Using a common language helps immensely when we try to communicate descriptive information. Please try to learn and use the terminology described here when making notes on features of bacterial colonies.
Bacteria grow tremendously fast when supplied with an abundance of nutrients. Different types of bacteria will produce different-looking colonies, some colonies may be colored, some colonies are circular in shape, and others are irregular. The characteristics of a colony (shape, size, pigmentation, etc.) are termed the colony morphology. Colony morphology is a way scientists can identify bacteria. In fact there is a book called Bergey's Manual of Determinative Bacteriology (commonly termed Bergey's Manual) that describes the majority of bacterial species identified by scientists so far. This manual provides descriptions for the colony morphologies of each bacterial species.
Although bacterial and fungi colonies have many characteristics and some can be rare, there are a few basic elements that you can identify for all colonies:(1)
Please note that 3 additional elements of morphology should be examined only in a supervised laboratory setting: consistency, emulsifiability, and odor.
Refer to the diagram below for illustrated examples of form, elevation, and margin:(2)
What Can Grow on a Nutrient Agar Plate?
- Bacteria: Each distinct circular colony should represent an individual bacterial cell or group that has divided repeatedly. Being kept in one place, the resulting cells have accumulated to form a visible patch. Most bacterial colonies appear white, cream, or yellow in color, and fairly circular in shape.
For example:
 |
| Bacillus subtilis(3) |
 |
| Proteus vulgaris(4) |
 |
| Staphylococcus aures(5) |
 |
| Streptococcus pyogenes(6) |
- Yeasts: Yeast colonies generally look similar to bacterial colonies. Some species, such as Candida, can grow as white patches with a glossy surface.
For example:
 |
| Candida Albicans) is a type of yeast that can grow on the surface of skin(7) |
 |
| Round yeast colonies(8) |
 |
| Pink yeast colonies(9) |
- Molds: Molds are actually fungi, and they often appear whitish grey, with fuzzy edges. They usually turn into a different color, from the center outwards. Two examples of molds are shown below:
 |
| Green Mold (Trichoderma harzianum)(10) |
 |
| Black Mold (Aspergillus nidulaus)(11) |
- Other Fungi: Moss green colonies, a white cloud, or a ring of spores can be attributed to the growth of Aspergillus, which is common in such fungal infections as athlete's foot. Here is an example of what Aspergillus looks like:(12)
 |
Finally, whenever a thorough, visual identification is not possible, examples of additional tests are gram stains (http://www.austincc.edu/microbugz/gram_stain.php), growths on selective media, and enzymatic tests.
Endnotes
(1) "Microbiology 101 Laboratory Manual." Washington State University. http://www.rlc.dcccd.edu/mathsci/reynolds/micro/lab_manual/colony_morph.html, accessed January 14, 2005.
(2) "Microbiology 101 Laboratory Manual." Washington State University. http://www.slic2.wsu.edu:82/hurlbert/micro101/pages/101lab4.html, accessed January 14, 2005.
(3) "Bacterial Colony Morphology." Austin Community College. http://www.austin.cc.tx.us/microbugz/03morphology.html, accessed January 14, 2005.
(4) "Bacterial Colony Morphology." Austin Community College. http://www.austin.cc.tx.us/microbugz/03morphology.html, accessed January 14, 2005.
(5) "Bacterial Colony Morphology." Austin Community College. http://www.austin.cc.tx.us/microbugz/03morphology.html, accessed January 14, 2005.
(6) "Bacterial Colony Morphology." Austin Community College. http://www.austin.cc.tx.us/microbugz/03morphology.html, accessed January 14, 2005.
(7) Silvermedicine. http://www.silvermedicine.org/Candidaalbicans.jpg, accessed January 14, 2005.
(8) Biology at the University of Cincinnati Clermont College. http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Yeast_Plate_Count/07_yeast_0.2mL_plate_P7201181.jpg, accessed January 14, 2005.
(9) Teachers Experiencing Antarctica and the Arctic. http://tea.rice.edu/Images/stoyles/stoyles_pinkJPG.JPG.jpg, accessed January 14, 2005.
(10) The Shroomery. http://www.shroomery.org/images/23418/green5.jpg, accessed January 14, 2005.
(11) The Shroomery. http://www.shroomery.org/images/23418/Aspergillus_nidulaus.jpg, accessed January 14, 2005.
(12) ETH Life International. http://www.ethlife.ethz.ch/images/aspergillus-l.jpg, accessed January 14, 2005.
Cells carrying the F plasmid are designated F+, and those lacking it are F−. The F plasmidgenes, which give the plasmid several important properties: contains approximately 100
Some properties of the fertility (F) factor of E. coli.
).
).
). However, a copy of F always remains behind in the donor cell. The recipient cell becomes converted into F+, because it now contains a circular F genome. The transfer of the F plasmid from F+− is rapid, so the F plasmid can spread like wildfire throughout a population from strainstrain. to F to F+ cells are usually inhibited from making contact with other F+ cells; therefore the F plasmid is not transferred from F+ to F+.
Integration of the F plasmid.
The transfer of E. coli chromosomal markers mediated by F. (a) Occasionally, the independent F factor combines with the E. coli chromosome. (b) When the integrated F transfers to another E. coli cell during conjugation, it carries along any E. coli DNA that is attached, thus transferring host chromosomal markers to a new cell. (c) In a population of F+ cells, a few cells will have F integrated into the chromosome; these few cells can transfer chromosomal markers. Therefore, when a population of F+ cells is mixed with a population of F− cells, a few F− cells will acquire markers from the donor. (d) Occasionally, the integrated F can leave the chromosome and return to the cytoplasm. In rare cases, F can carry host genes with it, incorporating them into the circular F, which is now termed an F′. The F′ can transfer these genes at high efficiency to other cells, because they are part of the F′ genome.
below and 9-5a on the following page. When this integration occurs, F can drive the transfer of the entire host chromosome into the recipient cell, along with its own integrated F DNA (Figure 9-5b).This last process, the associated transfer of F and host genes, has some interesting features. First, in any population of cells containing the F factor, F will integrate into the chromosome only in a small fraction of cells (Figure 9-5c
). These few cells can now transfer chromosomal alleles to a second strain. The transfer is detectable because donor and recipient alleles recombine to produce genetic recombinants that can be identified. Indeed, the observation of recombinants led to the initial discovery of geneconjugation (see Genetics in Process 9-1 on page 277). transfer by It is possible to isolate the rare cells in which the F factor is integrated into the host chromosomestrains derived from these cells. In such strains, every cell donates chromosomal alleles during F transfer, so the frequency of recombinants for these strains is much higher than it is for cells in the original population, where the F factor is not integrated in most cells. Therefore, strains with an integrated F factor are termed high frequency of recombination (Hfr) strains to distinguish them from normal F+strains, which contain only a few rare Hfr cells and thus display only a low frequency of recombination for the strain as a whole. Because they transfer chromosomal markers efficiently, Hfr strains are the ones used for genetic mapping, as we shall see later on. from the bacterial population and to cultivate pure
). This modified F, called F′ (pronounced “F prime”), can now transfer these specific host genes to a recipient (F−) cell in an infectious manner, in the same way that F is spread. Thus, the recipient cell now contains two copies of the same gene—one resident copy on its bacterial chromosome and one copy on the newly transferred cytoplasmic F′ factor.Conjugation allows genes from two different parental cells to come together in the same cell and hence provides an opportunity for recombination to occur. Hence mapping analysis is possible.
Transfer of single-stranded fragment of donor chromosome, and recombination with recipient chromosome. note: double crossovers can occur in any location; those shown are examples.
