Evolution of bacterial phosphoglycerate mutases: non-homologous isofunctional enzymes undergoing gene losses, gains and lateral transfers

Background

The glycolytic phosphoglycerate mutases exist as non-homologous isofunctional enzymes (NISE) having independent evolutionary origins and no similarity in primary sequence, 3D structure, or catalytic mechanism. Cofactor-dependent PGM (dPGM) requires 2,3-bisphosphoglycerate for activity; cofactor-independent PGM (iPGM) does not. The PGM profile of any given bacterium is unpredictable and some organisms such as Escherichia coli encode both forms.

Methods/Principal Findings

To examine the distribution of PGM NISE throughout the Bacteria, and gain insight into the evolutionary processes that shape their phyletic profiles, we searched bacterial genome sequences for the presence of dPGM and iPGM. Both forms exhibited patchy distributions throughout the bacterial domain. Species within the same genus, or even strains of the same species, frequently differ in their PGM repertoire. The distribution is further complicated by the common occurrence of dPGM paralogs, while iPGM paralogs are rare. Larger genomes are more likely to accommodate PGM paralogs or both NISE forms. Lateral gene transfers have shaped the PGM profiles with intradomain and interdomain transfers apparent. Archaeal-type iPGM was identified in many bacteria, often as the sole PGM. To address the function of PGM NISE in an organism encoding both forms, we analyzed recombinant enzymes from E. coli. Both NISE were active mutases, but the specific activity of dPGM greatly exceeded that of iPGM, which showed highest activity in the presence of manganese. We created PGM null mutants in E. coli and discovered the ΔdPGM mutant grew slowly due to a delay in exiting stationary phase. Overexpression of dPGM or iPGM overcame this defect.

Conclusions/Significance

Our biochemical and genetic analyses in E. coli firmly establish dPGM and iPGM as NISE. Metabolic redundancy is indicated since only larger genomes encode both forms. Non-orthologous gene displacement can fully account for the non-uniform PGM distribution we report across the bacterial domain.     

Cloning and nucleotide sequences of the genes encoding triose phosphate isomerase, phosphoglycerate mutase, and enolase from Bacillus subtilis.

The Bacillus subtilis genes tpi, pgm, and eno, encoding triose phosphate isomerase, phosphoglycerate mutase (PGM), and enolase, respectively, have been cloned and sequenced. These genes are the last three in a large putative operon coding for glycolytic enzymes; the operon includes pgk (coding for phosphoglycerate kinase) followed by tpi, pgm, and eno. The triose phosphate isomerase and enolase from B. subtilis are extremely similar to those from all other species, both eukaryotic and prokaryotic. However, B. subtilis PGM bears no resemblance to mammalian, fungal, or gram-negative bacterial PGMs, which are dependent on 2,3-diphosphoglycerate (DPG) for activity. Instead, B. subtilis PGM, which is DPG independent, is very similar to a DPG-independent PGM from a plant species but differs from the latter in the absolute requirement of B. subtilis PGM for Mn2+. The cloned pgm gene has been used to direct up to 25-fold overexpression of PGM in Escherichia coli; this should facilitate purification of large amounts of this novel Mn(2+)-dependent enzyme. Inactivation of pgm plus eno in B. subtilis resulted in extremely slow growth either on plates or in liquid, but growth of these mutants was enhanced by supplementation of media with malate. However, these mutants were asporogenous with or without malate supplementation.     

Gene dosage effect on the expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis and Bacillus megaterium

Significant differences in expression of the delta-endotoxin genes cryA1 and cryA2 of Bacillus thuringiensis subsp. kurstaki were observed in B. subtilis and B. megaterium. The cryA1 gene was expressed when present on a high-copy-number (hcn) vector in B. megaterium but not in B. subtilis. The cryA2 gene was expressed in both hosts, but at a higher level in B. megaterium. Expression of the cryA2 gene in B. megaterium was better from a hcn vector than from a low copy number vector. Inhibition of sporulation was observed when the toxin genes were present on hcn plasmids in B. subtilis while no such effect was evident in B. megaterium. In addition, there was a significant reduction in copy numbers in both B. subtilis and B. megaterium when delta-endotoxin genes or a spoVG promoter-containing fragment of DNA were cloned into hcn plasmids.     

