Jacqueline A. Greenwood

Molecular analysis of the lac operon encoding the binding‐protein‐dependent lactose transport system and β‐galactosidase in Agrobacterium radiobacter

The genes coding for the binding‐protein‐dependent lactose transport system and β‐galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose‐binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP‐binding protein (lacK) and β‐galactosidase (lacZ). The operon is transcribed in the order lacEFGZK, The operon is controlled by an upstream regulatory region containing putative ‐35 and ‐10 promoter sites, an operator site, a CRP‐binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild‐type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding‐protein‐dependent transport systems in bacteria.

Environmental Regulation of the Methanol Oxidase System of Methylophilus methylotrophus

Summary: Methylophilus methylotrophus was grown in continuous culture at different dilution rates under either methanol or oxygen limitation. Methanol dehydrogenase and cytochrome oxidase aa 3 were maximally repressed in oxygen-limited cultures and also in methanol-limited cultures at dilution rates close to mUmax, whereas cytochrome oxidase co was maximally repressed in methanol-limited cultures and also in oxygen-limited cultures at dilution rates close to μmax. The observed changes in the activity and concentration of methanol dehydrogenase were accompanied by approximately parallel changes in the whole cell respiration rate with ethanol, formaldehyde or acetaldehyde, but not with methanol. These and other results suggest that whole cell methanol oxidase activity reflects respiration from both methanol and its oxidation product, formaldehyde, and is regulated via repression–derepression of methanol dehydrogenase and the cytochrome oxidases. The methanol oxidase system is thus precisely tuned to the concentration of methanol or oxygen in the culture such that the latter is able to maintain a rate of respiration that satisfies the energy demands of the imposed growth rate.

Binding-protein-dependent sugar transport by Agrobacterium radiobacter and A. tumefaciens grown in continuous culture

Binding-protein-dependent sugar transport has been investigated in Agrobacterium radiobacter and A. tumefaciens. A. radiobacter contained two high-affinity glucose-binding proteins (GBP1 and GBP2) that additionally bound D-galactose (KD 0.26 microM) and D-xylose (KD 0.04 microM) respectively and were involved in the transport of these sugars. Partial sequencing of GBP1 and GBP2 showed that GBP2 exhibited significant homology with both the arabinose-binding protein (ABP) and the galactose-binding protein (GalBP) from Escherichia coli, whereas GBP1 exhibited significant homology only with ABP. Antiserum raised against GBP1 cross-reacted with GBP1 but not with GBP2, and vice versa. Anti-GBP1 and anti-GBP2 also cross-reacted with proteins corresponding to GBP1 and GBP2 respectively in A. tumefaciens, but little or no cross-reaction was observed with selected members of the Enterobacteriaceae, Rhizobiaceae and Pseudomonadaceae families grown under glucose limitation. GBP1 was less strongly repressed than GBP2 following batch growth of A. radiobacter on various carbon sources. The growth of A. radiobacter for more than approximately 10 generations in continuous culture under galactose or xylose limitation (D 0.045 h-1) led to the emergence of new strains which exhibited increased rates of glucose/galactose or glucose/xylose uptake, and which respectively hyperproduced GBP1 (strain AR18a) or GBP2 (strain AR9a). Similarly, growth of A. tumefaciens for more than approximately 15 generations under glucose or galactose limitation produced new strains which exhibited increased rates of glucose/xylose or glucose/galactose uptake and which respectively hyperproduced proteins analogous to GBP2 (strain AT9) or GBP1 (strain AT18a). It is concluded that growth of Agrobacterium species under carbon-limited conditions leads to the predictable emergence of new strains which specifically hyperproduce the transport system for the limiting nutrient. The GBP1-dependent system of A. radiobacter is unique amongst these transport systems in that the mutations that lead to its hyperproduction under carbon limitation render it least susceptible to repression by excess glucose during ammonia limitation, with the result that succinoglucan exopolysaccharide is produced from glucose at an enhanced rate.

