensitive, while lipid-facing regions were more tolerant. Sensitive sites mapped onto a Cx32 channel model, revealing interactions important for voltage gating and the pore. TM1 of
1. The document summarizes the discovery of bacterial conjugation through experiments by Lederberg and Tatum in 1946. They found that genetic information could be transferred between bacterial cells through direct contact.
2. It describes the F factor, a conjugative plasmid found in E. coli that carries genes for sex pilus formation and plasmid transfer. The F factor allows for conjugation between F+ donor cells and F- recipient cells.
3. Transposable genetic elements like insertion sequences (IS elements) and transposons are also discussed. IS elements contain transposase genes and inverted repeats, while transposons can contain additional genes and be flanked by IS elements or encode their own transposition enzymes.
This document summarizes Richard Rodriguez's final lab report on analyzing the L34P mutation in the gap junction protein Cx31 using site-directed mutagenesis. Key findings include:
1) The L34P mutation is associated with Erythrokeratoderma vairabilis (EKV) skin disorder and causes the disease through a recessive inheritance pattern by preventing formation of normal gap junctions between keratinocytes.
2) Site-directed mutagenesis was used to introduce the L34P mutation into a Cx31 plasmid, which was then transformed into E. coli cells. Over 200 colonies grew on each transformation plate.
3) The mutated Cx31 plasmid will be used
The F plasmid is a bacterial fertility factor that allows for bacterial conjugation. It was discovered by Esther Lederberg through experiments mapping lambda phage in E. coli. The F plasmid contains genes that encode a type IV secretion system for DNA transfer between bacterial cells during direct contact. It has origins of transfer and replication that facilitate this process. The F plasmid serves as a model for bacterial conjugation and was crucial for understanding horizontal gene transfer between bacteria. Fosmids and bacterial artificial chromosomes (BACs) are cloning vectors based on the F plasmid that are important tools for sequencing genomes.
Identification and Characterization of Saccharomyces cerevisiae Cdc6 DNA-bind...gan-navi
This document summarizes a study that identified Saccharomyces cerevisiae Cdc6 as a DNA-binding protein. It was found that purified Cdc6 protein can bind double-stranded DNA with a dissociation constant of about 1 x 10-7 M. The minimal DNA-binding domain was mapped to the N-terminal 47 amino acids of Cdc6. Mutating the 29KRKK region abolished Cdc6's DNA-binding activity and its ability to complement cdc6 mutant cells. The results suggest Cdc6 may initiate DNA replication by interacting with DNA at replication origins through its DNA-binding activity.
This document describes a study that identified amino acid residues in Tn5 transposase that are involved in binding both donor DNA and target DNA during transposition. The researchers generated mutant versions of Tn5 transposase and found that some mutations altered target insertion specificity, while others reduced formation of the synaptic complex (which binds donor DNA). They concluded that some amino acid residues contact both donor DNA and target DNA, defining a "bifunctional DNA binding region" that positions these DNAs sequentially during the transposition process. This provides new insights into the protein-DNA interactions that underlie the cut-and-paste mechanism of Tn5 transposition.
This document discusses three mechanisms of gene transfer in prokaryotes: conjugation, transformation, and transduction. Conjugation involves the transfer of genetic material between bacteria via cell-to-cell contact through sex pili. Transformation refers to the uptake of naked DNA by competent bacterial cells. Transduction is the transfer of DNA from one bacterium to another via bacteriophages. The document provides historical context and methodological details for each of these gene transfer mechanisms.
Kou Molecular and Cellular Biology 2003 3186-3201Jordan Irvin
This document summarizes a study analyzing mutations along the crystallographic dimer interface of the yeast TATA binding protein (TBP). 24 amino acids within the dimer interface were mutated. The mutants showed impaired dimerization, TAF1 binding, and TATA binding in vitro, consistent with structural models. In vivo, the mutants displayed phenotypes including low TBP levels, transcriptional derepression, slow growth, and synthetic toxicity with a TAF1 deletion, correlating with dimer instability. The results provide further support that TBP dimerization is physiologically important for negative gene regulation.
This document summarizes research characterizing the mumps virus nucleocapsid-binding domain (NBD) protein via fluorescence spectroscopy and circular dichroism. Researchers created a variant of the mumps NBD protein called F366W by introducing a tryptophan mutation using site-directed mutagenesis. This allowed stability measurements using fluorescence spectroscopy. The mutated protein was expressed in E. coli and purified. Fluorescence spectroscopy and circular dichroism were used to monitor the protein's tertiary structure as it was subjected to thermal and chemical denaturation conditions. Results from these experiments provided insights into the stability and folding of the mumps NBD protein.
1. The structure of GUN4 from Chlamydomonas reinhardtii was determined to 3.5 Angstrom resolution. GUN4 is involved in chlorophyll biosynthesis and plastid-to-nucleus signaling.
2. The structure consists of six molecules in the asymmetric unit and is predominantly alpha-helical. The structure is similar to previously solved cyanobacterial GUN4 structures, with the main differences being in several variable loops.
3. The conformational dynamics of these loops seen across GUN4 structures provides insight into how GUN4 binds protoporphyrin and magnesium protoporphyrin during chlorophyll biosynthesis.
Genetic material
Anant Mohan Sharma
All cell have the capability to give rise to the cell and
the encoded information in living cell is passed from
one generation to another. The information encoded
material is the genetic material or hereditary material
of the cell.
The genetic material is long sequence of nucleic
acids that contain the genetic instruction. Nucleic
acid are macromolecules in the form of DNA or
RNA.
Experimental evidences
Griffith’s experiment
Avery, MacLeod &McCarty experiment
Hershey & Chase experiment
RNA as genetic material
DNA structure
Z- DNA
V. Sasisekharan RL model
Types of RNA
B-DNA, Z-DNA, A-DNA, stability of dsDNA helix, DNA denaturation, factors affecting Tm ,GC content, ionic strength, DNA as a genetic material, Griffith’s experiment, Hershey-chase experiment
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Este documento describe varios métodos para estimar la transpiración de plantas, incluyendo lisímetros, potómetros e intercambio de gases. Los lisímetros miden la pérdida de peso de una planta en un contenedor sellado, mientras que los potómetros usan un indicador de burbujas de aire para medir el flujo de agua a través de ramas cortadas. Los sistemas de intercambio de gases usan analizadores infrarrojos de gases para medir la ganancia neta de vapor de agua que sale de las hojas.