).
). (Note that, although such integrative exchanges can be considered to be double crossovers in the formal genetic sense, at the DNA level the mechanism is a single integration event in which a long donor segment replaces the equivalent segment in the recipient.) Gene transfer and recombination provide the key to mapping the bacterial chromosome. There are two main methods: mapping by interrupted conjugation, which produces a low-resolution map of large parts of the genome, and mapping by recombinant frequency, which produces a higher-resolution map of a smaller region.
In mapping by interrupted conjugation, the Hfr and F− cells are mixed, and conjugation− cells are sampled to determine which donor alleles have entered. This sampling is accomplished by using a kitchen blender to separate the joined cells, resulting in interrupted conjugation. After separation, the Hfr cells are selectively killed, and the remaining F− cells, the exconjugants, are tested to see which of the donor alleles have entered and stably recombined with the endogenote. The times at which various donor alleles firstexconjugants are calculated. If a donor allele a+ enters the recipient at 5 minutes after union and allele b+ enters at 8 minutes, then the two genes are said to be 3 minutes apart on the chromosome. The map units in this case are minutes. Like the maps based on crossover frequencies, these linkage maps are purely genetic constructions. Although the amount of DNA corresponding to a minute is now known, when the method was first devised this was not the case. proceeds. Then, at fixed times, the F appear in the
Let’s analyze a typical cross in which the order and map position of the genes under study are not known. In this particular cross, the genes by which the parents differ will be azi (resistance or sensitivity to sodium azide), gal (ability or inability to utilize galactose as an energy source), lac (ability or inability to utilize lactose as an energy source), and ton (resistance or sensitivity to bacteriophage T1). A streptomycin-sensitivity allele (strs) in the Hfr and a streptomycin-resistance allele (strr) in the recipient are used to selectively kill the Hfr cells after conjugation. Selective killing is accomplished by adding streptomycin to the mixture of cells after interrupting the conjugation. It is advantageous if such an Hfr “contraselecting” allele enters close to last, because then it will only rarely enter the F−; in other words, it should be close to the integrated F factor. Hence the position of the contraselected gene must have been established in previous experiments. The parents of the cross under consideration here are as follows, where the unmapped genes are written in alphabetical order:
Interrupted-mating conjugation experiments with E. coli. F− cells that are strr are crossed with Hfr cells that are strs. The F− cells have a number of mutations (indicated by the genetic markers azi, ton, lac, and gal) that prevent then from carrying out specific metabolic steps. However, the Hfr cells are capable of carrying out all these steps. At different times after the cells have been mixed, samples are withdrawn, disrupted in a blender to break conjugation− cells to grow and to be tested for their ability to carry out the four metabolic steps. (a) A plot of the frequency of recombinants for each metabolic marker as a function of time after mating. Transfer of the donor allele for each metabolic step depends on how long conjugation is allowed to continue. (b) A schematic view of the transfer of markers over time. (Part a modified from E. L. Wollman, F. Jacob, and W. Hayes, Cold Spring Harbor Symposia on Quantitative Biology 21, 1956, 141.) between cells, and plated on media containing streptomycin. The antibiotic kills the Hfr cells but allows the F
. A linkage map can be constructed for the E. coli chromosome from interrupted-mating studies, by using the time at which the donor alleles first appear after mating. The units of distance are given in minutes; arrowhead at left indicates the direction of transfer of the donor alleles. . The azir gene is the first to be detected, entering at 8 minutes, followed by tonr, lac+, and gal+ in that order. Therefore not only is gene order on the chromosome map established, but map distances in minutes also are obtained, as shown in Figure 9-8.
, that alleles transferred early are found in a high percentage of F− exconjugants, but the late alleles are found in only a small proportion. The reason for this difference is either that transfer spontaneously stops or that the chromosome breaks. However, this result does not affect the time-of-entry calculations.
Circularity of the E. coli chromosome. (a) Through the use of different Hfr strains (H, 1, 2, 3, 312) that have the fertility factor inserted into the chromosome at different points and in different directions, interrupted-mating experiments indicate that the chromosome is circular. The mobilization point (origin) is shown for each strain. (b) The linear order of transfer of markers for each Hfr strain; arrowheads indicate the origin and direction of transfer.
. experiments, in which parental Interrupted-mating experiments provide a rough set of gene locations over the entire map. As we learned, the genes are mapped by time of entry. In such experiments, the exogenote must integrate by a double recombination event, but the mapping method is not based on any measurement of recombinant frequencies. However, to provide a higher-resolution method for measuring the sizes of smaller map distances, recombinant frequencies are used.
Suppose that we undertake an experiment to map three genes—met, arg, and leu—by recombinant frequency. To measure recombination between these genes, we must set up a merozygote that is heterozygous for all three. This can be accomplished if we can establish which gene enters last by an interruptedconjugation analysis. The Hfr allele of the last-entering gene is selected among the F− exconjugants. Then, knowing that we have selected the last gene, we know that the other two must also have been in the merozygote. If we know from interrupted-conjugation experiments that the gene order is met first followed by arg and then leu, the merozygote in a cross of Hfr met+ arg+ leu+ × F− met arg leu must have been as follows:
Mapping by recombination in E. coli. After a cross, selection is made for the leu+ marker, which is donated late. The early markers (arg+ and met+) may or may not be inserted, depending on the site where recombination between the Hfr fragment and the F− chromosome occurs.
. We know that a double crossover must have occurred to integrate leu+: one crossover is at the left of the leu gene, but the other can be in various positions at the right. Hence the genotype that arises from recombination between leu and arg will be leu+ arg− met−; so the percentage of bacteria with this genotype in the leu+ exconjugants will give us our recombinant frequency value for the leu-to-arg interval. The leu+ exconjugants arising from recombination between met and arg will be leu+ arg+ met−. The percentage of bacteria with this leu+ subgenotype will provide the recombinant frequencies and hence the map distances between the genes. frequency of 1 percent. In practice, this calculation is done by measuring the proportion of the total events needed to produce these
). These recombinants would be relatively rare. Let us consider some data from this cross. The percentages of the three main genotypesleu+ exconjugants are: obtained after testing
From these results, we can conclude that the leu–arg distance is 4 map units and that the arg–met distance is 9 map units.
Time-of-entry measurements in interrupted conjugation can generate a broad-scale map of the bacterial chromosome. Recombinant frequencies among exconjugants can be used in fine-scale mapping.
Origin and reintegration of the F′ factor—in this case, F′ lac. (a) F is inserted in an Hfr strainton and lac+ alleles. (b,c) Abnormal “outlooping” and separation of F occurs to include the lac locus, producing the F′-lac+ particle. (d) An F lac+ / lac− partial diploidis produced by the transfer of the F′-lac particle to an F− lac− recipient. (From G. S. Stent and R. Calendar, Molecular Genetics, 2d ed. Copyright © 1978 by W. H. Freeman and Company, New York.) between the
, in some cases, the excision event is not a precise reversal of the original insertion, and a part of the bacterial chromosome is incorporated into the liberated plasmid. Figure 9-11 shows incorporation of a nearby lac gene into the plasmid, but the precise gene incorporated depends on where the F factor had originally integrated in the particular Hfr. Such plasmids carrying bacterial genes are called F′. They are named for the gene that they carry: F′-lac, as in the case illustrated in Figure 9-11, or F′-gal, F′-trp, and so forth. An F′ can be obtained by looking for rapid infectious transfer of a gene that is normally transferred late on the chromosome of the particular Hfr strain used. If an F′ plasmid is transferred upon conjugation with an F− strain, the recipients generated are stable merozygotes, carrying a complete bacterial genome plus a donor fragment on the autonomously replicating plasmid. The process of creating a merozygote by an F′ element is called sexduction or F′-duction. Stable partial diploids are useful in bacterial genetics because they can be used for genetic studies usually possible only in a diploid cell, such as determinationlac+ donor is used to create an F′-lac+ plasmid and this plasmid− recipient that carries the allele lac−, then the partial diploidis heterozygous lac+ / lac−, and these cells can be used to determine which allele is dominant (lac+ of dominance. For example, if a is transferred to an F turns out to be dominant in this case).
