The evolutionary history of the mitochondrial COX2 locus has been used to infer the phylogenetic relationships among 15 peronosporomycete and a hyphochytriomycete species. This molecular phylogenetic analysis at both the ordinal and generic levels provides strong evidence for the recognition of the Saprolegniomycetidae and the Peronosporomycetidae as natural groups, and for the monophyly of the Saprolegniales, Leptomitales and Pythiales. A three amino acid insertion/deletion event (indel) has been identified as a putative synapomorphy for the Saprolegniales. Parsimony mapping of 12 morphological and biochemical characters on the COX2 molecular phylogeny yields an hypothesis for peronosporomycete ancestral states and shared-derived features.

Alleles of the var1 locus on yeast mitochondrial DNA specify the size of var1 ribosomal protein. We report the nucleotide sequence of a var1 allele that determines the smallest var1 protein. It contains an open reading frame of 396 codons, which we identify as the structural gene for var1 protein. The var1 protein specified by this allele has an amino acid composition in close agreement with that predicted by the DNA sequence. The var1 coding region is highly unusual: it is 89.6% AT and contains a 46 bp GC-rich palindromic cluster that accounts for 38% of the total GC residues. Our results strongly suggest that like mammalian mitochondria but unlike those from Neurospora, yeast mitochondria use AUA as a methionine codon. Comparison with the sequence of a var1 allele specifying a larger protein suggests that some size polymorphism of var1 protein results from in-frame insertions of a variable number of AAT (Asn) codons.

A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1,4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone. The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA. The menA gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the Escherichia coli genome between cytR and glpK. DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of amenA mutant. Reverse-phase high-performance liquid chromatography analysis of quinones extracted from theorf-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the menA product into theorf-complemented menA mutant. The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.

The formation of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC), the first identified intermediate in the menaquinone biosynthetic pathway, requires two reactions. They are the decarboxylation of alpha-ketoglutarate by an alpha-ketoglutarate decarboxylase, which results in the formation of succinic semialdehyde-thiamine PPi (TPP) anion, and the addition of the succinic semialdehyde-TPP anion to isochorismate carried out by the enzyme SHCHC synthase. Evidence is provided to support the conclusion that both enzymatic activities are encoded by an extended menD gene which is capable of generating a bifunctional 69-kDa protein. Consistent with the requirement for TPP in the decarboxylation of alpha-ketoglutarate, the translated amino acid sequence contains the characteristic TPP-binding motif present in all well-characterized TPP-requiring enzymes.