Practice Linkage and Mapping in Bacteria Solution 2

2. The development of antibiotic resistant bacteria is becoming a big problem. Knowing this you isolate bacteria from a local hospital and grow them on medium containing a variety of antibiotics. You find one particularly interesting one that is resistant to both kanamycin and tetracycline. You perform transformation mapping to determine the relative distance between these two loci. To perform this experiment you isolate DNA from the resistant bacteria and transform bacteria that are sensitive to both antibiotics. You then grow the resulting bacteria on medium containing no antibiotics, kanamycin only, tetracycline only, and kanamycin plus tetracycline. Your results are shown in the table below.

medium containing	number of colonies	genotype
no antibiotics	>1000
+ kanamycin	90 [that fail to grow on +tet/+kan]
+ tetracycline	72 [that fail to grow on +tet/+kan]
+ kanamycin + tetracycline	9

a. Fill in the last column of the table by noting the genotype for each group of bacteria.

b. What is the cotransfer index for the kanamycin and tetracycline resistance genes?

c. If the genes had been closer together, how might the numbers of colonies for each group have been different?

d. If the two genes had actually been the same exact gene (not 2 different loci) how might the numbers of colonies for each group have been different?

medium containing	explanation	number of colonies	genotype
no antibiotics		>1000
+ kanamycin	grows only on + kanamycin, not + kan/+ tet	90
+ tetracycline	grows only on + tetracyline, not + kan/+ tet	72
+ kanamycin + tetracycline	grows on any medium	9

a. Fill in the last column of the table by noting the genotype for each group of bacteria.

Let's use kan^R for resistant and kan^S for sensitive to kanamycin. Let's use tet^R for resistant and tet^S for sensitive to tetracyline. These are our two alleles for the gene encoding kanamycin resistance and our two alleles for the gene encoding tetracyline resistance.

So now let's move to the table...

Since we don't have any antibiotics in the "no antibiotics" medium, we can not tell what genotype they are.

medium containing	explanation	number of colonies	genotype
no antibiotics		>1000	?

If the bacteria have acquired the ability to grow on medium containing kanamycin, then they have received and incorporated the kanamycin resistance gene (kan^R) in place of their original kanamycin sensitive allele (kan^S). However, we also know that these 90 bacteria do not have the ability to grow in the presence of tetracycline. Therefore, they still contain the tetracyline sensitive allele (tet^S).

medium containing	explanation	number of colonies	genotype
no antibiotics		>1000	?
+ kanamycin	grows only on + kanamycin, not + kan/+ tet	90	kan^R tet^S

Just like the answer above, if the bacteria have acquired the ability to grow on medium containing tetracyline, then they have received and incorporated the tetracycline resistance gene (tet^R) in place of their original tetracyline sensitive allele (tet^S). However, we also know that these 72 bacteria do not have the ability to grow in the presence of kanamycin. Therefore, they still contain the kanamycin sensitive allele (kan^S).

medium containing	explanation	number of colonies	genotype
no antibiotics		>1000	?
+ kanamycin	grows only on + kanamycin, not + kan/+ tet	90	kan^R tet^S
+ tetracycline	grows only on + tetracyline, not + kan/+ tet	72	kan^S tet^R

If the bacteria have acquired the ability to grow on medium containing both kanamycin and tetracyline, then they have received and incorporated both the tetracycline resistance gene (tet^R) in place of their original tetracyline sensitive allele (tet^S) and the kanamycin resistance gene (kan^R) in place of their original kanamycin sensitive allele (kan^S).

medium containing	explanation	number of colonies	genotype
no antibiotics		>1000	?
+ kanamycin	grows only on + kanamycin, not + kan/+ tet	90	kan^R tet^S
+ tetracycline	grows only on + tetracyline, not + kan/+ tet	72	kan^S tet^R
+ kanamycin + tetracycline	grows on any medium	9	kan^R tet^R

b. What is the cotransfer index for the kanamycin and tetracycline resistance genes?

To calculate the cotransfer index we need to figure out which colonies represent double transformants. These will be those colonies that received and incorporated both traits by transformation (kan^R and tet^R). We can tell which ones these are by looking at our genotypes. The last row has this genotype (kan^R tet^R).

The single transformants are also apparent by looking at the genotypes and finding those that have only acquired one of the two traits. These are kan^R with tet^S (90 colonies) and kan^S with tet^R (72 colonies).

Now just fill in the formula: double transformants / ( double transformants + single transformants )

9 / ( 9 + 90 + 72 ) = 0.0526 or 0.06

c. If the genes had been closer together, how might the numbers of colonies for each group have been different?

If two genes are closer together they will be transferred more often together and less often separately. So, we would expect the number of single transformants to be less and the number of double transformants to be greater. For example, if we had seen 35 colonies for each of the single transformants and 101 for the double transformants, we would have calculated the following cotransfer index.

101 / ( 101 + 35 + 35 ) = 0.5906 or 0.59

d. If the two genes had actually been the same exact gene (not 2 different loci) how might the numbers of colonies for each group have been different?

This is the most extreme case and actually does happen with antibiotic resistance. Maybe the gene encodes a pump used to pump the antibiotic back out of the bacteria. Anyway, making part c as extreme as we could make it, we would see that the "2 genes" are always transferred together and never are transferred apart. For example, if we had seen no colonies for each of the single transformants and 171 colonies for the double transformants, we would have calculated the following cotransfer index.

171 / ( 171 + 0 + 0) = 1