Este documento describe los conceptos de ósmosis y presión osmótica. Explica cómo la ósmosis afecta a las células al permitir el paso de agua a través de la membrana celular dependiendo de si el medio es hipotónico, isotónico o hipertónico. También define la presión osmótica como la presión necesaria para detener el flujo de agua a través de una membrana semipermeable entre dos soluciones de diferente concentración.
Este documento presenta los siete pasos para elaborar un póster científico de manera efectiva: 1) Planificar, 2) Componer, 3) Elaborar, 4) Revisar, 5) Imprimir, 6) Trasladar, y 7) Presentar. Explica cada paso en detalle, incluyendo aspectos como las preguntas para la planificación, los elementos de diseño como colores y letras, y consejos para la composición, elaboración y presentación del póster. El objetivo es producir un póster atractivo visualmente que comunique de man
La esferocitosis hereditaria es una enfermedad hereditaria causada por deficiencias moleculares en proteínas de la membrana eritrocitaria como la espectrina, ankirina y banda 3. Esto causa fragilidad osmótica de los eritrocitos y anemia hemolítica. Se hereda de forma autosómica dominante y su prevalencia es de 1 en 2000 personas. Los síntomas van desde asintomáticos hasta anemia severa, requiriendo tratamiento con ácido fólico y esplenectomía en casos graves.
El documento describe el medio de cultivo R2A Agar, recomendado para el recuento de microorganismos heterótrofos en aguas tratadas. R2A Agar es un medio de bajo contenido nutricional que estimula el crecimiento de bacterias estresadas y tolerantes al cloro. Proporciona instrucciones sobre su uso, composición, almacenamiento, siembra e interpretación de resultados.
Este documento presenta las recomendaciones conjuntas de la EFLM y COLABIOCLI para la extracción de muestras de sangre venosa. El procedimiento de venopunción tiene gran variabilidad entre centros y países y es responsable de un alto porcentaje de errores preanalíticos. La guía establece recomendaciones detalladas para cada fase del proceso (preextracción, extracción y postextracción) basadas en evidencia y consenso de expertos. Su implementación requiere formación del personal, estandarización de los procedimientos y auditorías
Este documento proporciona directrices para realizar correctamente la prueba de sensibilidad antibiótica conocida como antibiograma de discos. Describe la importancia de usar cepas de control estandar para garantizar resultados confiables y la selección apropiada de antibióticos para probar dependiendo del microorganismo. También cubre aspectos técnicos como el almacenamiento y uso adecuado de los discos antibióticos.
analisis microbiologico de productos farmaceuticos no esterilesIPN
Este documento describe los análisis microbiológicos requeridos para productos farmacéuticos no estériles. Explica las diferentes formas farmacéuticas no estériles y los microorganismos permitidos. Detalla los métodos para realizar recuentos microbianos cuantitativos y cualitativos de organismos mesofílicos aerobios, hongos y levaduras, y la detección de microorganismos específicos. Además, presenta los medios de cultivo y condiciones requeridas para cada microorganismo.
microorganismos mesofílicos aerobios en aliemntosIPN
Este documento describe el recuento de organismos mesofílicos aerobios en alimentos. Explica que estos microorganismos crecen entre 20-37°C en presencia de oxígeno y son indicadores útiles. Detalla los procedimientos para preparar diluciones de muestras de alimentos y realizar el recuento en placa siguiendo las normas oficiales mexicanas. El objetivo es conocer las características de estos microorganismos y su importancia para evaluar el control sanitario de los alimentos.
Chlamydia es un género de bacterias intracelulares obligadas que incluye especies que infectan humanos y animales. Tienen una forma de cuerpo elemental infecciosa y una forma de cuerpo reticulado no infecciosa. Chlamydophila abortus causa abortos en ovejas, cabras y otros rumiantes al infectar la placenta, mientras que Chlamydophila felis causa conjuntivitis y queratitis en gatos. Ambas especies se transmiten principalmente a través de secreciones reproductivas y oculares, respectivamente. Su
separacion de fosfolipidos por cromatografia de capa finaIPN
Este documento describe dos técnicas para analizar lípidos en la yema de huevo. La primera es la cromatografía en capa fina para separar fosfolípidos, mostrando sus ventajas sobre la cromatografía en papel. La segunda es el método de Lieberman-Burchard para determinar la concentración de colesterol, basado en la reacción del colesterol con anhídrido acético y ácido sulfúrico para formar un complejo de color verde cuantificable. Se aplicaron ambos métodos para
Dinámica poblacional de Mammillaria humboldtii una cactácea endémica de México IPN
Esta investigación estudió la dinámica poblacional de la cactácea Mammillaria humboldtii en la Reserva de la Biosfera Barranca de Metztitlán en México. Los resultados mostraron que la población de M. humboldtii está disminuyendo a una tasa del 20% anual debido a la alta mortalidad, bajo reclutamiento de plántulas, germinación deficiente de semillas y dependencia de plantas nodrizas. Además, el saqueo ilegal de adultos también está afectando negativamente a la
Este documento describe Clostridium septicum, una bacteria anaerobia que causa edema maligno y braxy en ganado ovino y bovino. C. septicum es un bacilo gram positivo que forma esporas y produce la toxina alfa, la cual causa daño tisular. El edema maligno se presenta después de inyecciones o heridas e involucra hinchazón, dolor y muerte rápida. El braxy afecta el abomaso de corderos después de comer alimento congelado, presentando depresión, fiebre y muerte. La
This document discusses the genus Clostridium, including C. novyi which causes several diseases in livestock. C. novyi type A causes swollen head syndrome in sheep and goats, characterized by edema around the eyes, face and neck. Left untreated it is lethal. C. novyi type B causes black disease or necrotic hepatitis in sheep, which progresses rapidly and can kill animals within 4-6 hours. It causes necrosis in the liver. Vaccines include toxoids and cultures of C. chauvoei, C. perfringens types C and D, C. septicum, C. novyi, and C. sordelli to help prevent these diseases.
