Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci

Abstract

The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.

FIG. 1

Biosynthesis of IPP via the mevalonate pathway.

FIG. 2

Alignment of the conserved regions of the mevalonate pathway enzymes from gram-positive bacteria, B. burgdorferi, P. mevalonii, and eukaryotes. Bold type indicates that amino acids are identical in all proteins. The asterisks indicate amino acids proposed to function in catalysis and substrate binding. Dashes indicate gaps introduced into the sequence to optimize the alignment. Numbers refer to amino acid positions. In all alignments, abbreviations are as follows: EFA, E. faecalis; EFM, E. faecium; SEP, S. epidermidis; SHA, S. haemolyticus; SAU, S. aureus; SPN, S. pneumoniae; SPY, S. pyogenes; BBO, B. burgdorferi (AE001169). In HMG-CoA synthase, abbreviations are as follows: SCA, Staphylococcus carnosus (U450157); HUM, human cytoplasmic enzyme (Q01581); MIT, human mitochondrial enzyme (NP005509); SAC, S. cerevisiae (P54839). In HMG-CoA reductase, abbreviations are as follows: PME, P. mevalonii (P13702). In mevalonate kinase, abbreviations are as follows: HUM, human (NP000422); SAC, S. cerevisiae (NP000422). In phosphomevalonate kinase, abbreviations are as follows: SAC, S. cerevisiae (P24521). In mevalonate diphosphate decarboxylase, abbreviations are as follows: HUM, human (AAC50440); SAC, S. cerevisiae (AAC49252).

FIG. 3

Phylogenetic trees of HMG-CoA synthase (A), HMG-CoA reductase (B), mevalonate and phosphomevalonate kinase (C), and mevalonate diphosphate decarboxylase (D). Trees were constructed using the NJ method as implemented by the program NEIGHBOR of the PHYLIP 3.57c package (16). The scale bar represents 0.1 expected amino acid replacements per site as estimated by the program PROTDIST using the Dayhoff PAM-120 substitution matrix. Branches containing or linking low-G+C gram-positive coccal species are colored red, those of eukaryotes are blue, while those of archaea and other bacteria are black. Numbers at the branching points represent the percent occurrence in 1,000 random bootstrap replications of MP and NJ analyses. Values less than 50% are not shown or are indicated by a dash (–), while nodes supported in more than 90% of bootstrap replications for both methods are marked with an asterisk (∗). Nodes separating classes I and II of HMG-CoA reductase as well as the mevalonate (mvaK1) and phosphomevalonate (mvaK2) kinases are indicated.

FIG. 4

Organization of the mevalonate pathway genes in gram-positive cocci and B. burgdorferi. Numbers above the genes indicate the number of nucleotides between genes, and numbers below indicate the length of each gene. ypgA has homology to a gene clustered with carotenoid biosynthetic genes in E. herbicola (22).

FIG. 5

Chromosomal analysis of the S. pneumoniae mvaS null mutant. (A) The chromosome of the mvaS null mutant contains an allelic replacement of mvaS (dotted block arrow, 1,194 bp) from nucleotides 166 to 1161 with the ermAM gene (gray block arrow). The translationally coupled downstream gene (mvaA) and the putative upstream promoter (dotted arrow) remain intact in the mutant. Oligonucleotide primers (small arrows) were designed to hybridize within the ermAM gene and to regions flanking mvaS and were used in PCRs against chromosomal DNA template prepared from the mutant. (B) Reactions 1, 2, 4, and 5 generated characteristic PCR products (shown), which could not be amplified with chromosomal DNA from the wild-type parent strain (not shown). Reactions 3 and 6 generated control fragments, which could be amplified in the mutant and parent strains. (C) Wild-type and mutant chromosomal DNA was restricted with HinfI, subjected to agarose gel electrophoresis, and probed with labeled PCR fragment 6 (bold line). The predicted band sizes are 696 bp in the mutant and 1396 bp in the wild-type strain, since the left HinfI site (labeled 1 in panel A) is deleted in the mutant and replaced by the ermAM gene containing another HinfI site (labeled 2).