You are studying a pathogenic bacterium that infects your favorite fruit, wild Maine blueberries, causing them to become mushy. The virulence factor responsible is an enzyme called pectinase (because it degrades pectin - which is the substance that allows fruit, when cooked, to become jam). Under nearly all the laboratory conditions you've tested, the bacterium only produces very small amounts of pectinase. It makes lots of pectinase i) when growing on ripe blueberries and ii) when the bacteria are in culture at very high cell density but only when also supplied with pectin (hint: These two phenotypes already suggest at least two complementary ways in which the pectinase expression is regulated that follow paradigms we have studied this semester). You are interested in understanding how this bacterium regulates virulence and so take a classical genetic approach of mutagenesis, using a base analog (consider the type of mutations a base analog makes).
You isolate the following two general classes of mutants (with some subclasses):
Class 1: Pec- (never makes pectinase)
Some of these mutants can be complemented in trans:
A) There are four complementation groups with this phenotype for which the wild-type gene is trans-dominant
B) Only one of the four complementation groups can be complemented by crossfeeding, and it is complemented by any of the other (three) complementation groups
Some of the mutants cannot be complemented in trans:
C) One group of mutants is cis-acting/cis-dominant (but trans recessive to the wild-type so it doesn't block expression of the wild-type operon in a merodiploid)
D) There is one group of mutants that is trans-acting and trans-dominant.
Class 2: Pec++ (over-produces pectinase)
E) One class of mutants no longer needs pectin in the agar for pectinase production
F) One class of mutants still needs pectin to produce pectinase, but no longer needs to be at high cell density and this mutation is trans-acting and trans-dominant.
Using what you know about the paradigm systems we've studied, you are well prepared to describe the probable operon/regulon (26 points total).
A. Make a list of the paradigm model systems (for gene regulation) we have studied in class (and that might be at play above or similar to the above), and briefly describe the kinds of regulation you would see in each. You must describe a minimum of 2 systems. This will help you focus upon the kinds of mutants in this test question by comparison (5 points).
B. How many (structural or enzymatic) genes do you think are involved in the actual degradation of pectin (not its regulation)? Explain how you came to this conclusion drawing from examples in class (3 points).
C. Do you think negative regulation is involved? Explain what evidence (for or against) brought you to this conclusion (3 points).
D. Do you think positive regulation is involved? Explain what evidence (for or against) brought you to this conclusion drawing upon examples from class (3 points).
E. For each mutant phenotype above (A through F), briefly describe what the probable mutation is (for most, one word describing the gene or function of the gene or genetic element will suffice) (12 points).
Class I
A)
B)
C)
D)
Class II
E)
F)