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El documento describe el diagnóstico de pielonefritis infecciosa bovina en una zona entre Puebla y Veracruz en México. El diagnóstico se estableció basado en observaciones clínicas y pruebas de laboratorio que incluyeron el aislamiento e identificación del agente causal, Corynebacterium renale. Las lesiones patológicas incluyeron inflamación, hemorragias y úlceras en la vejiga urinaria.
This presentation was provided by Rebecca Benner, Ph.D., of the American Society of Anesthesiologists, for the second session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session Two: 'Expanding Pathways to Publishing Careers,' was held June 13, 2024.
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Beyond Degrees - Empowering the Workforce in the Context of Skills-First.pptxEduSkills OECD
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Philippine Edukasyong Pantahanan at Pangkabuhayan (EPP) CurriculumMJDuyan
(𝐓𝐋𝐄 𝟏𝟎𝟎) (𝐋𝐞𝐬𝐬𝐨𝐧 𝟏)-𝐏𝐫𝐞𝐥𝐢𝐦𝐬
𝐃𝐢𝐬𝐜𝐮𝐬𝐬 𝐭𝐡𝐞 𝐄𝐏𝐏 𝐂𝐮𝐫𝐫𝐢𝐜𝐮𝐥𝐮𝐦 𝐢𝐧 𝐭𝐡𝐞 𝐏𝐡𝐢𝐥𝐢𝐩𝐩𝐢𝐧𝐞𝐬:
- Understand the goals and objectives of the Edukasyong Pantahanan at Pangkabuhayan (EPP) curriculum, recognizing its importance in fostering practical life skills and values among students. Students will also be able to identify the key components and subjects covered, such as agriculture, home economics, industrial arts, and information and communication technology.
𝐄𝐱𝐩𝐥𝐚𝐢𝐧 𝐭𝐡𝐞 𝐍𝐚𝐭𝐮𝐫𝐞 𝐚𝐧𝐝 𝐒𝐜𝐨𝐩𝐞 𝐨𝐟 𝐚𝐧 𝐄𝐧𝐭𝐫𝐞𝐩𝐫𝐞𝐧𝐞𝐮𝐫:
-Define entrepreneurship, distinguishing it from general business activities by emphasizing its focus on innovation, risk-taking, and value creation. Students will describe the characteristics and traits of successful entrepreneurs, including their roles and responsibilities, and discuss the broader economic and social impacts of entrepreneurial activities on both local and global scales.
LAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UPRAHUL
This Dissertation explores the particular circumstances of Mirzapur, a region located in the
core of India. Mirzapur, with its varied terrains and abundant biodiversity, offers an optimal
environment for investigating the changes in vegetation cover dynamics. Our study utilizes
advanced technologies such as GIS (Geographic Information Systems) and Remote sensing to
analyze the transformations that have taken place over the course of a decade.
The complex relationship between human activities and the environment has been the focus
of extensive research and worry. As the global community grapples with swift urbanization,
population expansion, and economic progress, the effects on natural ecosystems are becoming
more evident. A crucial element of this impact is the alteration of vegetation cover, which plays a
significant role in maintaining the ecological equilibrium of our planet.Land serves as the foundation for all human activities and provides the necessary materials for
these activities. As the most crucial natural resource, its utilization by humans results in different
'Land uses,' which are determined by both human activities and the physical characteristics of the
land.
The utilization of land is impacted by human needs and environmental factors. In countries
like India, rapid population growth and the emphasis on extensive resource exploitation can lead
to significant land degradation, adversely affecting the region's land cover.
Therefore, human intervention has significantly influenced land use patterns over many
centuries, evolving its structure over time and space. In the present era, these changes have
accelerated due to factors such as agriculture and urbanization. Information regarding land use and
cover is essential for various planning and management tasks related to the Earth's surface,
providing crucial environmental data for scientific, resource management, policy purposes, and
diverse human activities.
Accurate understanding of land use and cover is imperative for the development planning
of any area. Consequently, a wide range of professionals, including earth system scientists, land
and water managers, and urban planners, are interested in obtaining data on land use and cover
changes, conversion trends, and other related patterns. The spatial dimensions of land use and
cover support policymakers and scientists in making well-informed decisions, as alterations in
these patterns indicate shifts in economic and social conditions. Monitoring such changes with the
help of Advanced technologies like Remote Sensing and Geographic Information Systems is
crucial for coordinated efforts across different administrative levels. Advanced technologies like
Remote Sensing and Geographic Information Systems
9
Changes in vegetation cover refer to variations in the distribution, composition, and overall
structure of plant communities across different temporal and spatial scales. These changes can
occur natural.
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Making of a Nation.
From the NZ Wars to Liberals,
Richard Seddon, George Grey,
Social Laboratory, New Zealand,
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BÀI TẬP BỔ TRỢ TIẾNG ANH 8 CẢ NĂM - GLOBAL SUCCESS - NĂM HỌC 2023-2024 (CÓ FI...
articulo 3. conjugación articulo
1. Vol. 162, No. 2
DNA Transfer Occurs During a Cell Surface Contact Stage of F Sex
Factor-Mediated Bacterial Conjugation
MITRADAS M. PANICKER AND EDWIN G. MINKLEY, JR.*
Department ofBiological Sciences, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213
Received 17 September 1984/Accepted 4 February 1985
Donor bacteria containing JCFL39, a temperature-sensitive traD mutant of the F sex factor, were used at the
nonpermissive temperature to accumulate stable mating pairs with recipient cells. At this stage in conjugation,
extracellular F pili were removed by treatment with 0.01% sodium dodecyl sulfate. Upon then shifting to the
permissive temperature for JCFL39, transfer of the F plasmid was observed. The mating pairs that were
accumulated with JCFL39 at the nonpermissive temperature were readily observed by electron microscopy in
wall-to-wall contact with the recipient bacteria. These results demonstrate that the traD product, which is
known to be required in transferring DNA to a recipient bacterium, acts after the stage at which extracellular
F pili are required. In addition, we concluded that DNA transfer takes place while donor and recipient cells are
in surface contact and not necessarily through an extended F pilus as envisioned in some models of bacterial
conjugation.