The Relationship between Glucose Transport and the Production of Succinoglucan Exopolysaccharide by Agrobacterium radiobacter

Agrobacterium radiobacter NCIB 11883 was grown in ammonia-limited continuous culture at low dilution rate with glucose as the carbon source. Under these conditions the organism produced an extracellular succinoglucan polysaccharide and transported glucose using the same periplasmic glucose-binding proteins (GBP1 and GBP2) as during glucose-limited growth. Transition from glucose- to ammonia-limited growth was accompanied by a very rapid decrease in glucose uptake capacity, whereas the glucose-binding proteins were diluted out much more slowly (t1/2 approximately 1 h and 14 h respectively). Although the rate of glucose uptake and the concentrations of GBP1 and GBP2 were much lower during ammonia limitation, the activities of enzymes involved in the early stages of glucose metabolism and in the production of succinoglucan precursors were essentially unchanged. Glucose transport was also investigated in two new strains of A. radiobacter which had been isolated following prolonged growth under glucose limitation. Glucose uptake by strain AR18 was significantly less repressed during ammonia limitation compared with either the original parent strain or strain AR9, and this was reflected both in its relatively high concentration of GBP1 and in its significantly higher rate of succinoglucan synthesis. Flux control analysis using 6-chloro-6-deoxy-D-glucose as an inhibitor of glucose transport showed that the latter was a major kinetic control point for succinoglucan production. It is concluded that glucose uptake by A. radiobacter, particularly via the GBP1-dependent system, is only moderately repressed during ammonia-limited growth and that the organism avoids the potentially deleterious effects of accumulating excess glucose by converting the surplus into succinoglucan.

An outer-membrane porin inducible by short- chain amides and urea in the methylotrophic bacterium Methylophilus methylotrophus

The fmdA and fmdB genes encoding formamidase and a putative regulatory protein, respectively, from the methylotrophic bacterium Methylophilus methylotrophus were recloned with additional flanking DNA (pSW1). fmdC, encoding a weakly hydrophilic protein containing an N-terminal signal sequence, was identified upstream of fmdAB. The derived amino acid sequence of mature FmdC (M(r) 39204) showed that it was rich in beta-sheet and aromatic amino acids, and exhibited significant similarities to several outer-membrane porins from other bacteria. Cell fractionation studies showed that the protein was located in the outer membrane. Mature FmdC was purified and shown to consist of a single type of subunit (M(r) 40,000) with the predicted N-terminal amino acid sequence (GATISF-). SDS-PAGE and Western blotting of cells grown in continuous culture under various conditions showed that mature FmdC was induced by formamide, acetamide and urea, repressed by excess ammonia, and over-expressed during prolonged growth under formamide limitation. It is concluded that mature FmdC is a porin involved in the transport of short-chain amides and urea through the outer membrane of M. methylotrophus under conditions where these nitrogen sources are present at very low concentration.

Binding-protein-dependent glucose transport by Agrobacterium radiobacter grown in glucose-limited continuous culture.

Agrobacterium radiobacter NCIB 11883 was grown in glucose-limited continuous culture at low dilution rate. Whole cells transported glucose using an energy-dependent mechanism which exhibited an accumulation ratio greater than 2000. Three major periplasmic proteins were purified and their potential role as glucose-binding proteins (GBP) were investigated using equilibrium dialysis. Two of these, GBP1 (Mr 36,500) and GBP2 (Mr 33,500), bound D-glucose with high affinity (KD 0.23 and 0.07 microM respectively), whereas the third protein (Mr 30,500) showed no binding ability. Competition experiments using various analogues showed that those which differed from glucose at C-6 (e.g. 6-chloro-6-deoxy-D-glucose and 6-deoxy-D-glucose) variably decreased the binding of glucose to both GBP1 and GBP2, whereas those which differed at C-4 (e.g. D-galactose) were only effective with GBP1. The rate of glucose uptake and the concentration of the glucose-binding proteins increased in parallel during prolonged growth under glucose-limitation due to the emergence of new strains in which GBP1 (e.g. strain AR18) or GBP2 (e.g. strain AR9), but not both, was hyperproduced and accounted for at least 27% of the total cell protein. It is concluded that A. radiobacter synthesizes two distinct periplasmic binding proteins which are involved in glucose transport, and that these proteins are maximally derepressed during growth under glucose limitation.

Environmental Regulation of Methanol and Formaldehyde Metabolism by Methylophilus methylotrophus

Summary: Methylophilus methylotrophus was grown in continuous culture at different dilution rates under either methanol or oxygen limitation. Methanol dehydrogenase and formate oxidation were repressed by the presence of high concentrations of methanol in the growth medium, whereas hexulose phosphate synthase was constitutive and the glucose-6-phosphate and 6-phospho-gluconate dehydrogenases were regulated only by the growth rate. Chemical analysis and 13C NMR spectroscopy showed that the oxidation of methanol was accompanied by the spillage of 0 to 50% of the input carbon as formaldehyde and dihydroxyacetone, the extent of which reflected the methanol dehydrogenase: hexulose phosphate synthase activity ratio and/or the energy status of the cells. It is concluded that the observed regulation of key enzymes of methanol and formaldehyde metabolism by the growth environment occurs in order to achieve the required in situ rates of energy conservation and carbon flux demanded by the imposed growth rate, and that the spillage of formaldehyde and dihydroxyacetone reflects limitations on metabolism imposed by kinetic and/or metabolite pool effects.