SOURCE: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mga&part=A1391
Cells carrying the F plasmid are designated F+, and those lacking it are F−. The F plasmidgenes, which give the plasmid several important properties: contains approximately 100
Some properties of the fertility (F) factor of E. coli.
).
).
). However, a copy of F always remains behind in the donor cell. The recipient cell becomes converted into F+, because it now contains a circular F genome. The transfer of the F plasmid from F+− is rapid, so the F plasmid can spread like wildfire throughout a population from strainstrain. to F to F+ cells are usually inhibited from making contact with other F+ cells; therefore the F plasmid is not transferred from F+ to F+.
Integration of the F plasmid.
The transfer of E. coli chromosomal markers mediated by F. (a) Occasionally, the independent F factor combines with the E. coli chromosome. (b) When the integrated F transfers to another E. coli cell during conjugation, it carries along any E. coli DNA that is attached, thus transferring host chromosomal markers to a new cell. (c) In a population of F+ cells, a few cells will have F integrated into the chromosome; these few cells can transfer chromosomal markers. Therefore, when a population of F+ cells is mixed with a population of F− cells, a few F− cells will acquire markers from the donor. (d) Occasionally, the integrated F can leave the chromosome and return to the cytoplasm. In rare cases, F can carry host genes with it, incorporating them into the circular F, which is now termed an F′. The F′ can transfer these genes at high efficiency to other cells, because they are part of the F′ genome.
below and 9-5a on the following page. When this integration occurs, F can drive the transfer of the entire host chromosome into the recipient cell, along with its own integrated F DNA (Figure 9-5b).This last process, the associated transfer of F and host genes, has some interesting features. First, in any population of cells containing the F factor, F will integrate into the chromosome only in a small fraction of cells (Figure 9-5c
). These few cells can now transfer chromosomal alleles to a second strain. The transfer is detectable because donor and recipient alleles recombine to produce genetic recombinants that can be identified. Indeed, the observation of recombinants led to the initial discovery of geneconjugation (see Genetics in Process 9-1 on page 277). transfer by It is possible to isolate the rare cells in which the F factor is integrated into the host chromosomestrains derived from these cells. In such strains, every cell donates chromosomal alleles during F transfer, so the frequency of recombinants for these strains is much higher than it is for cells in the original population, where the F factor is not integrated in most cells. Therefore, strains with an integrated F factor are termed high frequency of recombination (Hfr) strains to distinguish them from normal F+strains, which contain only a few rare Hfr cells and thus display only a low frequency of recombination for the strain as a whole. Because they transfer chromosomal markers efficiently, Hfr strains are the ones used for genetic mapping, as we shall see later on. from the bacterial population and to cultivate pure
). This modified F, called F′ (pronounced “F prime”), can now transfer these specific host genes to a recipient (F−) cell in an infectious manner, in the same way that F is spread. Thus, the recipient cell now contains two copies of the same gene—one resident copy on its bacterial chromosome and one copy on the newly transferred cytoplasmic F′ factor.Conjugation allows genes from two different parental cells to come together in the same cell and hence provides an opportunity for recombination to occur. Hence mapping analysis is possible.
Transfer of single-stranded fragment of donor chromosome, and recombination with recipient chromosome. note: double crossovers can occur in any location; those shown are examples.
).
). (Note that, although such integrative exchanges can be considered to be double crossovers in the formal genetic sense, at the DNA level the mechanism is a single integration event in which a long donor segment replaces the equivalent segment in the recipient.) Gene transfer and recombination provide the key to mapping the bacterial chromosome. There are two main methods: mapping by interrupted conjugation, which produces a low-resolution map of large parts of the genome, and mapping by recombinant frequency, which produces a higher-resolution map of a smaller region.
In mapping by interrupted conjugation, the Hfr and F− cells are mixed, and conjugation− cells are sampled to determine which donor alleles have entered. This sampling is accomplished by using a kitchen blender to separate the joined cells, resulting in interrupted conjugation. After separation, the Hfr cells are selectively killed, and the remaining F− cells, the exconjugants, are tested to see which of the donor alleles have entered and stably recombined with the endogenote. The times at which various donor alleles firstexconjugants are calculated. If a donor allele a+ enters the recipient at 5 minutes after union and allele b+ enters at 8 minutes, then the two genes are said to be 3 minutes apart on the chromosome. The map units in this case are minutes. Like the maps based on crossover frequencies, these linkage maps are purely genetic constructions. Although the amount of DNA corresponding to a minute is now known, when the method was first devised this was not the case. proceeds. Then, at fixed times, the F appear in the
Let’s analyze a typical cross in which the order and map position of the genes under study are not known. In this particular cross, the genes by which the parents differ will be azi (resistance or sensitivity to sodium azide), gal (ability or inability to utilize galactose as an energy source), lac (ability or inability to utilize lactose as an energy source), and ton (resistance or sensitivity to bacteriophage T1). A streptomycin-sensitivity allele (strs) in the Hfr and a streptomycin-resistance allele (strr) in the recipient are used to selectively kill the Hfr cells after conjugation. Selective killing is accomplished by adding streptomycin to the mixture of cells after interrupting the conjugation. It is advantageous if such an Hfr “contraselecting” allele enters close to last, because then it will only rarely enter the F−; in other words, it should be close to the integrated F factor. Hence the position of the contraselected gene must have been established in previous experiments. The parents of the cross under consideration here are as follows, where the unmapped genes are written in alphabetical order:
Interrupted-mating conjugation experiments with E. coli. F− cells that are strr are crossed with Hfr cells that are strs. The F− cells have a number of mutations (indicated by the genetic markers azi, ton, lac, and gal) that prevent then from carrying out specific metabolic steps. However, the Hfr cells are capable of carrying out all these steps. At different times after the cells have been mixed, samples are withdrawn, disrupted in a blender to break conjugation− cells to grow and to be tested for their ability to carry out the four metabolic steps. (a) A plot of the frequency of recombinants for each metabolic marker as a function of time after mating. Transfer of the donor allele for each metabolic step depends on how long conjugation is allowed to continue. (b) A schematic view of the transfer of markers over time. (Part a modified from E. L. Wollman, F. Jacob, and W. Hayes, Cold Spring Harbor Symposia on Quantitative Biology 21, 1956, 141.) between cells, and plated on media containing streptomycin. The antibiotic kills the Hfr cells but allows the F
. A linkage map can be constructed for the E. coli chromosome from interrupted-mating studies, by using the time at which the donor alleles first appear after mating. The units of distance are given in minutes; arrowhead at left indicates the direction of transfer of the donor alleles. . The azir gene is the first to be detected, entering at 8 minutes, followed by tonr, lac+, and gal+ in that order. Therefore not only is gene order on the chromosome map established, but map distances in minutes also are obtained, as shown in Figure 9-8.
, that alleles transferred early are found in a high percentage of F− exconjugants, but the late alleles are found in only a small proportion. The reason for this difference is either that transfer spontaneously stops or that the chromosome breaks. However, this result does not affect the time-of-entry calculations.