Conjugation is the process whereby DNA is transferred
from a donor to a recipient bacterium by a mechanism that
involves contact between the cells. Most conjugation studies
have been performed with gram-negative bacteria and in
particular have centered on Escherichia coli and its sex
factor, F. A central feature of F-mediated conjugation is the
function of the F pilus, a hairlike extracellular filament that
is produced in one or few copies by an F plasmid-containing
donor bacterium (9). Although there is strong evidence that
the F pilus is essential for the formation of the initial contact
between a donor and a recipient bacterium (4, 7), there is
still a degree of uncertainity as to the role that this organelle
plays in conjugative DNA transfer. The earliest observations
(5, 16) indicated the possibility of direct cell surface contact
between conjugating bacteria, but since these studies pre-
dated the discovery of F pili, no critical experiments were
performed at that time to distinguish between possible roles
for the sex pilus. Brinton's studies on F pili led him to
propose a class of models in which the F pilus is directly
involved in conjugative DNA transfer (6). However, no
direct evidence that demonstrates an association between F
pili and DNA that is being transferred conjugatively has
been reported in the literature. As an alternative, Curtiss
(10) and Marvin and Hohn (18) have suggested that F pili
might function by retracting and thereby drawing the donor
and recipient cell surfaces together, at which point DNA
transfer would occur. This idea is central to the currently
favored model for conjugative transfer by F-like plasmids.
The central features of this model have most recently been
reviewed by Willetts and Skurray (26) and are presented in
Fig. 1.
The model envisions conjugation as proceeding through a
series of ordered stages of cell surface and DNA metabolism
events. Much of the evidence for this model is based upon
the phenotypes of F plasmid mutants that are deficient in
transfer (tra) (26). Mutants in traA,L,E,K,B,V,W,C,U, F,H,
or the first part of traG do not synthesize F pili and are
defective in all stages of conjugation. Mutants in traN and
the second part of traG synthesize F pili and make unstable,
but not stable (shear-resistant), cell surface contacts (17).
*
Corresponding author.
Recipient bacteria which lack the outer membrane ompA
protein are also unable to form stable mating pairs (21).
Mutants in traM,D,I,Z, (and probably tra 1) are piliated and
are able to form stable mating pairs (26); the gene products
have been implicated in donor conjugative DNA synthesis
and transfer (15).
A shortcoming of these genetic studies is that they do not
permit a definitive ordering of the inferred stages of conju-
gation. As noted above, one area of particular concern is the
placement ofthe DNA transfer step at a time when the donor
and recipient cell surfaces are in contact, and at which point
extended F pili are presumably no longer required. Perhaps
the best attempt to demonstrate this point involved treating
mating mixtures with low concentrations of the detergent
sodium dodecyl sulfate (SDS), which depolymerizes F pilus
filaments (24). Achtman et al. (3) found that SDS treatment
did not cause disaggregation of preformed mating pairs that
included an Hfr donor, and that the number of recombinants
continued to increase during further incubation in the pres-
ence of SDS. This result is clearly consistent with the notion
that extended F pili are not essential for DNA transfer, once
cell surface contact is established. However, stable mating
pairs were not isolated as an intermediate, nor was subse-
quent DNA transfer demonstrated. The use of recombinant
formation as the assay for DNA transfer further introduced
a complication not present in an F plasmid mating.
From a genetic point of view, the most powerful method
of analyzing the order of events in a biological pathway is the
one devised by Jarvik and Botstein (13). Their test employs
a pair of conditional mutants, one temperature sensitive and
the other cold sensitive; by appropriate temperature shift
experiments it is possible to determine unambiguously the
relative times at which the corresponding gene functions are
required. To apply this approach to F-mediated bacterial
conjugation, we have employed SDS and an F lac traD(Ts)
mutant as the requisite pair of conditional blocks. As in the
experiments of Achtman et al. (3), SDS was used to elimi-
nate extended F pilus filaments. Analysis of the phenotypes
of the various tra mutants that affect conjugative DNA
metabolism suggests that the traD product plays a direct role
in DNA transfer (15). We have used SDS and the traD(Ts)
mutant to demonstrate that the traD product can act in
584
JOURNAL OF BACTERIOLOGY, May 1985, p. 584-590
0021-9193/85/050584-07$02.00/0
Copyright X 1985, American Society for Microbiology
2. CELL SURFACE CONTACT DURING BACTERIAL CONJUGATION
Disaggregatio Recipient
and Donor SD
tra expression sensitivivt Pilus binding
DNA troD function Pilus
transfer Nal sensitivity retraction
Stable mating pair Unstable mating pair
Stabilization
FIG. 1. Model for conjugative transfer by F-like plasmids. The
figure is modified from Willetts and Skurray (26) and highlights the
involvement of F pili in the cell surface contact portion of the mating
cycle and the requirement of traD function for DNA transfer. F pili
are extremely sensitive to low concentrations of SDS (24), and
extended F pili filaments quickly dissappear from the cell surface of
bacteria treated with the detergent, thereby effectively destroying
donor activity (3). The details of conjugative DNA metabolism have
been considerably simplified in the figure, and interested readers
should consult the review by Willetts and Wilkins (27). For simplic-
ity, the donor bacterium is indicated as rod-shaped, whereas the
recipient is spherical. The F sex factor is indicated as a circle in the
donor bacterium, and chromosomal DNA is not shown for either
bacterium.
conjugation at a stage after that requiring the function of an
extended F pilus. The result further strengthens the sugges-
tion (3, 26) that DNA transfer normally occurs during a stage
in conjugation in which the donor and recipient cell surfaces
are in actual contact.
MATERIALS AND METHODS
Plasmids and strains. The plasmids used were JCFLO (F
lac tra+), JCFL8 [F lac traD8(Am)], and JCFL39 [F lac
traD39(Ts)] from the collection of Achtman et al. (4). These
were used in a JC3272 background (F- lac gal his trp lys Ar
str) to give the donor strains JC3273, JC6129, and JC6140,
respectively (4). The recipient strain used was XK1502 (F-
AlacUJ69 nalA) (19).