Agrobacterium radiobacter and related organisms take up fructose via a binding-protein-dependent active-transport system

Washed cells of Agrobacterium radiobacter prepared from a fructose-limited continuous culture (D 0.045 h-1) transported D(-)[U-14C]fructose in a linear manner for up to 4 min at a rate several-fold higher than the rate of fructose utilization by the growing culture. D(-)[U-14C]Fructose transport exhibited a high affinity for fructose (KT < 1 microM) and was inhibited to varying extents by osmotic shock, by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and by unlabelled sugars (D-fructose/D-mannose > D-ribose > D-sorbose > D-glucose/D-galactose/D-xylose; no inhibition by D-arabinose). Prolonged growth of A. radiobacter in fructose-limited continuous culture led to the selection of a novel strain (AR100) which overproduced a fructose-binding protein (FBP) and showed an increased rate of fructose transport. FBP was purified from osmotic-shock fluid using anion-exchange fast protein liquid chromatography (FPLC). The monomeric protein (M(r) 34,200 by SDS-PAGE and 37,700 by gel-filtration FPLC) bound D-[U-14C]-fructose stoichiometrically (1.17 nmol nmol FBP-1) and with high affinity (KD 0.49 microM) as shown by equilibrium dialysis. Binding of D-[U-14C]fructose by FBP was variably inhibited by unlabelled sugars (D-fructose/D-mannose > D-ribose > D-sorbose; no inhibition by D-glucose, D-galactose or D-arabinose). The N-terminal amino acid sequence of FBP (ADTSVCLI-) was similar to that of several sugar-binding proteins from other species of bacteria. Fructose transport and FBP were variably induced in batch cultures of A. radiobacter by growth on different carbon sources (D-fructose > D-ribose/D-mannose > D-glucose; no induction by succinate). An immunologically similar protein to FBP was produced by Agrobacterium tumefaciens and various species of Rhizobium following growth on fructose. It is concluded that fructose is transported into A. radiobacter and related organisms via a periplasmic fructose/mannose-binding-protein-dependent active-transport system, in contrast to the phosphotransferase system used by many other species of bacteria.

Isolation of novel strains of Agrobacterium radiobacter with altered capacities for lactose metabolism and succinoglucan production

SUMMARY: Agrobacterium radiobacter NCIB 11883 was grown in continuous culture at low dilution rate under lactose limitation. Washed cells rapidly transported lactose [and its non-metabolizable analogue MTG (methyl β-d-thiogalactoside)] via a lactose-binding protein (LBP)-dependent uptake system, and subsequently hydrolysed the lactose using a highly active β-galactosidase composed of two identical subunits of M r approximately 86000. Growth under these conditions for <40 generations led to the selection of a novel strain (AR50) which overexpressed both LBP and β-galactosidase, and exhibited lactose (MTG) uptake and β-galactosidase activities that were two to three times those of the wild-type organism. Both enzymes were expressed constitutively in strain AR50, in contrast to the wild-type organism, but remained subject to catabolite repression (particularly by galactose). Southern blotting of restricted DNA from the two organisms using an oligonucleotide probe for the structural gene for LBP showed a 2·7-fold amplification in strain AR50, together with a deletion of at least 1·7 kb which may be part of a regulatory gene. The wild-type organism and strain AR50 exhibited similar lactose (MTG) uptake rates during growth under ammonia limitation, and also synthesized an exocellular succinoglucan polysaccharide at only marginally different rates [q p 0·21--0·24 g h−1 (g cells)−1]; both organisms exhibited a flux control coefficient for lactose uptake on succinoglucan production of > 0·45, indicating that lactose uptake is a major kinetic control point for polysaccharide production. Growth of strain AR50 under ammonia limitation for > 40 generations led to the selection of another novel strain (AR60) which exhibited a decreased q p [0·16 g h−1 (g cells)−1]. Washed cells of strain AR60 exhibited significantly lower rates of lactose (MTG) uptake than strain AR50, an observation which was commensurate with the rate of polysaccharide production being predominantly controlled by the rate of lactose uptake, but β-galactosidase activity was substantially higher. Both the lactose uptake system and β-galactosidase were expressed constitutively in strain AR60, but catabolite repression of β-galactosidase was much weaker than in the wild-type organism or strain AR50.

Expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

The classical glycolytic pathway contains an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, with NADP-dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP.