Degree Name

Master of Science (MS)

Semester of Degree Completion

1986

Thesis Director

Russell Carlson

Abstract

The outer membrane and periplasmic proteins of Rhizobium trifolii ANU 843 and its mutants ANU 851 and ANU 845(pRt150) were isolated and analyzed by polyacrylamide gel electrophoresis (PAGE). ANU 843 is the wild type strain which possesses all the characteristics necessary for nitrogen fixation. ANU 851 has a transposon (Tn5) inserted into the nodD gene of the root hair curling region of the symbiotic plasmid. It has lost its ability to cause root hair curling and therefore can not form nodules and can not fix nitrogen. ANU 845(pRt150) harbors only the 5.5 kb root hair curling region of the pSym which is comprised of the genes nodD, A, B, C. The band profile of the outer membrane proteins from the three strains are similar. These proteins have molecular weights of 129, 48, 42, 38, 34,21, and 20 kDa. In the case of the periplasmic proteins, ANU 843 and 851 exhibit the same protein profile except for a 23.4 kDa protein present in 851 but missing in 843. ANU 845(pRt150), on the other hand, has a 38 kDa protein which is absent in both 843 and 851. Also it is missing the 22 and 28 kDa protein found in both 843 and 851.

A strain which contains the 14 kb nodulation region, ANU 845(pRt032), was exposed to inducing materials in order to study the expression of the nodulation genes. The optimum conditions for induction were determined by using a mu-lac mutant. ANU 845(pRt032)121 has a mu-lac inserted in nodA. When the root hair curling genes are expressed, mu-lac is simultaneously expressed, which leads to the production of B-galactosidase. Induction is thus measured by the extent of B-galactosidase activity. The maximum induction occurs at early log phase of bacterial growth.

The outer membrane, periplasmic and cytoplasmic proteins, extracellularpolyssacharides (EPS), and lipoplysaccharides (LPS) of induced and noninduced ANU 845(pRt032) were isolated and analyzed. The PAGE results indicate induction of the 30, 29, 19, 17, and 16 kDa outer membrane proteins. Several periplasmic proteins seemed to be induced by apigenin, with the major differences noted in the 87, 65, 56, 48, 32, and 20 kDa proteins. The profile of the cytoplasmic proteins of the induced and noninduced are similar except that there is an additional 19 kDa protein and an enhancement of the 18.6 kDa protein in the induced.

The EPS compositions of the induced and noninduced are similar, suggesting that EPS may not play a role in the root hair curling process. Some small differences were noted on the neutral components of the LPSs. Further work involving structural analysis needs to be done to learn the significance of those differences.

Download

Included in

Chemistry Commons

Share

COinS