Mating efficiency determinations at 32 and 42°C. JC6140,
JC3273, and XK1502 were grown in duplicate at 32 and 42°C
as standing overnight cultures in 10 ml of LB broth in 125-ml
Erlenmeyer flasks. Samples (0.1 ml) of the donor cultures
(JC6140 or JC3273) were subcultured into 2.4 ml of LB broth
and allowed to stand for 2 h at either temperature. Then
0.4-ml samples of these donor cultures were gently mixed
with 0.4 ml of the standing overnight cultures of XK1502
(grown at 32 or 42°C), and the mating mixture was allowed to
stand for 2 hours at 32 or 42°C. At the start of mating,
samples were diluted and plated onto lactose-MacConkey
agar plates containing 100 ,ug of streptomycin sulfate per ml
for donor cell counts. At the end of mating, samples of the
mating mixture were diluted and plated onto lactose-minimal
agar plates containing 20 ,ug of nalidixic acid per ml to
determine the number of transconjugants.
Kinetics of inactivation and reactivation of traD39. To
follow the time course of inactivation of the traD39 product
in terms of ability to transfer, a standing overnight culture of
JC6140 donor bacteria was grown at 32°C, diluted 25-fold
into 10 ml of LB broth in a 125-ml Erlenmeyer flask, and
then shaken gently at 32°C in a water bath. When the optical
density at 550 nm (OD550) of the culture reached 0.4, a 0.4-ml
sample was mixed with 0.4 ml of a standing overnight culture
of XK1502 recipients that had been grown at 32°C, and the
bacteria were allowed to mate for 30 min at 32°C with gentle
agitation. Meanwhile, the remainder of the donor culture
was shifted to 42°C, and a second 0.4-mi sample was
immediately added to 0.4 ml of an XK1502 culture that had
been grown as a standing culture overnight at 42°C; these
were allowed to mate for 30 min at 42°C. At various intervals
after the temperature shift, samples of JC6140 were taken,
and the mating procedure was repeated. Whenever the
OD550 of the donor culture reached 0.8, it was diluted
twofold with prewarmed LB broth to maintain exponential
growth. Matings were stopped after 30 min by vortexing and
chilling on ice. The mixtures were then diluted and plated
onto lactose-minimal agar plates containing nalidixic acid to
assay for transconjugants and onto lactose-MacConkey agar
plates containing streptomycin sulfate for donor counts.
Reactivation of traD39 was followed in a similar manner,
using a culture of JC6140 grown overnight at 42°C and then
shifted to 32°C.
Temperature shift experiments. JC6140 (donor) and
XK1502 (recipient) bacteria were grown as 2.5-ml standing
overnight cultures at 42°C in 18- by 150-mm culture tubes. A
0.1-ml sample of the JC6140 culture was subcultured into 2.5
ml of LB broth in a similar culture tube and incubated for 1
h at 42°C. A 0.25-ml sample of this donor culture was then
added to 0.25 ml of the standing overnight culture of
XK1502, gently mixed, and incubated for 30 min at 42°C
without shaking. At this time, the cultures were diluted
10-fold into LB broth containing SDS at 0.01%, and nalidixic
acid at 20 ,ug/ml was added to some of the mating mixtures.
The cultures were then incubated for an additional 2 h at
either 42 or 32°C. The numbers of transconjugants were
obtained by plating various dilutions onto lactose-minimal
agar plates containing nalidixic acid at 20 ,ug/ml.
Electron microscopy. JC6140 and XK1502 strains were
grown as 2.5-ml standing overnight cultures in LB broth in
18- by 150 mm tubes at 42°C. Each of these was diluted
25-fold into LB broth and grown for 1 h at 42°C. Samples of
the donor, recipient, or an equal mixture of donor and
recipient were incubated for 20 min at 42°C, after which 9
volumes of LB broth containing 0.01% SDS and 1% glutar-
aldehyde (freshly prepared) was added to each tube with
gentle swirling. The cultures were incubated at 42°C for an
additional 20 min and centrifuged for 10 min at 5,100 x g,
and the cell pellets were suspended in 0.5 ml of saline. Cells
were prepared for electron microscopy by spotting a sample
onto a 300-mesh, 0.2% Formvar carbon-coated grid. The
grids were stained with 1% aqueous uranyl acetate and
examined in a Phillips 300 electron microscope at 60 kV.
Two grids per sample were used, and greater than 200 cells
were examined to determine the degree of aggregation.
Western blot analysis. Anti-traD protein (TraDp) immune
serum was raised in New Zealand White female rabbits by
using purified TraDp (manuscript in preparation). Individual
strains for the immunoblot were grown as standing overnight
cultures in LB broth and then diluted 50-fold into LB broth
to obtain exponentially growing cells. Samples of 3 ml were
taken when the OD550 reached 0.6 to 0.8. Cells were then
collected by centrifugation and suspended in SDS-gel sam-
ple buffer and incubated in boiling water for 3 min. Equiva-
lent amounts of material were loaded onto an 11- by 14-cm
585VOL. 162, 1985
3. 586 PANICKER AND MINKLEY
9.5% SDS-polyacrylamide slab gel and electrophoresed as
described previously (19). Western blot analysis was per-
formed by a modification of the procedure of Burnette (8).
The proteins were electrophoretically transferred onto an
11.5- by 14.5-cm sheet of nitrocellulose at 50 mA overnight
in a Hoeffer Transphor apparatus with a transfer buffer of 25
mM Tris-hydrochloride (pH 8.4)-192 mM glycine-20% meth-
anol. The nitrocellulose sheet was washed with distilled
water and then incubated for 1 h with 50 ml of 3% bovine
serum albumin in PBSa (10 g of NaCl, 0.25 g of KCI, 2.71 g
of Na2HPO4. 7H20, and 0.25 g of KH2PO4 per liter of
distilled water with a final pH of 7.6), followed by another
1-h incubation in 1% bovine serum albumin-1% normal goat
serum in PBSa. Immunoglobulin G (IgG)-enriched anti-
TraDp serum was used at a 1:50 dilution in blotting buffer
(see below), and 2 ml of serum per each cm of width of gel
lane was allowed to incubate for 2 h. This was followed by
four washes of 15 min each with blotting buffer. Anti-rabbit
Fc goat antibody conjugated to horseradish peroxidase was
used at a dilution of 1:200 in blotting buffer, and 2 ml per gel
lane was incubated with the paper for 2 h. The nitrocellulose
sheet was then washed three times with blotting buffer for 15
min each followed by three 5-min washes with PBSa.
