Abstract
Glycolysis is one of the most important metabolic pathways in heterotrophic organisms. Several genes encoding glycolytic enzymes are essential in many bacteria even under conditions when neither glycolytic nor gluconeogenic activities are required. In this study, a screening for in vivo interaction partners of glycolytic enzymes of the soil bacterium Bacillus subtilis was used to provide a rationale for essentiality of glycolytic enzymes. Glycolytic enzymes proved to be in close contact with several other proteins, among them a high proportion of essential proteins. Among these essential interaction partners, other glycolytic enzymes were most prominent. Two-hybrid studies confirmed interactions of phosphofructokinase with phosphoglyceromutase and enolase. Such a complex of glycolytic enzymes might allow direct substrate channeling of glycolytic intermediates. Moreover we found associations of glycolytic enzymes with several proteins known or suspected to be involved in RNA processing and degradation. One of these proteins, Rny (YmdA), which has so far not been functionally characterized, is required for the processing of the mRNA of the glycolytic gapA operon. Two-hybrid analyses confirmed the interactions between the glycolytic enzymes phosphofructokinase and enolase and the enzymes involved in RNA processing, RNase J1, Rny, and polynucleotide phosphorylase. Moreover RNase J1 interacts with its homologue RNase J2. We suggest that this complex of mRNA processing and glycolytic enzymes is the B. subtilis equivalent of the RNA degradosome. Our findings suggest that the functional interaction of glycolytic enzymes with essential proteins may be the reason why they are indispensable.
Highlights
Glycolysis is one of the most important metabolic pathways in heterotrophic organisms
The proteins were analyzed by SDSPAGE, and the interaction partners were identified by mass spectrometry. Those potential interaction partners that were detected with 10 or more peptides were regarded as significant in this study. This procedure was performed for PFK, FBA, triose-phosphate isomerase (TPI), glyceraldehyde-3phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM), and ENO
The Essential Protein YmdA Is Involved in the Maturation of the gapA Operon mRNA—Using the Strep-protein interaction experiment (SPINE) approach, we identified several essential interaction partners for the glycolytic enzymes
Summary
(FBA) of the upper part and the five genes tpiA, gapA, pgk, pgm, and eno encoding the triose-phosphate isomerase (TPI), GAPDH, PGK, phosphoglycerate mutase (PGM), and ENO of the lower part of glycolysis are essential in B. subtilis (see Fig. 1) [5, 6]. This is a striking observation because the systematic gene inactivation was performed in complex growth medium (Luria-Bertani medium containing glucose) in which neither glycolysis nor gluconeogenesis are expected to be necessary. It turned out that several glycolytic enzymes, i.e. PFK, ENO, and PGM, form a complex in vivo
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