Circularity of the E. coli chromosome. (a) Through the use of different Hfr strains (H, 1, 2, 3, 312) that have the fertility factor inserted into the chromosome at different points and in different directions, interrupted-mating experiments indicate that the chromosome is circular. The mobilization point (origin) is shown for each strain. (b) The linear order of transfer of markers for each Hfr strain; arrowheads indicate the origin and direction of transfer.
. experiments, in which parental Interrupted-mating experiments provide a rough set of gene locations over the entire map. As we learned, the genes are mapped by time of entry. In such experiments, the exogenote must integrate by a double recombination event, but the mapping method is not based on any measurement of recombinant frequencies. However, to provide a higher-resolution method for measuring the sizes of smaller map distances, recombinant frequencies are used.
Suppose that we undertake an experiment to map three genes—met, arg, and leu—by recombinant frequency. To measure recombination between these genes, we must set up a merozygote that is heterozygous for all three. This can be accomplished if we can establish which gene enters last by an interruptedconjugation analysis. The Hfr allele of the last-entering gene is selected among the F− exconjugants. Then, knowing that we have selected the last gene, we know that the other two must also have been in the merozygote. If we know from interrupted-conjugation experiments that the gene order is met first followed by arg and then leu, the merozygote in a cross of Hfr met+ arg+ leu+ × F− met arg leu must have been as follows:
Mapping by recombination in E. coli. After a cross, selection is made for the leu+ marker, which is donated late. The early markers (arg+ and met+) may or may not be inserted, depending on the site where recombination between the Hfr fragment and the F− chromosome occurs.
. We know that a double crossover must have occurred to integrate leu+: one crossover is at the left of the leu gene, but the other can be in various positions at the right. Hence the genotype that arises from recombination between leu and arg will be leu+ arg− met−; so the percentage of bacteria with this genotype in the leu+ exconjugants will give us our recombinant frequency value for the leu-to-arg interval. The leu+ exconjugants arising from recombination between met and arg will be leu+ arg+ met−. The percentage of bacteria with this leu+ subgenotype will provide the recombinant frequencies and hence the map distances between the genes. frequency of 1 percent. In practice, this calculation is done by measuring the proportion of the total events needed to produce these
). These recombinants would be relatively rare. Let us consider some data from this cross. The percentages of the three main genotypesleu+ exconjugants are: obtained after testing
From these results, we can conclude that the leu–arg distance is 4 map units and that the arg–met distance is 9 map units.
Time-of-entry measurements in interrupted conjugation can generate a broad-scale map of the bacterial chromosome. Recombinant frequencies among exconjugants can be used in fine-scale mapping.
Origin and reintegration of the F′ factor—in this case, F′ lac. (a) F is inserted in an Hfr strainton and lac+ alleles. (b,c) Abnormal “outlooping” and separation of F occurs to include the lac locus, producing the F′-lac+ particle. (d) An F lac+ / lac− partial diploidis produced by the transfer of the F′-lac particle to an F− lac− recipient. (From G. S. Stent and R. Calendar, Molecular Genetics, 2d ed. Copyright © 1978 by W. H. Freeman and Company, New York.) between the
, in some cases, the excision event is not a precise reversal of the original insertion, and a part of the bacterial chromosome is incorporated into the liberated plasmid. Figure 9-11 shows incorporation of a nearby lac gene into the plasmid, but the precise gene incorporated depends on where the F factor had originally integrated in the particular Hfr. Such plasmids carrying bacterial genes are called F′. They are named for the gene that they carry: F′-lac, as in the case illustrated in Figure 9-11, or F′-gal, F′-trp, and so forth. An F′ can be obtained by looking for rapid infectious transfer of a gene that is normally transferred late on the chromosome of the particular Hfr strain used. If an F′ plasmid is transferred upon conjugation with an F− strain, the recipients generated are stable merozygotes, carrying a complete bacterial genome plus a donor fragment on the autonomously replicating plasmid. The process of creating a merozygote by an F′ element is called sexduction or F′-duction. Stable partial diploids are useful in bacterial genetics because they can be used for genetic studies usually possible only in a diploid cell, such as determinationlac+ donor is used to create an F′-lac+ plasmid and this plasmid− recipient that carries the allele lac−, then the partial diploidis heterozygous lac+ / lac−, and these cells can be used to determine which allele is dominant (lac+ of dominance. For example, if a is transferred to an F turns out to be dominant in this case).
SOURCE: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mga&part=A1391
This exercise will take up the lab period for 3 weeks. In the first week you will count the number of bacteria in a culture of E. coli by serial dilution and plating. This experiment will familiarize you with the basic microbiological procedures you will need for the actual conjugation experiment to be done in the second period. The third period will be devoted to counting the plates resulting from the conjugation experiment.
In the main experiment you will map 3 genes in E. coli by determining the time that each gene enters the recipient during conjugation. The genes are his, pro, and trp; they are biosynthetic genes for histidine, proline, and tryptophan. The Hfr (donor) is his+ pro+ trp+ and the F- (recipient) is his- pro- trp-.
It is also necessary to select against the Hfr itself (so only the F- exconjugants grow). To do this, streptomycin is used to kill the Hfr's. Thus, the F- is str-R (resistant to streptomycin) and the Hfr is str-S (streptomycin sensitive).
In the original work, conjugation was stopped by putting the bacteria in a Waring blender. This isn't so practical for us, so we are using an alternative system, where naladixic acid stops the conjugation by killing the Hfr. The Hfr is thus nal-S and the F- is nal-R (otherwise, the naladixic acid would kill the recipient).
The actual cross can be written:
Hfr: his+ pro+ trp+ str-S nal-S x F-: his- pro- trp- str-R nal-R
To count the number of bacteria containing the various markers, the bacteria are plated onto selective media. The plates contain minimal medium plus two of the three required nutrients. For example, to count the number of his+ bacteria (ignoring the other two markers), plates containing proline and tryptophan are used. Since no histidine is present, his+ bacteria will grow but his- bacteria won't. Since tryptophan and proline are in the medium, both trp+ and trp- bacteria, and pro+ and pro- bacteria will grow. These plates will only determine the number of his+ bacteria.
There are 3 types of selective media used. The plates can be distinguished by colored stripes on the side.
1. proline/ tryptophan plates for counting his+
2. proline/ histidine plates for counting trp+
3. histidine/ tryptophan plates for counting pro+
In addition, a fourth type of plate containing minimal medium only is used as a control. Nothing should grow on these plates.
All plates also contain streptomycin and naladixic acid to kill the Hfr's and stop conjugation, as discussed above.
Conjugation takes place over a period of time (100 minutes under good conditions), with different markers entering the F- at different times. Thus, this experiment involves mixing the Hfr with the F-, then stopping conjugation at various time points and plating the bacteria on selective media.
TITERING A CULTURE
The first week's experiment is designed to familiarize you with basic microbiological lab procedures. You will be given an E. coli culture, perform serial dilutions on it, then plate the bacteria and fianlly count them. This procedure is called "titering" a culture. See Figure 1 for a visual depiction of the procedure.
1. Serial dilutions.
a. Take 9 culture tubes containing 9 ml of water. Arrange them in a row. Using a pipet, sterilely transfer 1 ml of bacteria from the original culture into the first tube. Vortex at low speed or slap the bottom of the tube to mix. This tube now contains bacteria diluted 10x (10-1).
b. Using a fresh pipet, sterilely transfer 1 ml from the 10-1 tube into the next tube. Mix. This tube is 100x less concentrated than the original culture (10-2).
c. Repeat this process until all the tubes have been used. use a fresh pipet every time! The final tube is 10-9 as concentrated as the original culture.