Peroxidase buffer containing the chromogenic substrate 4-
chloro-1-napthol (see below) was then added onto the nitro-
cellulose. After the bands developed, the paper was rinsed in
distilled water and allowed to dry at room temperature.
Materials. Blotting buffer was 150 mM NaCl-5 mM
EDTA-50 mM Tris-hydrochloride (pH 7.4)-0.25% gel-
atin-0.05% Tween 20. Peroxidase buffer was prepared by
mixing 43.2 ml of distilled water, 10.8 ml of PBSa, and 0.2 ml
of 30% hydrogen peroxide; just before use 6 ml of a 6%
solution of 4-chloro-1-napthol in methanol (freshly prepared)
was added.
Nitrocellulose (BA83) was from Schleicher & Schuell Co.
BSA, fraction V, was from Boehringer Mannheim Biochemi-
cals, Indianapolis, Ind. Normal goal serum and horseradish
peroxidase-conjugated anti-rabbit Fc goat IgG were from
Cappel Laboratories, Cochranville, Pa. Glutaraldehyde was
from Ladd Research Industries, Burlington, Vt. SDS was
from Pierce Chemical Co., Rockford, Ill. 4-Chloro-1-napthol
was from Sigma Chemical Co., St. Louis, Mo.
RESULTS
Further characterization of JCFL39. The plasmid JCFL39
was isolated by Achtman et al. (4) as a nonsuppressible
transfer-defective derivative of a wild-type F lac (JCFL0),
and the mutation defect was mapped in the F traD gene
(traD39) (25). In their standard 40-min mating, JCFL39 had
a transfer efficiency of 10-2 at 42°C and 100 at 32°C, relative
to JCFLO as 100 (4). As a first step in using their mutant, we
determined the transfer efficiency of JCFL39 under the
conditions that would be used in the temperature shift
experiments. The data in Table 1 show that in transfer from
a JC3272 host into an XK1502 recipient, JCFL39 was sixfold
less efficient than JCFLO at 32°C and 7.8 x 104-fold less
efficient than JCFL0 at 42°C. Thus there is a 3.2 x 104-fold
difference in transfer ability by JCFL39 at 32 and 42°C.
To carry out the temperature shift experiments, it was also
necessary to determine the rate at which a JCFL39-
containing donor regains activity when shifted from 42 to
32°C. To increase the sensitivity of this kinetics experiment,
a shortened mating time of 30 min was used. The mating
efficiency of JCFL39 increased exponentially upon shifting
to the permissive temperature, rising approximately 100-fold
TABLE 1. Mating efficiencies of F lac( plasmids at 32 and 42°C
No. of No. of
Donor Temp tcC)donors Nscon- Mating
plasmid introducedTjugants efficiency'introduced obtained"
JCFLO 32 1.5 x 107 1.8 x 107 120
JCFLO 42 3.5 x 107 1.6 x 107 46
JCFL39 32 2.5 x 107 4.8 x 106 19
JCFL39 42 4.2 x 107 2.5 x 102 5.2 x 10-4
JCFLO is F lac tra'. JCFL39 is F lac traD39(Ts).
The recipient was XK1502. Lac' Nal' colonies were scored as transconju-
gants after mating for 2 h at the indicated temperature.
' Efficiency of mating was calculated as number of transconjugants ob-
tained per 100 donors.
in 4 h (Fig. 2A). At this point it had not yet reached the value
for cells grown continuously at 32°C. The rate of initial
increase corresponded to a doubling every 35 min, compared
with the doubling time of 50 to 60 min for growth ofthe cells.
Based on this result, we chose a 2-h mating period for the
temperature shift experiment described below (and the ex-
periment of Table 1).
As a corollary to this experiment, we determined the rate
of inactivation of traD39 activity when a JCFL39 donor was
shifted from 32 to 42°C. The kinetics differed substantially
from those of activation (Fig. 2B). The initial rise in mating
efficiency was reproducible and could be due to conjugative
transfer being inherently more efficient at 42°C. This was
followed by a rapid exponential decline in activity during the
first hour (t412 of 8 to 10 min) and perhaps a slower decline
thereafter. The value reached after 2 to 4 h was in agreement
with the mating efficiency for a JCFL39 donor grown con-
tinuously at 42°C. Thus 95 to 99% of the traD39 product
appears to be rapidly inactivated as a single species upon
temperature shift; there may be a residual active fraction
that is diluted out more slowly.
Stage-specific involvement of traD in bacterial conjugation.
We were now in a position to ask whether the stage in
conjugation at which extended F pili function could be
physically separated from the stage at which conjugative
DNA transfer occurs, by using SDS addition and the tem-
perature-sensitive traD39 allele as the requisite pair of
conditional blocks. To do this, JCFL39-containing donors
and an Lac-, nalidixic acid-resistant recipient, both grown
continuously at 42°C, were mixed and incubated for 30 min
at 42°C. SDS was then added at 0.01% to remove extracel-
lular F pili (3) and prevent the further formation of stable
mating pairs. One portion of the culture was shifted to 32°C,
and another was maintained at 42°C. After 2 h, these
cultures were assayed for Lac' Nalr transconjugants. The
traD product can indeed be reactivated and can function
after the addition of SDS; there was a greater than 100-fold
increase in transconjugants at the permissive temperature
relative to continued incubation at the nonpermissive tem-
perature (Table 2). In a control experiment, SDS was
present throughout the initial 30-min period (Table 2). This
demonstrated the importance of the preincubation step and
confirmed the SDS sensitivity of a stage in conjugation at
which extracellular F pili act in the formation of a stable
mating pair. Nalidixic acid inhibits DNA gyrase (22) and is
known to stop DNA transfer immediately when added to a
mating mixture (12), but will not affect the recipient that is
nalidixic acid resistant. Consistent with the role of traD in
DNA transfer, when nalidixic acid was added at the time of
the temperature shift, an increase in transconjugants was not
observed in the presence of SDS at either 42 or 32°C (Table
J. BACTERIOL.
4. CELL SURFACE CONTACT DURING BACTERIAL CONJUGATION
(A)
0
0
0 50 100 150 200 250
10-1
0
c
0
0
0
0
CL
0
C)c
ot
c
CG
0
F04-
6
z
(B)
0
100 150 200 250
minutes minutes
FIG. 2. (A) Kinetics of reactivation of traD39(Ts) activity in an exponentially growing culture of JC6140 shifted from 42 to 32°C (B)
Kinetics of inactivation of traD39(Ts) activity in an exponentially growing culture of JC6140 donors shifted from 32 to 42°C. Cultures were
grown and transconjugants were assayed as described in the text. The square in panel B is the value obtained for a 30-min mating between
JC6140 and XK1502 grown continuously and mated at 32°C.
