Molecular analysis of two fructokinases involved in sucrose metabolism of enteric bacteria

Abstract

Sucrose-positive derivatives of Escherichia coli K-12, containing the plasmid pUR400, and of Klebsiella pneumoniae hydrolyse intracellular sucrose 6-phosphate by means of an invertase into D-glucose 6-phosphate and free D-fructose. The latter is phosphorylated by an ATP-dependent fructokinase (gene scrK of an scr regulon) to D-fructose 6-phosphate. The lack of ScrK does not cause any visible phenotype in wild-type strains of both organisms. Using genes and enzymes normally involved in D-arabinitol metabolism from E. coli C and K. pneumoniae, derivatives of E. coli K-12 were constructed which allowed the identification of scrK mutations on conventional indicator plates. Cloning and sequencing of scrK from sucrose plasmid pUR400 and from the chromosome of K. pneumoniae revealed an open reading frame of 924 bp in both cases--the equivalent of a peptide containing 307 amino acid residues (Mr 39 and 34 kDa, respectively, on sodium dodecyl sulphate gels). The sequences showed overall identity among each other (69% identical residues) and to a kinase from Vibrio alginolyticus (57%) also involved in sucrose metabolism, lower overall identity (39%) to a D-ribose-kinase from E. coli, and local similarity to prokaryotic, and eukaryotic phosphofructokinases at the putative ATP-binding sites.

Genes for D-arabinitol and ribitol catabolism from Klebsiella pneumoniae

“…pneumoniae KAY2026 (Sprenger & Lengeler, 1984), one of the strains used by Lin, Hartley and others in their evolutionary studies (references in Mortlock, 1982). The DNA was treated with HirtdIII before being cloned into the medium-copy-number plasmid pSU18 and trans- constitutively (Tanaka et al, 1967;Lengeler, 1975 ;Aulkemeyer et al, 1991;Heuel et al, 1997). Among several thousand transformants containing HindIII DNA fragments, one Dal' (strong) Mtl' (weak) but Rtlcolony was found.…”

Molecular Analysis of Sucrose Metabolism of Erwinia amylovora and Influence on Bacterial Virulence

“…The corresponding genes were named scrK, scrY, scrA, scrB, and scrR in analogy to other scr regulons. In the region preceding scrK, a 6-bp palindrome (TAAACC/CGTTTA) was detected, similar to the palindrome TAAACC/CCTTTA identified as the binding site of the repressor ScrR in pUR400 and K. pneumoniae (5). The genes scrY, scrA, and scrB are organized in an operon structure with an intergenic space of 18 nucleotides between scrY and scrA and an overlap of scrA and scrB (ATGA), which has also been described for related genes and may indicate translational coupling (42).…”

“…In these bacteria, the uptake of sucrose is mediated via the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS), yielding sucrose 6-phosphate, which is cleaved by an intracellular hydrolase into glucose 6-phosphate and fructose (45,49). The scr regulons of K. pneumoniae and pUR400 consist of four structural genes: scrK codes for an ATP-dependent fructokinase (5), scrY codes for a sucrose-specific porin of the outer membrane (30), scrA codes for enzyme II scr of the PTS, and scrB codes for an intracellular ␤-D-fructofuranoside fructohydrolase (EC 3.2.1.26), which cleaves sucrose 6-phosphate into ␤-D-fructose and ␣-D-glucose 6-phosphate (51). The regulon is controlled by the negative regulator ScrR (34) and is induced in medium containing sucrose, fructose, or raffinose (45,46).…”

Gene expression and characterization of 2-keto-3-deoxy-gluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of Serratia marcescens KCTC 2172

“…Consensus pattern 2 was shown to be involved in ATP binding ability (Aulkemeyer et al 1991). KDGK evidences significant homology, and approximately 70% identity with the KDG kinase of Yersinia pseudotuberculosis.…”