2. Plating.
a. Pipet 1/10 ml of the 10-9 tube onto a agar plate.
b. Flame the glass hockey stick to sterilize it.
c. Spread the bacteria out in an even layer with the hockey stick.
d. Repeat with the 10-8, 10-7, and 10-6 tubes. DON'T plate the higher concentrations.
e. Allow the plates to dry before putting them in the incubator.
3. Counting.
a. After the plates have incubated overnight you can count them. The TA may have put them in the refrigerator for storage .
b. Choose plates with 30-300 colonies on them to count.
c. Using a marker, touch the back of the plate on top of every colony as you count it.
4. Figuring the bacterial concentration in the original tube.
a. The concentration of bacteria is measured as the number of bacteria per ml.
b. The number of colonies on a plate is equal to the number of bacteria that were in 1/10 ml of the tube you got them from. Thus, the number of bacteria per ml in that tube is 10 times the count.
c. To get the concentration in the original tube, multiply the concentration for the plate you counted by the dilution factor for that tube. For example, if you counted a plate from the 10-6 tube, multiply the concentration by 106.
d. Example: You count 84 colonies on the 10-7 plate. Since 1/10 of a ml was put on the plate, the concnetration of bacteria in the 10-7 tube is 840 per ml. Multiplying by the dilution factor, you find the original culture had 840 x 107 bacteria per ml. This could be better written as 8.4 x 109.
CONJUGATION
This experiment will be started in the second lab period. This experiment will take 2 hours or more, so be prepared in advance and get started right away. The basic procedure is to mix the two strains of bacteria and allow them to grow at 37o. At different time points, a small sample is removed and put into a naladixic acid buffer to stop conjugation. Then samples of this are spread onto the proper Petri plates at various dilutions. The bacteria are allowed to grow for 48 hr, and then the resulting colonies are counted. The number of colonies for each marker is plotted versus time, and from this, the order of genes can be determined. See Figure 2. Each student will be assigned one or two time points to take care of. Each time point will be tested by several students in each lab period, and all the class's data will be pooled and passed out to everyone.
Here is the basic protocol to be followed at your assigned time points:
1. Mix 1 ml of Hfr with 9 ml of F-. Immediately put them in the 37o water bath and start the timer. This should be done at the very start of class. (This step is done in groups).
2. At the indicated time intervals (0, 20, 40, 60, 80, and 100 minutes) pipet 0.2 ml of bacteria into the tube containing 1.8 ml of naladixic acid buffer. Vortex for 30 sec. Immediately put the rest of the bacteria back in the water bath.
3. a. Pipet 0.1 ml of the naladixic acid bacteria on the plates for that time point. This is the "10-1" dilution, meaning that the tube of bacteria in naladixic acid is 1/10 as concentrated as the conjugation tube (remember you did a 1/10 dilution by putting 0.2 ml of bacteria into 1.8 ml of naladixic acid). Since you only plate 1/10 of a ml, the number of colonies that you count on the plate will be equal to 1 /100 of the number of bacteria per ml in the conjugating culture. There are four types of plate--do one of each type.
b. Serial dilution: put 1 ml of the naladixic acid bacteria into 9 ml of dilution buffer (tubes containing 9 ml of dilution buffer are available for you). Then plate 0.1 ml of this onto the four types of plate as in step 3. This is the "10-2" dilution--the number of colonies is 1/1000 of the bacteria per ml.
4. Streak out the controls (Hfr alone and F- alone) at some point during the experiment--after things settle down a bit. Do one plate of each type with each of the 2 strains. The exact time isn't that critical for this part. Nothing should grow on these plates. (This step is also done in groups).
5. Let the plate incubate upside down at 37o for 2 days. The TA will then move the plates into the refrigerator.
6. Count the number of large colonies on each plate, using a marker on the plastic to be sure you get them all. Ignore air bubbles--if you use the colony counters you will easily see that air bubbles are clear while colonies are opaque white. It is essential that you get a count for each plate type at each time point: don't use "TNTC" (too numerous to count) for both dilutions. Hand in your data to the TA, including your name, the counts for each type of plate, and the time points you did.
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Data Analysis:
You will be given data for all the time points from the entire class. Your job is to examine this data, determine the mean and the standard deviation of the counts for each time point, plot the data on a graph, and determine the order of the genes on the chromosome.
1. For each marker, convert the colony counts to cells/ml. Only use the count on one plate, either 10-1 or 10-2 for each person's data--use the plate with the highest count. This is generally the 10-1 plate, but sometimes there will be so many colonies that this plate will be uncountable. In this case, use the 10-2 plate.
2. For each marker, plot all the available data for each time point. Look at the data points and decide whether any of them are obviously a mistake. BE VERY CAUTIOUS about throwing out any data points !!! Use all the data unless some particular point is very wrong, for example, if one point has 1/10 or 10 times as many colonies as any other. Generally you should use all the data, but you must exercise your judgement here to find data that is clearly attributable to experimental error.
3. Determine the mean and the standard deviation for each marker at each time point. The mean is simply the average number: the sum of all counts for that marker and time point divided by the number of data points. The standard deviation is: 
where Xi is each individual count (for that time point and marker), m is the mean, and n is the number of counts. The means to take the sum for all data points.
Many people prefer an alternate formula for calculating standard deviation: 
To use this formula, you need to add up the sum of all your observations ( 
), then square that sum ( 
), and also add up the sum of the squares of all your observations ( 
). Using this formula is faster becasue it avoids having to subtract the mean from each indiovidula observation.
4. Plot the data. The x-axis is time in minutes, and the y-axis is cells/ml. The mean for each time point should be plotted, and error bars should extend above and below the mean point for a distance of one standard deviation. For instance, if the mean at 60 min is 52 cells/ml and the standard deviation is 13, there should be a point at 52 cells/ml with a vertical bar extending from 65 (= 52 + 13) to 39 (= 52 - 13).
5. Draw a line through the means. You can smooth the line if you like. The line will not be straight--it is basically logarithmic. Extrapolate the lines back to 0 to determine the initial time of entry for each marker. You will end up with one graph with 3 lines on it. ((Advanced analysis--plot a regression line for the data after linearizing it by using the logarithm of time))
Historical Background
The search for bacterial conjugation stemmed directly from the desire to study genetics in bacterial systems.
EDWARD TATUM & HIS MUTANTS
George Beadle and Edward Tatum, working with the fungus Neurospora had shown that mutants could be blocked in different steps of the same biochemical pathway. This led to their famous "one gene - one enzyme" hypothesis.
Beadle, writing in 1945, recognized the dilemma of establishing this hypothesis in bacteria:
| The genetic definition of a gene implies sexual reproduction. It is only through segregation and recombination of genes during meiosis and fusion of gametes that the gene exhibits its unitary property. In bacteria, for example, in which cell reproduction is vegetative, there are presumably units functionally homologous with the genes of higher organisms, but there is no means by which these can be identified by the techniques of classical genetics. |
Beadle |
Tatum |
We should also note that Beadle and Tatum's work was revolutionary for their use of nutritional mutants of Neurospora as conditional mutants. They were awarded the The Nobel Prize in Physiology or Medicine in 1958 "for their discovery that genes act by regulating definite chemical events"
Tatum, nevertheless, tried to see if he could extend his nutritional studies to E. coli K12. He isolated a large number of double mutant strains. This was a significant advance because he demonstrated the first real proof of heredity in bacteria -- the mutant phenotype persisted over generations.
