Biochemical and genetic analysis of the Bacteroides ovatus galactomannan utilization system

I constructed the E. coli-Bacteroides shuttle vector, pVAL-1. This was the first in a series of new cloning vehicles that made it easier to clone Bacteroides DNA in E. coli and mobilize it from E. coli to Bacteroides species.My first approach to identifying non-enzymatic components of the galactomannan utilization system was to attempt to clone genes encoding the known enzymes and then look for linked galactomannan utilization genes.Since the properties of the different $\alpha$-galactosidases were different enough to make them useful indicators of what the bacteria were utilizing in vivo, I surveyed disaccharidase activities in B. ovatus obtained from ceca of colonized germfree mice. None of the disaccharidase activities were very high. This raised the question of whether the high level of induction seen in laboratory medium is ever achieved in vivo.Since the cloning approach had not yielded the desired galactomannan utilization genes, I next tried to locate these genes by transposon mutagenesis. The galactomannanases can be induced by glucose as well as galactomannans, and the level of activity in cells grown on glucose was comparable to that in cells grown on guar gum. Applying this finding to the mutants, I demonstrated that the transposon insertions had not affected the galactomannanases.The polypeptide profiles of the mutants M5 and M7 were compared to the parental wild type B. ovatus strain using two dimensional gel analysis. This revealed two membrane polypeptides in M5 and M7 affected by the transposon insertion. In each case, one was now constitutive and the other was overexpressed.Two 10 kb segments of B. ovatus chromosomal DNA that overlapped the M4/M7 and M5 loci, respectively, were cloned in E. coli. The transposon insertion sites in M4 and M7 were mapped to within 0.5 kb of each other. The promoter has been localized to within 1.4 kb. Results indicated that there are two separate galactomannan associated transcriptional units within the 10 kb HindIII fragment. (Abstract shortened with permission of author.)U of I OnlyETDs are only available to UIUC Users without author permissio