Novel Activities of Glycolytic Enzymes in Bacillus subtilis

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. Glycolysis is a central metabolic pathway that appeared early in the evolution of life (1). Major functions of the glycolytic pathway are the generation of precursors for anabolic reactions and the conservation of energy that is needed to fuel all other cellular processes. The glycolytic pathway is conventionally divided into two parts: (i) the upper part, also referred to as the preparatory phase of glycolysis because the reactions of this part consume energy to convert the incoming sugars to triose phosphates, and (ii), the lower part or payoff phase that is characterized by the net gain of energy and the formation of reduction equivalents. Although glycolysis is highly conserved from archaea and bacteria to man not all organisms use this pathway for the oxidization of glucose. Escherichia coli is able to oxidize glucose via glycolysis, but the Entner-Doudoroff and the pentose phosphate pathways may replace the preparatory phase. In contrast to E. coli, the Entner-Doudoroff pathway is not present in Bacillus subtilis (2). Interestingly the enzymes of the upper glycolytic part seem to be completely absent in many archaea (3, 4). These few examples show that there is a high plasticity in how archaea and bacteria can feed glucose into the triose phosphate part of glycolysis. This plasticity is in good agreement with the observation that...

5′ End-independent RNase J1 Endonuclease Cleavage of Bacillus subtilis Model RNA

“…Our hypothesis to explain the extreme stability of 5Ј-SSSϩNotI RNA in the RNase J1 mutant strain, which involves resistance to RNase J1 5Ј-exonuclease activity, cannot apply to RNase Y, as the latter enzyme has only endonuclease activity. Rather, the recent evidence that RNase Y exists in a degradosome complex that includes RNase J1 (18,32) may suggest that the effect of depleting RNase Y on trp leader RNA decay is an indirect one, resulting from a negative effect on RNase J1 activity. We believe that the close correlation of half-lives for all four constructs in the RNase J1 and RNase Y mutant strains lends credence to this hypothesis.…”

“…Involvement of RNase Y in trp Leader RNA Decay-When our model for RNase J1-mediated decay of trp leader RNA was first formulated, we were not aware of the existence of another essential B. subtilis endoribonuclease, RNase Y (18,19). There are indications that RNase Y, like RNase E in E. coli, is a central element of an RNA degradosome complex that includes RNase J1 and PNPase (18,32).…”

“…There are indications that RNase Y, like RNase E in E. coli, is a central element of an RNA degradosome complex that includes RNase J1 and PNPase (18,32). We investigated the effect of reducing RNase Y expression on trp leader RNA decay, using a strain in which RNase Y expression is under IPTG-inducible control and is grown in 1 mM ITPG, as above for the RNase J1 mutant strain.…”

“…The role that the more recently discovered RNase Y (18,19), which is thought to play an even larger role than RNase J1 in bulk mRNA turnover (19), plays in trp leader RNA was also assessed. In addition, derivatives of trp leader RNA were constructed to show that the mechanism of trp leader RNA decay was not dependent on its unique characteristics, most notably TRAP binding, which suggests that trp leader RNA can serve as a model for general RNA decay mechanisms.…”

The Blueprint of a Minimal Cell: MiniBacillus

“…Conflicting reports are available for RNases in B. subtilis. The endoribonuclease RNase Y and the 5=-to-3= exonuclease RNase J1 have long been considered to be essential, whereas a recent report suggests that these proteins are dispensable (34,(67)(68)(69). However, the strong expression of the corresponding genes suggests that they should be part of a minimal cell.…”