Some of the strains he obtained were:
Strain Auxotroph for: 58-161 methionine & biotin 58-278 phenylalanine & biotin 58-309 cysteine & biotin 58-336 isoleucine & biotin 58-580 thiamine & biotin 58-741 histidine & biotin 58-2651 proline & biotin 679-183 proline & threonine 679-662 glutamic acid & threonine 679-680 leucine & threonine
Unknown to him, the real importance in these mutants lay in the fact that mutant 679-680 was genetically different from all of the others. We will see later that it plays a critical role in Lederberg's discovery of conjugation.
JOSHUA LEDERBERG & THE DISCOVERY OF CONJUGATION
In 1945, Joshua Lederberg decided to use nutritional mutants to look for evidence of mating in bacteria:
| He conceived the use of nutritional mutants as a means of searching for mating in bacteria. With the prototrophic recovery approach in mind, he devised a possible experiment. "The basic protocol ... entailed the use of a pair of nutritional mutants, say A+B- and A-B+. If crossing occurred, one could plate out billions of cells in a selective medium ... one should be able to find even a single A+B+ recombinant. |  |
| Text from "The Emergence of Bacterial Genetics" by T.D. Brock, p81. |
He isolated 2 E. coli mutants: one was met-pro+; the other was met+pro-. He tried a mating experiment but he found no evidence for mating.
When Lederberg heard that Tatum had isolated double mutant strains of E. coli, he wanted to try these:
| Such double mutants were of special value since their reversion to prototrophy should be extremely low. Another value perceived by Lederberg for obtaining Tatum's strains was the following: In the event that mating types or sterility factors were present in Escherichia coli, mating might not occur. Self-incompatibility was common in fungi, and one of the best ways of obtaining distinct mating types was to use independent isolates. this idea was expressed in Lederberg's first letter to Tatum: "It should ... be advantageous to use stocks of heterogeneous origin in the event that there exist mating types, sterility factors, etc." This idea, of course, proved to be true. However, Lederberg had no way of knowing that Tatum's Escherichia coli K-12 would be one of the rare strains containing an F-plasmid and that this plasmid had been lost in the threonine- leucine- double mutant (679-680). |
| Text from "The Emergence of Bacterial Genetics" by T.D. Brock, p81. |
The basic experiment to demonstrate conjugation was as follows:
[G10-4]
This protocol selects for prototrophs. The only cells which can grow are cells that no longer have any nutrient requirements.
The results were as follows:
Since no colonies grew on plates 1 or 2, the colonies on plate 3 could not have arisen as a result of reversion. However, the frequency with which colonies were seen on plate 3 explains why Lederberg's initial experiment was unsuccessful. This frequency is the same as the natural mutation rate. Thus any experiments with single mutants is unable to distinguish between prototroph recovery due to reversion and prototroph recovery due to any other reason.
Although the experiment seems convincing, a number of alternative explanations need to be excluded:
Could the cells have been transformed?
Might cells of one strain have lysed allowing their DNA to be taken up by cells of the other strain?
This possibility was ruled out by using the FILTRATES of each culture. No recovery or prototrophs was found indicating that free DNA was not involved.
Could cross-feeding have been responsible?
Might cells of one strain have secreted metabolic intermediates that were used for growth by the other?
This possibility was ruled out by the U-TUBE EXPERIMENT. This experiment demonstrated that physical contact between the cells was required.
Tom Brock in "The Emergence of Bacterial Genetics" lists three reasons why Lederberg's "simple experiment was considered so brilliant":
Once he had demonstrated the existence of genetic mating in bacteria (E. coli), Lederberg thought that he would be able to use the technique to construct a complete genetic map of E. coli. He soon ran into difficulties interpreting the results. Fundamentally, the problem was that he did not really know or understand the real mechanism underlying mating in bacteria. That understanding was provided by the work of Bill Hayes.
BILL HAYES & THE DISCOVERY OF FERTILITY
Hayes studied the kinetics of bacterial mating not just the end results of mating to develop an insight into the process. Just like Lederberg, he used a streptomycin selection procedure.
He did a series of mating experiments, all of which pointed to the conclusion that a successful mating depended on the continued viability of one parent only and that mating was unidirectional.
EXPT #1A.
Consider a mating experiment with following strains:
- strain A is met-strS
- strain B is thr-leu-strR
- grow cultures of both strains, mix the cultures, and plate on minimal media plates containing streptomycin.
In this experiment, prototrophic recombinants are recovered at a normal frequency.
EXPT #1B.
Consider the same experiment with the same two strains EXCEPT that the streptomycin resistances are reversed:
- strain A is met-strR
- strain B is thr-leu-strS
- grow cultures of both strains, mix the cultures, and plate on minimal media plates containing streptomycin
This time, no prototrophs are recovered -- this is a sterile cross.
Comparing the results of these two experiments, it seems that the continued viability of strain B is essential for successful recovery of prototrophs.
EXPT #2.
In this experiment, both strain A and strain B are strS.
Streptomycin is used to kill one of the two strains. Treatment with streptomycin does not cause the cell to lyse. Streptomycin treated cells are able to carry out conjugation for a short while after treatment.
- If strain A is treated with streptomycin prior to mixing the two cultures, then normal prototrophic recovery is observed.
- If strain B is treated with streptomycin prior to mixing the two cultures, then a sterile cross results.
Once again, the continued viability of strain B is essential.
These experimental observations led to the suggestion that strain A was a DONOR strain and that strain B was a RECIPIENT strain.
Fertility
It was soon shown that FERTILITY, the ability to act as a donor, was a genetic trait.
We denote donor strains as F+ and recipient strains as F-. We can summarize the results of bacterial crosses between F+ and F- strains as follows:
| | This cross is FERTILE. Progeny are F+ Transfer of fertility occurs at a much higher frequency than the transfer of bacterial markers which occurs at a low frequency. |
| | This cross is STERILE. |
| | This cross is FERTILE and it occurs at a very low efficiency. |
The discovery of fertility in bacteria revolutionized the field of bacterial genetics and provided the tools that were necessary to move genes from one bacterial cell to another. Jim Watson heard about Hayes work at a small meeting on microbial genetics in Pallanza in 1952. In his book "The Double Helix", Watson describes the impact of Hayes' announcement of his discovery:
| Bill's appearance was the sleeper of the three-day gathering: before his talk no one except Cavalli-Sforza knew he existed. As soon as he had finished his unassuming report, however, everyone in the audience knew that a bombshell had exploded in the world of Joshua Lederberg. In 1946, Joshua, then only twenty, burst upon the biological world by announcing that bacteria mated and showed recombination. Since then he had carried out such a prodigious number of pretty experiments that virtually no one except Cavalli dared work in the same field. |
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The Fertility Factor -- The F Plasmid
We now know that the fertility factor or F factor is a very large (94,500 bp) circular dsDNA plasmid; it is generally independent of the host chromosome.
The F factor controls its own replication. It has two origins of replication: oriV is the origin for bidirectional replication; oriS is the origin for unidirectional replication. The F factor also has genes that regulate DNA synthesis so that its copy number is kept at a low level; and, genes that regulate the partition into the daughter cells after E. coli divides.
The F factor is self-mobilizable -- it can transfer itself to other cells. This requires the functions coded by a large number of transfer (tra and trb) genes. Transfer initiates at a special origin of transfer, oriT, and proceeds via a rolling circle mechanism of replication. DNA is transfered through pili which form on the surface of a donor cell and can attach to the surface of a recipient cell. Pili cannot attach to other donor cells due to the presence of the proteins coded by the traS and traT genes -- this phenomenon is called surface exclusion.
