The Characterization and Cloning of a Gluconate (gnt) Operon of Bacillus subtilis

Abstract

The enzymes involved in gluconate utilization in Bacillus subtilis seemed to be gluconate permease and gluconate kinase. Several mutants unable to grow on gluconate were isolated. The mutations they harboured (gnt) were clustered between iol-6 and $2'~-74 on the B. subtilis chromosome (a tentative map order of gnt-f0, gnt-4, gnt-26, gnt-23 and gnt-9 was obtained). The gnt-f0 mutation seemed to be located within the structural gene of the kinase, and the gnt-23 and gnt-26 mutations seemed to be within that of the permease. An EcoRI fragment (4.5 MDal) containing an intact gluconate (gnt) operon consisting of these two structural genes was cloned in phage $105 by prophage transformation and was mapped physically. The physical location of the mutations coincided with their order on the genetic map. The HindIII-A fragment (2.4 MDal), which corrects all the gnt mutations, was subcloned in plasmid pC194. The fragment contained the structural genes for the gluconate permease and kinase, but not the regulatory region of the gluconate operon. I N T R O D U C T I O NAfter entering the Bacillus subtilis cell, gluconate is phosphorylated to gluconate 6-phosphate, which, because B. subtilis lacks the Entner-Doudoroff pathway, can only be metabolized through the pentose cycle. Therefore, only two enzymes may be needed for the utilization of gluconate as a carbon source; one is gluconate permease, which transports gluconate into the cell, and the other is gluconate kinase, which phosphorylates gluconate. Both enzymes are induced in response to gluconate and their induction is repressed in the presence of rapidly metabolizable carbohydrates such as glucose (Dowds et al., 1978;Nihashi & Fujita, 1984). We have tentatively reported the cloning of the genes for gluconate utilization in a temperate phage, pl1 (Fujita et d., 1983). In this paper, we communicate the genetic mapping and characterization of several gnt mutations that render the cell unable to grow on gluconate and we also report the recloning of an EcoRI fragment cloned in pl 1 in another temperate phage, $105. This fragment encodes an intact gluconate operon. To our knowledge, this work is the first example of the use of a B. suhtilis host -vector system to clone an intact catabolic operon of this species. M E T H O D SBactrriu, phugr und plu.smicis. Strain 6001 5 (trpC2 mrtC7) is our standard strain. Among Gnt-mutants, the isolation of strain 61656 (Aigj hisAl IruAB metB5 trpC2), YF127 (grit-4 trpC2 metC7) and YF029 (gnt-9purA16 IrttAX metB.5 hisA.3) has already been reported (Fujita & Freese, 1981 ;Fujita & Fujita, 1983). Strain YFI 58 (gnt-4 recE4 trpC2) was constructed from YF127 by congression using the DNA of strain lA423 (IrtiAB thr-5 urgAI5 recE4 r-m-), which was obtained from the Bacillus Genetic Stock Center, Ohio State University, Columbus, USA. Strain YF160 (pit-10 try<'? n~r C 7 )was isolated from strain 60015 that had been treated with t Present address:

The gluconate operon gnt of Bacillus subtilis encodes its own transcriptional negative regulator.

“…Strain 61656 (Aigf trpC2 metBS leuA8 hisAl) with a deletion of the entire gnt operon was isolated and genetically characterized as described previously (6,9). The isolation and properties of strain YF160 [gntK10 (formerly gnt-10) trpC2 metC7I affecting the gluconate kinase gene were described previously (3). A 4105 derivative (0105gnt') containing an EcoRI fragment (7.0 kb) that carries the entire gnt operon was constructed by prophage transformation as described previously (3).…”

“…The isolation and properties of strain YF160 [gntK10 (formerly gnt-10) trpC2 metC7I affecting the gluconate kinase gene were described previously (3). A 4105 derivative (0105gnt') containing an EcoRI fragment (7.0 kb) that carries the entire gnt operon was constructed by prophage transformation as described previously (3). Promoter probe vector pPL603B was a gift from D. Rothstein.…”

“…We have characterized and cloned the gluconate (gnt) operon of B. subtilis, which includes the structural genes for gluconate permease and gluconate kinase (3). Recently, we reported the entire nucleotide sequence of the gnt operon (4).…”

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

“…The operons involved in the catabolism of secondary carbon sources are as follows (the carbon sources in parentheses): gntRKPZ (gluconate), 16,35,36) xylAB (xylose), 9) iolABCDEFGHIJ (myo-inositol), 40,41,102) trePAR (trehalose), 25,47,103) galKT (galactose), 25) glpFK (glycerol), 33) glvARC (6-P--glucoside), 34) bglPH ( -glucoside), 26,27) yjlBCD-uxaC-yjmBCD-uxuA-yjmFexuTR-uxaBA (hexuronate), 25,48) xynPB ( -xyloside), 65) yxjC-scoAE-bdh ( -hydroxybutyrate), 25,49) ara-ABDLMNPQ-abfA (arabinose) 24,52,53) and abnA xsa (arabinose), 50) kdgRKAT (hexuronate), 25,42) and kduID (galacturonate). 43) The yxkF-msmX operon probably involved in the transport of unknown sugars has been found to be under CcpA-mediated CCR.…”

Mutations in yhiT enable utilization of exogenous pyrimidine intermediates in Salmonella enterica serovar Typhimurium

“…YhiV shows similarity to gene products involved in the catabolism of sugar-amino acid conjugates, specifically, the conversion of fructosamine 6-phosphates to glucose 6-phosphate and free amino acids in Bacillus subtilis (YurP; 58 %) (Wiame et al, 2004) and the conversion of mannopine to glutamine and mannose in Agrobacterium tumefaciens (MocD; 46 %) (Kim & Farrand, 1996). ORF2 (yhiU, STM3600) is predicted to encode a 31 kDa hydrophobic polypeptide, which displays 48 % similarity to the putative kinase MocE from A. tumefaciens (Kim & Farrand, 1996) and 54 % similarity to the kinase YurL of B. subtilis (Fujita et al, 1986;Wiame et al, 2004).…”