2). This indicates that DNA transfer had not yet taken place
during the preincubation at 42°C and must have occurred at
32°C when traD activity was recovered.
The transfer efficiency of JCFL39 in this experiment
(Table 2) was about 4. Although this value cannot be directly
related to either Table 1 or Fig. 2A, it does indicate that a
high percentage of JCFL39 donors that have potentially
regained traD activity at 32°C are, in fact, able to transfer
DNA to a recipient bacterium. This suggested efficient
formation of stable mating pairs during the 42"C preincuba-
tion period and the resistance of these mating pairs to
dissociation by SDS (3). Since SDS might be expected to
decrease the level of nonspecific aggregation of donor and
recipient cells (3), we decided to look in the electron
microscope at the mating bacteria that were accumulated by
the procedure in Table 2.
Electron microscopy of mating pairs. JCFL39-containing
donor bacteria were allowed to form mating aggregates with
TABLE 2. Transfer of JCFL39 in preformed stable mating pairs
at 32 and 42°C
Temp of Temp of No. of
initial Additions at 30 min second Nonof
incubation incubation" transconjugants
(OC) (OC) per ml
42 SDS 42 1.5 x lo,
42 SDS 32 3.9 x 105
42 SDS' 32 4.0 x 102
42 SDS, nalidixic acid 42 9.0 X 102
42 SDS, nalidixic acid 32 9.0 x 102
aAll second incubations were for 2 h.
b
All matings used JC6140 donors and XK1502 recipients and were per-
formed as described in the text. The number of donors introduced was 1.0 x
107 per ml.
'SDS was added to the mating mixture during the initial incubation.
recipient bacteria at the nonpermissive temperature. After
30 min of incubation at 42°C, SDS and glutaraldehyde were
added, and the culture was examined in the electron micro-
scope. Cells prepared in this manner were rarely seen in
contact in the individual donor and recipient cultures,
whereas in the mixed mating population virtually all of the
bacteria were found to be in some sort of aggregate with the
cells in surface contact (Fig. 3). In this experiment, it was
not possible to specifically identify the donor and recipient
bacteria in the mating aggregates, since they are morpholog-
ically similar. Thus the correlation of aggregated cells to
mating bacteria is based upon the observed statistical distri-
bution. The mating aggregates observed in this manner were
similar in appearance to those reported by Achtman et al.
(3). Although negative staining of whole cells readily dem-
onstrated the existence of such pairs, it was not able to
provide additional detail about the precise nature of the
region in wall-to-wall contact. That analysis will require
carefully prepared thin sections of mating pairs, such as
these, to be viewed in the electron microscope. Such an
analysis is in progress elsewhere (M. Durrenberger, M.S.
dissertation, Basel University, 1982; E. Kellenberger, per-
sonal communication).
Analysis of traD protein levels in JCFL39 donors. The
temperature-sensitive phenotype of the traD39 mutation
could result from either temperature-sensitive synthesis or
temperature-sensitive function of the traD product. The
rapid decline in activity detected in the experiment of Fig.
2B is consistent with temperature-sensitive function. How-
ever, the rate of increase in mating efficiency during traD39
activation parallelled cell growth (Fig. 2A) and suggests that
the functional traD39 product may have to be newly synthe-
sized. The level of traD product in F+ cells is very low, and
the protein has previously been observed only by using a
high-copy-number plasmid or A transducing phage carrying
0
o
c
0
0
0
0.
-
c
CS0
CP._.
0
C
cn
c
0
4-
6
z
587VOL. 162, 1985
5. 588 PANICKER AND MINKLEY
the gene in conjunction with a specific labeling protocol (14,
20). Since we now have available rabbit antiserum against
purified traD protein, we were able to determine directly the
level of TraDp in the JCFL39 donor at both the permissive
and nonpermissive temperatures, using Western blotting as a
highly sensitive assay. The results with JCFL39 containing
cells grown exponentially at 32°C and 42°C are shown in Fig.
4, along with F-, F lac tra+, and F lac traD8(Am) control
stains. The results indicated that the levels of TraDp in both
traD+ and traD39 cells were essentially identical at both
temperatures. In fact, there is an increase in the level of
TraDp in both types of cells grown at 42°C. This indicates
that the defect associated with the traD39 allele results from
a loss of function at the nonpermissive temperature, and not
temperature-sensitive synthesis of the protein.
DISCUSSION
These results provide direct evidence on the ordering of
two events in F sex factor-mediated bacterial conjugation.
With SDS addition and an F lac traD(Ts) mutant it was
possible to determine that the SDS-sensitive step in conjuga-
tin (the function of extended F pili) precedes the step at
which traD function is required. Further, we found that the
timing of traD function is coincident with sensitivity to
nalidixic acid, providing additional evidence for the direct
role of the traD product in DNA transfer. Combining these
results with electron microscopy of mating pairs, we con-
75]
50
25
u)
X 75
O.-
2 50
0
' 25
CLA
7
I.
1-
Donors alone
Recipient alone
v-
75- Mating mixture
Si
r) _2 3 4 5 >6
No. of Cells in Aggregate
FIG. 3. Histograms of the state of aggregation of JC6140 donor
bacteria, XK1502 recipient bacteria, or a mixture of the two, after
incubation for 30 mim at 420C. Cells were prepared and observed in
the electron microscope as described in the text. At least 200 cells
were examined to obtain each histogram. The vertical bars repre-
sent the number of cells in each type of aggregate as a percentage of
the total number of cells examined.