The transfer genes are all located within a 33.3 Kbp transfer region. The following table summarizes these genes and their functions:
| FUNCTION | GENES |
|---|---|
| | traA, traL, traE, traK, traB, traV, traC, traW, traU, traF, traQ, traH, traG |
| | traS, traT |
| | traN, traG |
| | traM, traY (exonuclease),traD, traI (helicase), traZ (exonuclease) |
| | finP, finO, traJ (positive regulator) |
The F factor also contains a number of Insertion Sequences (IS2, IS3a and IS3b) and the transposon Tn1000 (also known as gamma-delta).
| |
Transfer of F-factors
F factors transfer themselves from one cell to another. oriT is nicked by TraY and one of the 2 strands of the F plasmid is displaced as the TraI helicase unwinds it permitting it to be transferred to the recipient cell through the F-pilus. The transferred strand is covalently linked to the TraD protein during transfer. Once it has been transferred, the strand circularizes and second strand synthesis takes place.
Transfer of chromosomal DNA -- I
The progeny of an F+ x F- mating receive a complete copy of F with a very high frequency. Hence the progeny are also F+ or FERTILE. However, this does not explain how chromosomal markers can be transferred during a bacterial mating.
Chromosomal markers are transferred at a relatively low frequency -- much lower than the frequency with which fertility is transferred. How are chromosomal markers transferred?
The answer comes from another discovery made both by Bill Hayes and by Luigi Luca Cavalli-Sforza.
Hfr strains
Hayes and Cavalli-Sforza both discovered strains of E. coli that transferred chromosomal markers at a much higher frequency than normal. They called these HIGH FREQUENCY of RECOMBINATION strains or Hfr strains.
We can compare the behaviour of an Hfr strain with an F+ strain during a bacterial mating:
| | Progeny are F+ Low frequency of recombinants. |
| | Progeny are F- High frequency of recombinants. |
Understanding this behaviour of Hfr strains in constrast to that of F+ strains came from:
Campbell's suggestion was that both E. coli and the F plasmid were circular molecules and that recombination between them would generate a single larger circular molecule.
Now, if conjugational transfer were to start while the F plasmid was integrated, not only would the F-DNA be transferred but the chromosomal DNA into which it had integrated would also be transferred.
Transfer of such a large molecule would take a long time. The probability of an entire chromosome of E. coli being transferred would be extremely low. Furthermore, since oriT is in the middle of the integrated F-DNA, and since transfer is a unidirectional process, transfer of the F-DNA would not be complete until the entire bacterial chromosome had been transferred. Thus, the progeny of an Hfr x F- cross would almost always be F-.
However, since a large amount of bacterial DNA would be transferred during an Hfr x F- cross, there would be a high probability that recombination between the transferred chromosomal DNA and the recipient chromosomal DNA would occur. If the donor and recipient strains could be distinguished genetically, then the results of this recombination could be observed.
Transfer of chromosomal DNA -- II
We can distinguish between the Hfr state and an Hfr strain. Once recombination between the F plasmid and the host chromosome occurs, the cell is in an Hfr state. A plasmid that can reversibly integrate into the host chromosome is called an episome.
Integration of F plasmids can occur at many places. The F plasmid contains mobile genetic elements (3 Insertion Sequences and a Transposon) which also occur in the E. coli chromsome. Recombination between a chromosomal element and an F-plasmid element generates a strain in the Hfr state. Bacterial markers that are close to the site of F plasmid integration are transferred at a very high frequency.
Recombination between an F plasmid and the bacterial chromosome is readily reversible. Under normal circumstances, the F plasmid will excise itself before conjugational transfer occurs. Hfr strains were identified because, in essence, the F plasmid got stuck in the integrated Hfr state.
Transfer of bacterial markers in a typical conjugation experiment occurs because in any population of F+ cells, a small percentage will be in the integrated state. If conjugation occurs while the cell is in this state, there will be a high frequency of recombinants but the progeny will mostly be F-.
The Interrupted Mating Experiment
The mechanism of transfer of bacterial markers was demonstrated by the interrupted mating experiment of Elie Wollman and François Jacob.
Wollman adopted the idea of using a kitchen blender to disrupt the cell pairs that form during conjugation from the experiment of Hershey and Chase. The experimental design was simple:
The actual cross used in this experiment was the following:
HfrH was the Hayes Hfr strain. The results of this experiment showed that the bacterial markers were transferred in the order :
By using different Hfr strains -- in which F has integrated in different places -- it became both conceptually and practically possible to map genes in E. coli simply by measuring and comparing time of transfer.
Transfer of chromosomal DNA -- III
As we have noted, recombination between an F plasmid and the bacterial chromosome is readily reversible. Normally this process is quite precise, i.e. the F plasmid excises correctly.
Occasionally, though, this does not happen. Sometimes the F plasmid will excise incorrectly and, in doing so, it will take a small section of bacterial chromosome with it. The resulting F plasmid carries all of the genetic functions that it needs for conjugation but in addition it now carries a very specific bacterial marker. These type of modified F plasmids are denoted F' (F prime) factors.
[G10-3d]
F' factors are FERTILE. They transfer fertility at a normal frequency and they transfer some chromosomal markers or loci -- those that are now contained on the F plasmid -- at a similar high frequency. They transfer most chromosomal markers at the usual low frequency.
F' factors were discovered by Edward Adelberg. Their discovery was described by Elie Wollman to Tom Brock in "The Emergence of Bacterial Genetics" (p104):
| "Adelberg had brought back to Berkeley some of our Hfr strains. I spent the year 1958-59 in Berkeley -- finishing the writing of our book. Once Ed Adelberg came to me telling me that one of the Hfr strains had changed: the frequency of recombinants was less than expected, but all were donors of intermediate frequency. I suggested that, by comparison with HFT (Hfr) phage the sex factor had left its site accompanied by neighbouring genetic fragments. This was verified experimentally. Lwoff, who had come to visit, brought the news back to François Jacob who immediately used it for making partial Lac diploids. This is the history of F prime factors" |
F' factors were extremely important in the early days of molecular biology. They were, in fact, the first cloning vectors -- the conceptual groundwork for all modern cloning was provided by the use of F' factors.
F' factors have also been used to map the E. coli chromosome.
The following two methods can be used for isolating F' factors.
In both cases, a second mating will confirm that the marker must reside on an F' factor.
SOURCE: http://www.mun.ca/biochem/courses/3107/Lectures/Topics/conjugation.html
A biofilm is an aggregate of microorganisms in which cells are stuck to each other and/or to a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm EPS, which is also referred to as "slime," is a polymeric jumble of DNA, proteins and polysaccharides. Biofilms may form on living or non-living surfaces, and represent a prevalent mode of microbial life in natural, industrial and hospital settings . The cells of a microorganism growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which by contrast, are single-cells that may float or swim in a liquid medium. Microbes form a biofilm in response to many factors, which may include cellular recognition of specific or non-specific attachment sites on a surface, nutritional cues, or in some cases, by exposure of planktonic cells to sub-inhibitory concentrations of antibiotics. When a cell switches to the biofilm mode of growth, it undergoes a phenotypicregulated. shift in behavior in which large suites of genes are differentially
Formation of a biofilm begins with the attachment of free-floating microorganisms to a surface. These first colonists adhere to the surface initially through weak, reversible van der Waals forces. If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion structures such as pili.