Expression of a Bacillus megaterium sporulation-specific gene during sporulation of Bacillus subtilis

The gene for the Bacillus megaterium spore C protein, a sporulation-specific gene, has been transferred into Bacillus subtilis. The B. megaterium gene was expressed little, if at all, during log-phase and early-stationary-phase growth, but was expressed during sporulation with the same kinetics as and at a level similar to that of the analogous B. subtilis genes. This finding is most consistent with the regulation of this class of genes by a mechanism of positive control.     

Molecular dynamics study of interaction and substrate channeling between neuron‐specific enolase and B‐type phosphoglycerate mutase

Phosphoglycerate mutase (PGM) and enolase are consecutive enzymes in the glycolytic pathway. We used molecular dynamics simulation to examine the interaction of human B‐type PGM (dPGM‐B) and neuron‐specific enolase (NSE). Specifically, we studied the interactions of 31 orientations of these enzymes by means of the effective energy function implicit solvation method. Interactions between active regions of the enzymes occurred preferentially, although the strongest interactions appeared to be between the back side of NSE and the active regions of dPGM‐B. Cleavage of 2PG from dPGM‐B was investigated, and the Ser¹⁴–Leu³⁰ loop of dPGM‐B is suggested as a cleavage site and, likely, another entrance site of a ligand. Substrate channeling between the enzymes was observed when NSE with its active regions Leu¹¹–Asn¹⁶, Arg⁴⁹–Lys⁵⁹, and Gly¹⁵⁵–Ala¹⁵⁸ covered the Ser¹⁴–Leu³⁰ loop of dPGM‐B. Analyses of the results make us believe that the channeling between PGM and enolase “benefits” from weak interaction. The probability of formation of channeling favorable complex is estimated to be up to 5%, while functional interaction between NSE and dPGM‐B might be as high as 20%. NSE and dPGM‐B functional interaction seems not to be isotype specific. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Effect of decoyinine on the regulation of alpha-amylase synthesis in Bacillus subtilis.

Decoyinine, an inhibitor of GMP synthetase, allows sporulation in Bacillus subtilis to initiate and proceed under otherwise catabolite-repressing conditions. The effect of decoyinine on alpha-amylase synthesis in B. subtilis, an event which exhibits regulatory features resembling sporulation initiation, was examined. Decoyinine did not overcome catabolite repression of alpha-amylase synthesis in a wild-type strain of B. subtilis but did cause premature and enhanced synthesis in a mutant strain specifically blocked in catabolite repression of alpha-amylase synthesis. Decoyinine had no effect on alpha-amylase enzymatic activity. Thus, it appears that the catabolite control mechanisms governing alpha-amylase synthesis and sporulation in B. subtilis differ in their responses to decoyinine and hence must consist at least partially of separate components.     

The putative DNA translocase SpoIIIE is required for sporulation of the symmetrically dividing coccal species Sporosarcina ureae

The spoIIIE gene of Sporosarcina ureae encodes a 780-residue protein, showing 58% identity to the SpoIIIE protein of Bacillus subtilis, which is thought to be a DNA translocase. Expression of the S. ureae spoIIIE gene is able to restore sporulation in a B. subtilis spoIIIE mutant. Inactivation of the S. ureae spoIIIE gene blocks sporulation of S. ureae at stage III. Within the limits of detection, the sporulation division in S. ureae shows the same symmetry, or near symmetry, as the vegetative division (in contrast to the highly asymmetric location of the sporulation division for B. subtilis), and so it is inferred that SpoIIIE facilitates chromosome partitioning during sporulation, even when the division is not grossly asymmetric. It is suggested that chromosome partitioning lags behind division during sporulation but not during vegetative growth.     

A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination.

The Bacillus subtilis sleB gene, which codes for the enzyme homologous to the germination-specific amidase from Bacillus cereus, was cloned and its nucleotide sequence was determined. Sequence analysis showed that it had an open reading frame of 918 bp, coding for a polypeptide of 305 amino acids with a putative signal sequence of 29 residues. Enzyme activity was not found in germination exudate of B. subtilis spores, which differs from the case of B. cereus enzyme. A B. subtilis mutant with an insertionally inactivated sleB gene revealed normal behavior in growth and sporulation. However, the sleB mutant was unable to complete germination mediated by L-alanine.     

