Question

17. The soil bacterium Pseudomonas fluorescens (taxid:294) is heavy metal tolerant and can survive in industrial waste and degrade and detoxify diverse organic pollutants.

You obtain Pseudomonas fluorescens ATCC 13525 which is sensitive to ampicillin (Ap), streptomycin (Sm) and spectinomycin (Sp) but resistant to chloramphenicol (Cm) and nalidixic acid (Nx). This strain is also lacZnegative (devoid of β-galactosidase activity).

You also obtain a donor Escherichia coli strain containing an ampicillin (Ap) resistant suicidal plasmid carrying the minitransposon mTn5-lacZSmSp  (this mTn5 has a promoterless lacZ and expresses Sm and Sp resistance). This strain is not resistant to any other antibiotics.

Consider in this case that cells can develop resistance to each antibiotic gene through spontaneous mutation at the frequency of 1 in 10⁶ cells.

You conjugally mate 10⁹ cells of ATCC 13525 with 10⁹ cells of the E. coli donor strain to construct random mTn5 mutations and identify molecular determinants for cadmium resistance.

1. What combination of antibiotics would you use in the media to select specifically for ATCC 13525::mTn5-lacZSmSp transconjugants and prevent the growth of spontaneous antbiotic resistant mutants?                                                       

2. To prevent secondary mutations from plasmid insertion, you would need to ensure the transconjugant cells had lost the delivery plasmid. How would you confirm the delivery plasmid was no longer present in these mutants?                        (1 mark)

You then replica patch transconjugants onto media containing the chromogenic substrate X-Gal in the presence and absence of cadmium. The following table shows the phenotypes of selected transconjugants.

Transconjugant number	Colour of colony after growth (β-galactosidase expression)
	Media without cadmium	Media with cadmium
1	White	No growth
4	Deep blue	Deep blue
9	Deep blue	Light blue
11	White	Medium blue
12	Medium blue	Dark blue
17	White	Deep blue
20	Light blue	Deep blue

c. Which mutant(s) have insertions in genes essential for cadmium resistance?

d. Which mutant(s) have insertions in genes up-regulated by cadmium?

e. Which mutant(s) have insertions in genes down-regulated by cadmium?

f. Which mutant(s) have insertions in genes that are constitutively expressed?

g. Which mutant(s) have insertions in genes that are not expressed in both the absence and presence of cadmium?

h. The DNA flanking the mTn5-lacZSmSp mutation was cloned from the cadmium sensitive mutant and the following sequence was obtained for the coding strand:

                

         5’.. GGCAGCACCAGCAAGG mTn5-lacZSmSp CCAGCAAGGTCAGCA..3’

Using this sequence provided, reconstruct the wild-type sequence

i. Provided with the information that the 5’ second base (underlined) in the provided sequence is the first base of a codon, provide the wild-type amino acid sequence of this portion of the protein and identify the amino and carboxyl termini.

j. Using the established amino acid sequence and the BLAST algorithm, what is the E value and the accession number of the highest scoring hit that aligns in the NCBI GenBank database?

k. What can you deduce to be the function of the protein based on the alignments obtained using the BLAST algorithm? (1 mark)

Answer 1

a.) To grow ATCC 13525::mTn5-lacZmSp transconjugants, we can use streptomycin (Sm) and Spectinomycin (Sp). Pseudomonas fluorescens ATCC 13525 is sensitive to both of these antibiotics while plasmid that is carried by E.coli is resistant to both of these antibiotics.

So, those transconjugats will be able to survive which will be having that suicidal plasmid. Thus by using Sm and Sp, growth of ATCC 13525 can be halted.

We can not use Ampicillin (Ap) here. It will result of growth of unconjugated E. coli also.

b.) To confirm that the delivery plasmid is no longer present in mutants then there will be no growth of mutants on Cadmium containing media.

c.) Cadmium resistance gene is the result of random mutation in mTn5. This minitransposon contains the lacZ gene also. Therefore, if mutation will occur for Cadmium resistance then lac Z will not be able to code enzyme for X-Gal metabolism. Media containing cadmium with white colony are the mutants with Cadmium resistance gene.

d.) White colony mutants

Reason - In the presence of Cadmium , bacteria has to up-regulate the genes which can help it in survival in that antibiotic.

e.) light blue and medium blue

Reason - Those mutants which is having mutatation in mTn5 will be able to survive in Cadmium containing medium. And mutation in mTn5 leads to distrubance of lac-Z gene. Light blue and medium blue colony representing down - regulation of Lac-Z gene.