The first colonists facilitate the arrival of other cells by providing more diverse adhesion sites and beginning to build the matrix that holds the biofilm together. Some species are not able to attach to a surface on their own but are often able to anchor themselves to the matrix or directly to earlier colonists. It is during this colonization that the cells are able to communicate via quorum sensing using such products as AHL. Once colonization has begun, the biofilm grows through a combination of cell division and recruitment. The final stage of biofilm formation is known as development, and is the stage in which the biofilm is established and may only change in shape and size. This development of biofilm allows for the cells to become more antibiotic resistant.
There are five stages of biofilm development (see illustration at right).
Dispersal of cells from the biofilm colony is an essential stage of the biofilm lifecycle. Dispersal enables biofilms to spread and colonize new surfaces. Enzymes that degrade the biofilm extracellular matrix, such as dispersin B and deoxyribonuclease, may play a role in biofilm dispersal. Biofilm matrix degrading enzymes may be useful as anti-biofilm agents.cis-2-decenoic acid, is capable of inducing dispersion and inhibiting growth of biofilm colonies. Secreted by Pseudomonas aeruginosa, this compound induces dispersion in several species of bacteria and the yeast Candida albicans Recent evidence has shown that a fatty acid messenger,
Biofilms are usually found on solid substrates submerged in or exposed to some aqueoussolution, although they can form as floating mats on liquid surfaces and also on the surface of leaves, particularly in high humidity climates. Given sufficient resources for growth, a biofilm will quickly grow to be macroscopic. Biofilms can contain many different types of microorganism, e.g. bacteria, archaea, protozoa, fungi and algae; each group performing specialized metabolic functions. However, some organisms will form monospecies films under certain conditions.
Researchers from the Helmholtz Center for Infection Research have investigated the strategies used by biofilms. They discovered that biofilm bacteria apply chemical weapons in order to defend themselves against disinfectants and antibiotics, phagocytes and our immune system.
The lead researcher, Dr. Carsten Matz, began a serious investigation in order to find why phagocytes cannot annihilate the biofilm bacteria. He analyzed the marine bacteria, which defend themselves against the amoebae, the behavior of which copies the behavior of phagocytes. The amoebae behave in the sea just like the immune cells in human body: they search for and feed on the bacteria. When bacteria are alone and separated in the water, they become an easy catch for the attackers. However, when they attach to a surface and join other bacteria, the amoebae cannot assault them.
The researcher stated that biofilms may be seen as a source of new bioactive agents. When bacteria are organized in biofilms, they produce effective substances which individual bacteria are unable to produce alone.
The biofilm is held together and protected by a matrix of excreted polymeric compounds called EPS. EPS is an abbreviation for either extracellular polymeric substance or exopolysaccharide. This matrix protects the cells within it and facilitates communication among them through biochemical signals. Some biofilms have been found to contain water channels that help distribute nutrients and signalling molecules. This matrix is strong enough that under certain conditions, biofilms can become fossilized.
Bacteria living in a biofilm usually have significantly different properties from free-floating bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways. One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community. In some cases antibiotic resistance can be increased 1000 fold.
The concept that biofilms are more resistant to antimicrobials is not completely accurate. For instance the biofilm form of Pseudomonas aeruginosa has no greater resistance to antimicrobials, when compared to stationary phase planktonic cells. Although, when the biofilm is compared to logarithmic phase planktonic cells, the biofilm does have greater resistance to antimicrobials. This resistance to antibiotics in both stationary phase cells and biofilms may be due to the presence of persister cells.
Biofilms are ubiquitous. Nearly every species of microorganism, not only bacteria and archaea, have mechanisms by which they can adhere to surfaces and to each other.
Biofilms have been found to be involved in a wide variety of microbial infections in the body, by one estimate 80% of all infections. Infectious processes in which biofilms have been implicated include common problems such as urinary tract infections, catheter infections, middle-ear infections, formation of dental plaque, gingivitis, coating contact lenses, and less common but more lethal processes such as endocarditis, infections in cystic fibrosis, and infections of permanent indwelling devices such as joint prostheses and heart valves.. More recently it has been noted that bacterial biofilms may impair cutaneous wound healing and reduce topical antibacterial efficiency in healing or treating infected skin wounds.
It has recently been shown that biofilms are present on the removed tissue of 80% of patients undergoing surgery for chronic sinusitis. The patients with biofilms were shown to have been denuded of cilia and goblet cells, unlike the controls without biofilms who had normal cilia and goblet cell morphology. Biofilms were also found on samples from two of 10 healthy controls mentioned. The species of bacteria from interoperative cultures did not correspond to the bacteria species in the biofilm on the respective patient's tissue. In other words, the cultures were negative though the bacteria were present.
Biofilms can also be formed on the inert surfaces of implanted devices such as catheters, prosthetic cardiac valves and intrauterine devices.
New staining techniques are being developed to differentiate bacterial cells growing in living animals, e.g. from tissues with allergy-inflammations.
The achievements of medical care in industrialised societies are markedly impaired due to chronic opportunistic infections that have become increasingly apparent in immunocompromisedinfections remain a major challenge for the medical profession and are of great economic relevance because traditional antibiotic therapy is usually not sufficient to eradicate these infections. One major reason for persistence seems to be the capability of the bacteria to grow within biofilms that protects them from adverse environmental factors. Pseudomonas aeruginosa is not only an important opportunistic pathogen and causative agent of emerging nosocomial infections but can also be considered a model organism for the study of diverse bacterial mechanisms that contribute to bacterial persistence. In this context the elucidation of the molecular mechanisms responsible for the switch from planktonic growth to a biofilm phenotype and the role of inter-bacterial communication in persistent disease should provide new insights in P. aeruginosa pathogenicity, contribute to a better clinical management of chronically infected patients and should lead to the identification of new drug targets for the development of alternative anti-infective treatment strategies. patients and the aging population. Chronic
Dental plaque is the material that adheres to the teeth and consists of bacterial cells (mainly Streptococcus mutans and Streptococcus sanguis), salivary polymers and bacterial extracellular products. Plaque is a biofilm on the surfaces of the teeth. This accumulation of microorganisms subject the teeth and gingival tissues to high concentrations of bacterial metabolites which results in dental disease.
Legionella bacteria are known to grow under certain conditions in biofilms, in which they are protected against disinfectants. Workers in cooling towers, persons working in air conditioned rooms and people taking a shower are exposed to Legionella by inhalation when the systems are not well designed, constructed, or maintained.
Neisseria gonorrhoeae is an exclusive human pathogen. Recent studies have demonstrated that it utilizes two distinct mechanisms for entry into human urethral and cervical epithelial cells involving different bacterial surface ligands and host receptors. In addition it has been demonstrated that the gonococcus can form biofilms on glass surfaces and over human cells. There is evidence for formation of gonococcal biofilms on human cervical epithelial cells during natural disease and that outer membrane blebbing by the gonococcus is crucial in biofilm formation over human cervical epithelial cells.
Technological progress in microscopy, molecular genetics and genome analysis has significantly advanced our understanding of the structural and molecular aspects of biofilms, especially of extensively studied model organisms such as Pseudomonas aeruginosa. Biofilm development can be divided into several key steps including attachment, microcolony formation, biofilm maturation and dispersion; and in each step bacteria may recruit different components and molecules including flagellae, type IV pili, DNA and exopolysaccharides. The rapid progress in biofilm research has also unveiled several genetic regulation mechanisms implicated in biofilm regulation such as quorum sensing and the novel secondary messenger cyclic-di-GMP. Understanding the molecular mechanisms of biofilm formation has facilitated the exploration of novel strategies to control bacterial biofilms.