Characterization of cofactor-dependent and cofactor-independent phosphoglycerate mutases from Archaea

Phosphoglycerate mutases (PGM) catalyze the reversible conversion of 3-phosphoglycerate and 2-phosphoglycerate as part of glycolysis and gluconeogenesis. Two structural and mechanistically unrelated types of PGMs are known, a cofactor (2,3-bisphosphoglycerate)-dependent (dPGM) and a cofactor-independent enzyme (iPGM). Here, we report the characterization of the first archaeal cofactor-dependent PGM from Thermoplasma acidophilum, which is encoded by ORF TA1347. This ORF was cloned and expressed in Escherichia coli and the recombinant protein was characterized as functional dPGM. The enzyme constitutes a 46 kDa homodimeric protein. Enzyme activity required 2,3-bisphosphoglycerate as cofactor and was inhibited by vanadate, a specific inhibitor of dPGMs in bacteria and eukarya; inhibition could be partially relieved by EDTA. Histidine 23 of the archaeal dPGM of T. acidophilum, which corresponds to active site histidine in dPGMs from bacteria and eukarya, was exchanged for alanine by site directed mutagenesis. The H23A mutant was catalytically inactive supporting the essential role of H23 in catalysis of the archaeal dPGM. Further, an archaeal cofactor-independent PGM encoded by ORF AF1751 from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus was characterized after expression in E. coli. The monomeric 46 kDa protein showed cofactor-independent PGM activity and was stimulated by Mn²⁺ and exhibited high thermostability up to 70°C. A comprehensive phylogenetic analysis of both types of archaeal phosphoglycerate mutases is also presented.     

Analysis by fluorescence microscopy of the development of compartment-specific gene expression during sporulation of Bacillus subtilis.

The use of a fluorogenic substrate, 5-octanoylaminofluorescein-di-beta-D-galactopyranoside, for beta-galactosidase has made it possible to visualize enzyme activity in individual cells of sporulating populations of Bacillus subtilis by fluorescence microscopy. lacZ fusions to different sporulation-associated genes have been used to investigate the cell compartmentalization of gene expression during sporulation. A strain with a lacZ fusion to sspA, a gene which is transcribed by E-sigma G at a late stage of sporulation, displayed predominantly compartment-specific fluorescence. Expression of the early-expressed spoIIA locus, which includes the structural gene for sigma F, was seen not to be compartmentalized. Populations of strains with lacZ fusions to gpr and dacF, genes which are transcribed by E-sigma F at intermediate stages of sporulation, included some organisms showing uncompartmentalized fluorescence and others showing compartment-specific fluorescence; the proportion showing compartment-specific fluorescence increased in samples taken later in sporulation. Several possible explanations of the results obtained with gpr and dacF are considered. A plausible interpretation is that sigma F activity is initially not compartmentalized and becomes compartmentalized as sporulation progresses. The progression to compartmentalization does not require the activities of the sporulation-specific factor sigma E or sigma G but may require some product of sigma F activity.     

Expression in Bacillus subtilis of the 51- and 42-kilodalton mosquitocidal toxin genes of Bacillus sphaericus

A 3,080-base-pair KpnI-HindIII DNA fragment from Bacillus sphaericus 2362 coding for 51- and 42-kilodalton mosquitocidal proteins was cloned into Bacillus subtilis DB104 by using the vector pUB18. In B. subtilis these proteins were not detected during vegetative growth but were expressed during sporulation at levels comparable to those found in B. sphaericus.     

Expression in Bacillus subtilis of the 51- and 42-kilodalton mosquitocidal toxin genes of Bacillus sphaericus.

A 3,080-base-pair KpnI-HindIII DNA fragment from Bacillus sphaericus 2362 coding for 51- and 42-kilodalton mosquitocidal proteins was cloned into Bacillus subtilis DB104 by using the vector pUB18. In B. subtilis these proteins were not detected during vegetative growth but were expressed during sporulation at levels comparable to those found in B. sphaericus.