Gel electrophoretic analysis of Zymomonas mobilis glycolytic and fermentative enzymes: identification of alcohol dehydrogenase II as a stress protein

Abstract

The 13 major enzymes which compose the glycolytic and fermentative pathways in Zymomonas mobilis are particularly abundant and represent one-half of the soluble protein in exponential-phase cells. One-and two-dimensional polyacrylamide gel electrophoresis maps were developed for 12 of these enzymes. Assignments were made by comigration with purified proteins, comparison with overexpressed genes in recombinant strains, and Western blots (immunoblots). Although most glycolytic enzymes appeared resistant to turnover and accumulated in stationary-phase cells, the protein levels of pyruvate kinase, alcohol dehydrogenase I, and glucokinase declined. Alcohol dehydrogenase II was identified as a major stress protein and was induced both by exposure to ethanol and by elevated temperature (45°C). This enzyme, encoded by the adhB gene, is expressed from tandem promoters which share partial sequence identity with the Escherichia coli consensus sequence for heat shock proteins.

Mutational analysis of segmental stabilization of transcripts from the Zymomonas mobilis gap-pgk operon

“…In apparent compensation, threefold-greater amounts of the GAPDH protein than PGK protein are maintained in the cytoplasm (18,19). Neither GAP nor PGK is preferentially degraded, eliminating protein turnover as a means of maintaining differences in enzyme abundance (2). Although gap andpgk are transcribed as a single operon, measurements of functional message pools were in close agreement with the observed differences in protein abundance (30).…”

“…Native messages derived from the gap-pgk operon are very stable and encode highly expressed proteins (2,18,30). Our results with plasmid-borne copies of mutant operons indicate that the degradation of the bicistronic gap-pgk message proceeds according to the general model now emerging for bacterial mRNA (7,8,17,20,23,29): (i) 3' stem-loops are required to block degradation by abundant 3' to 5' exoribonucleases; (ii) nonrandom endoribonuclease cleavage serves to initiate message degradation; and (iii) for highly expressed genes, long 5' UTRs are present and contain secondary structures which are essential for stability.…”

Effect of Temperature Stress and Metal Ion Supplement on Ethanol Fermentation by Zymomonas mobilis

“…Tekanan suhu berakibat pada melambatnya laju pertumbuhan, viabilitas (Michel and Starka, 1986) dan kemampuan fermentatif pada sel mikroorganisme (Osman and, Ingram, 1985). Stres/tekanan suhu tersebut juga mengubah fluiditas dari membran plasma (Carey and Ingram, 1983;Moreau et al, 1997) serta menstimulasi respon-respon spesifik pada stress terkait (Michel and Starka, 1986;An et al, 1991;Barbosa et al, 1994). Lebih dari itu, tekanan akibat suhu di atas suhu optimal pertumbuhan, menyebabkan gangguan homeostasis ionik seluler, yang memicu penurunan aktivitas metabolik dan bahkan kematian sel (Thanonkeo et al, 2007 (Swings and De Ley, 1977).…”

Immunocytochemical localization of glycolytic and fermentative enzymes in Zymomonas mobilis

“…1). Antibodies to the 12 enzymes stained single dominant bands at the expected molecular weights (4 Antisera against phosphoglycerate mutase (PGM), phosphogluconate lactonase (PGL), and alcohol dehydrogenase II (ADHII) also stained minor bands, each of which appeared slightly smaller than twice the corresponding monomeric size. In the case of ADHII, both bands appear to contain authentic protein since both were also present in recombinant E. coli containing the minimal coding region for adhB.…”

“…Enzymes were purified to apparent homogeneity essentially as described previously (13,19,22,(25)(26)(27)31) and were used to prepare polyclonal antibodies in goats (9) or New Zealand White rabbits (4,18). All antisera produced a single sharp band in Ouchterlony assays when tested against protein extracts from Z. mobilis.…”