(J D c d e f g h
..,Tra Dp
FIG. 4. Western blot analysis of traD protein levels in exponen-
tially growing cells at 32 and 42°C. Anti-TraDp rabbit IgG and
horseradish peroxidase-conjugated sesond antibody were used to
visualize traD protein in an SDS-polyacrylamide slab gel of whole
cell protein (see the text). Lanes: a and b, JC3273 (F lac tra+) at 32
and 42°C, respectively; c and d, JC3272 (F-) at 32 and 42°C,
respectively; e and f, JC6140 [F lac traD39(Ts)] at 32 and 42°C,
respectively; g and h, JC6190 [F lac traD8(Am)] at 32 and 42°C,
respectively. Each lane contained the protein from approximately 3
x 108cells.
clude, as also proposed by others, that the role of F pili in
conjugation is the generation of stable mating pairs that are
in cell surface contact, and that once these are formed there
no longer exists a requirement for an extended F-pilus
filament. DNA transfer then occurs from donor to recipient
bacterium while the cell surfaces are in contact. Although it
is obvious that F pili play the essential role of first establish-
ing the cell surface contact between the mating bacteria, the
extended F pilus cannot be considered necessary in the
subsequent DNA transfer events.
Comparison of these results to earlier experiments high-
lights the advantages inherent in the Jarvik and Botstein
approach to ordering events in a biological pathway (13). To
obtain a high percentage of mating aggregates (70% of all
cells), Achtman et al. (3) used a 20-min incubation period
before adding SDS. However, to obtain reasonably high
levels of recombinant formation, they employed an HfrC
donor and assayed for the lac genes, which are transferred at
7 min (23). The use of a late marker could have provided
more convincing evidence, but the Hfr's gradient of trans-
mission would have reduced the number of recombinants
significantly. Even then, the basis of their experiment is
kinetics, and thus there was not a conclusive argumefnt for a
discrete stage in conjugation where an extended F pilus is
not required. We place greater confidence in the use of the
traD(Ts) mutant, where an intermediate is accumulated
which can be isolated and characterized and then shown to
proceed through the subsequent stages of conjugative trans-
fer.
Previous characterization of traD mutants placed the time
of traD action after the formation of a stable mating pair.
However, demonstration that a traD mutant can form a
stable mating pair does not rule out the possibility that traD
function is actually required at an earlier stage in conjugation
and that the observed stable mating pairs actually represent
an aborted or dead-end stage in conjugative transfer. In fact,
it has been reported that the F lac mutant JCFL60, which
carries a traD missense mutation, makes 2.5 times as many
F pili as does the parent wild-type F lac strain (2). Also, cells
carrying an F lac traD mutant are known to be resistant to
infection by the f2 class of bacteriophage. The phage parti-
cles adsorb to the sides of the F pilus, but no RNA
penetrates the cell (4). These additional traD phenotypes
suggest the validity of questioning whether the timing of
traD function may well be at an earlier stage in conjugation,
J. BACTERIOL.
I
4
6. CELL SURFACE CONTACT DURING BACTERIAL CONJUGATION
such as when an extended F pilus is present. However, as
far as conjugation is concerned, our experiment explicitly
rules out this possibility.
The traD product is an inner membrane protein of molec-
ular weight about 78,000 (14, 20; our unpublished observa-
tions). Since it is a membrane protein, the temperature
response of the traD39(Ts) protein is potentially interesting.
Western blot analysis showed that the protein is not subject
to temperature-sensitive synthesis. In temperature shift ex-
periments, the traD39 mutant protein showed rapid inacti-
vation at the nonpermissive temperature, but extremely slow
recovery of activity when shifted to the permissive temper-
ature. The recovery time of traD39 protein (more than 4 h to
recover maximal activity) is especially remarkable when
compared with the 30-min interval during which an entire
population of newly mated recipients becomes competent as
donor bacteria (unpublished observations). One possibility
for this discrepancy is that the traD39 protein synthesized at
42°C is irreversibly inactivated and that a tight regulation
over the level of tra protein expression results in a very low
rate of synthesis of active traD39 product in an existing
donor bacterium shifted to 32°C. A second possibility is that
inactive traD39 protein is incorporated into a cell structure
(for example, the basal body of the F pilus), and it takes
several generations of growth at 32°C to dilute out the
inactive traD molecules already present at these sites. A
third possibility is that even at the permissive temperature
most of the traD39 product synthesized is inactive; it may
then take many generations to accumulate a level of the
active form of the protein sufficient for conjugative transfer
to occur. This latter suggestion is consistent with the rather
poor transfer efficiency of JCFL39-containing donors at the
permissive temperature.
Experiments currently in progress in this laboratory sug-
gest that a function of the traD protein in conjugation may be
to serve as a membrane anchor for DNA helicase I, the
product of the F traI gene (1). In this way, the energy of ATP
hydrolysis used to unwind the F-plasmid circle and to
translocate the unwinding enzyme with respect to the DNA
being unwound would be directly converted into the motive
force for transport of a strand of the plasmid's DNA into the
recipient bacterium. The fact that we have shown that traD
function is required when the mating bacteria are in cell
surface contact is consistent with the requirements of this
model.
Although these experiments help to clarify certain aspects
of the stages involved in bacterial conjugation, there still
remain a number of steps (outlined in Fig. 1) for which there
is less than definitive evidence. First, there is as yet no
convincing evidence that F pili function via retraction to
bring the mating cells into cell surface contact. Second, little
is known about the nature of the conversion of an unstable
mating pair into a stable mating pair. And third, as pointedly
noted in a recent review, much remains to be learned about
the biochemistry of the tra products involved in conjugative
DNA metabolism (27).
Finally, these experiments make possible ah interesting
comparison of F-mediated conjugation to the conjugative
transfer of plasmids that occurs in gram-positive bacteria,
such as Streptococcus faecalis, where sex pili have not been
found. In S.faecalis, there is evidence that a sex pheromone
induces cell clumping and that plasmid DNA is transferred
while bacteria are in cell surface contact (11). Since we no
longer believe that the F pilus plays a direct role in DNA
transfer, a comparison can be made between the function of
the sex pilus found in gram-negative organisms and the cell
clumping induced by the action of the sex pheromone in
gram-positive bacteria. It is possible that the subsequent
DNA transfer events that occur may be more directly related
to each other.
ACKNOWLEDGMENTS
We thank Bonnie Chojnacki for her assistance with the electron
microscopy experiments and John Bodner for introducing us to the
Western blotting procedure.
This research was supported by Public Health Service grant
GM28925 from the National Institute of General Medical Sciences.
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