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Biochemical, Molecular, and Genetic Analyses of the Acetone Carboxylases from Xanthobacter autotrophicus Strain Py2 and Rhodobacter capsulatus Strain B10. Miriam K. Sluis, 2002.Acetone carboxylase is the key enzyme of bacterial acetone metabolism, catalyzing the condensation of acetone and CO2 to form acetoacetate . In this study, the acetone carboxylase of the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus was purified to homogeneity and compared to that of Xanthobacter autotrophicus strain Py2, the only other organism from which an acetone carboxylase has been purified . The biochemical properties of the enzymes were virtually indistinguishable, with identical subunit compositions (  2ß2 2 multimers of 85-, 78-, and 20-kDa subunits), reaction stoichiometries (CH3COCH3 + CO2 + ATP CH3COCH2COO- + H+ + AMP + 2Pi), and kinetic properties (Km for acetone, 8 µM; kcat = 45 min-1) . Both enzymes were expressed to high levels (17 to 25% of soluble protein) in cells grown with acetone as the carbon source but were not present at detectable levels in cells grown with other carbon sources . The genes encoding the acetone carboxylase subunits were identified by transposon mutagenesis of X . autotrophicus and sequence analysis of the R . capsulatus genome and were found to be clustered in similar operons consisting of the genes acxA (ß subunit), acxB ( subunit), and acxC ( subunit) . Transposon mutagenesis of X . autotrophicus revealed a requirement of
 54 and a
54-dependent transcriptional activator (AcxR) for acetone-dependent growth and acetone carboxylase gene expression . A potential
54-dependent promoter 122 bp upstream of X . autotrophicus acxABC was identified . An AcxR gene homolog was identified 127 bp upstream of acxA in R . capsulatus, but this activator lacked key features of
54-dependent activators, and the associated acxABC lacked an apparent
54-dependent promoter, suggesting that
54 is not required for expression of acxABC in R . capsulatus. These studies reveal a conserved strategy of ATP-dependent acetone carboxylation and the involvement of transcriptional enhancers in acetone carboxylase gene expression in gram-negative acetone-utilizing bacteria .
Metabolism of Fructooligosaccharides by Lactobacillus paracasei 1195. Handan Kaplan, 2003.Fermentation of fructooligosaccharides (FOS) and other oligosaccharides has been suggested to be an important property for the selection of bacterial strains used as probiotics . However, little information is available on FOS transport and metabolism by lactic acid bacteria and other probiotic bacteria . The objectives of this research were to identify and characterize the FOS transport system of Lactobacillus paracasei 1195 . Radiolabeled FOS was synthesized enzymatically from [3H]sucrose and purified by column and thin-layer chromatography, yielding three main products: glucose (G)  -1,2 linked to two, three, or four fructose (F) units (GF2, GF3, and GF4, respectively) . FOS hydrolysis activity was detected only in cell extracts prepared from FOS- or sucrose-grown cells and was absent in cell supernatants, indicating that transport must precede hydrolysis . FOS transport assays revealed that the uptake of GF2 and GF3 was rapid, whereas little GF4 uptake occurred . Competition experiments showed that glucose, fructose, and sucrose reduced FOS uptake but that other mono-, di-, and trisaccharides were less inhibitory . When cells were treated with sodium fluoride, iodoacetic acid, or other metabolic inhibitors, FOS transport rates were reduced by up to 60%; however, ionophores that abolished the proton motive force only slightly decreased FOS transport . In contrast, uptake was inhibited by ortho-vanadate, an inhibitor of ATP-binding cassette transport systems . De-energized cells had low intracellular ATP concentrations and had a reduced capacity to accumulate FOS . These results suggest that FOS transport in L . paracasei 1195 is mediated by an ATP-dependent transport system having specificity for a narrow range of substrates .
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