Purification and characterization of osmoregulatory betaine aldehyde dehydrogenase of Escherichia coli

The osmoregulatory NAD-dependent betaine aldehyde dehydrogenase (betaine aldehyde:NAD oxidoreductase, EC 1.2.1.8), of Escherichia coli, was purified to apparent homogeneity from an over-producing strain carrying the structural gene for the enzyme (betB) on the plasmid vector pBR322. Purification was achieved by ammonium sulfate fractionation of disrupted cells, followed by affinity chromatography on 5'-AMP Sepharose, gel-filtration and ion-exchange chromatography. The amino acid composition was determined. The dehydrogenase was found to be a tetramer with identical 55 kDa subunits. Both NAD and NADP could be used as cofactor for the dehydrogenase, but NAD was preferred. The dehydrogenase was highly specific for betaine aldehyde. None of the analogs tested functioned as a substrate, but several inhibited the enzyme competitively. The enzyme was not activated by salts at concentrations encountered during osmotic upshock, but it was salt tolerant, retaining 50% of maximal activity at 1.2 M K+. It is inferred that salt tolerance is an essential property for an enzyme participating in the cellular synthesis of an osmoprotectant.     

Enhanced stress tolerance in Escherichia coli and Nicotiana tabacum expressing a betaine aldehyde dehydrogenase/choline dehydrogenase fusion protein

In Escherichia coli the osmoprotective compound glycine betaine is produced from choline by two enzymes; choline dehydrogenase (CDH) oxidizes choline to betaine aldehyde and then further on to glycine betaine, while betaine aldehyde dehydrogenase (BADH) facilitates the conversion of betaine aldehyde to glycine betaine. To evaluate the importance of BADH, a BADH/CDH fusion enzyme was constructed and expressed in E. coli and in Nicotiana tabacum. The fusion enzyme displayed both enzyme activities, and a coupled reaction could be measured. The enzyme was characterized regarding molecular weight and the dependence of the enzyme activities on environmental factors (salt, pH, and poly(ethylene glycol) addition). At high choline concentrations, E. coli cells expressing BADH/CDH were able to grow to higher final densities and to accumulate more glycine betaine than cells expressing CDH only. The intracellular glycine betaine levels were almost 5-fold higher for BADH/CDH when product concentration was related to CDH activity. Also, after culturing the cells at high NaCl concentrations, more glycine betaine was accumulated. On medium containing 20 mM choline, transgenic tobacco plants expressing BADH/CDH grew considerably faster than vector-transformed control plants.     

Immunolocalization of betaine aldehyde dehydrogenase in porcine kidney.

Polyclonal anti-BADH serum was raised in rabbits against native BADH purified from porcine kidney. The antiserum cross-reacted strongly with BADH purified from kidney, Amaranthus palmierii, and Pseudomona aeuroginosa (1:1000), and weakly with Amaranthus hypochondriacus L (1:100). Antibodies bound to purified native kidney BADH in immunoblots showed a major band of an apparent molecular mass of 340 kDa and a subunit with an apparent molecular mass of 52 kDa. Data on activity assays showed higher activity in cortex sections (81.3 nmol/min/mg protein) than in medulla sections (21.3 nmol/min/mg protein). Immunolocalization of BADH in kidney tissue sections showed that BADH is found in cortex and medulla. In inner medulla, the enzyme was mainly localized in cells surrounding the tubules. Western blot analysis on extracts from the cortex and medulla sections showed higher concentration of BADH protein in cortex than in medulla. These results were in accordance with immunolocalization and activity analysis.     

Overproduction and nucleotide sequence of the respiratory D-lactate dehydrogenase of Escherichia coli.

Recombinant DNA plasmids containing the gene for the membrane-bound D-lactate dehydrogenase (D-LDH) of Escherichia coli linked to the promoter PL from lambda were constructed. After induction, the levels of D-LDH were elevated 300-fold over that of the wild type and amounted to 35% of the total cellular protein. The nucleotide sequence of the D-LDH gene was determined and shown to agree with the amino acid composition and the amino-terminal sequence of the purified enzyme. Removal of the amino-terminal formyl-Met from D-LDH was not inhibited in cells which contained these high levels of D-LDH.     

Overproduction of the MotA protein of Escherichia coli and estimation of its wild-type level.

The motA gene of Escherichia coli was placed under the control of a high-level promoter, that of the tryptophan operon of Serratia marcescens. In the presence of the inducer beta-indoleacrylic acid, MotA was synthesized at greatly elevated levels and inserted without apparent limit into the inner membrane. Growth and motility were impaired, but not drastically so, indicating that MotA by itself does not act as a proton ionophore. Antibody raised against the overproduced protein was used to estimate that a wild-type cell contained 600 +/- 250 copies of MotA. This number is more than would be needed to surround each flagellar basal body with a single circlet of MotA protein; possible interpretations of the result are discussed. The antibody was also used to establish that the MotA protein of Salmonella typhimurium has a similar molecular weight to that of E. coli and is immunologically cross-reactive with it; functional complementation of S. typhimurium motA mutants by the E. coli gene was established.     

Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase.

Mutants of Escherichia coli resistant to allyl alcohol were selected. Such mutants were found to lack alcohol dehydrogenase. In addition, mutants with temperature-sensitive alcohol dehydrogenase activity were obtained. These mutations, designated adhE, are all located at the previously described adh regulatory locus. Most adhE mutants were also defective in acetaldehyde dehydrogenase activity.     

Glyceraldehyde 3-phosphate dehydrogenase mutants of Escherichia coli.

We describe glyceraldehyde 3-phosphate dehydrogenase mutants of Escherichia coli. The gene (gap) is at approximately 34 min, with the transductional order gap-fadD-eda. One gap mutant is temperature sensitive and has a heat-labile enzyme. Another is amber.     

O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli.

O(6)-Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3H-labeled O(6)-methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase.     

Isolation of cDNA and enzymatic properties of betaine aldehyde dehydrogenase from Zoysia tenuifolia

We isolated cDNAs encoding betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) from the salt-tolerant Poaceae, Zoysia tenuifolia by polymerase chain reactions. Zoysia betaine aldehyde dehydrogenase 1 (ZBD1) is 1892bp long and codes for 507 amino acids. The deduced amino acid sequence of ZBD1 is 88% similar to the sequence of rice BADH. Ten cDNA clones were isolated from a cDNA Library of salt-treated Z. tenuifolia by using the ZBD1 fragment as a probe. The proteins coded in some clones were more homologous to BBD2, the cytosolic BADH of barley, than to ZBD1. To investigate their enzymatic properties, ZBD1 and spinach BADH were expressed in Escherichia coli and purified. The optimal pH of ZBD1 was 9.5, which was more alkaline than that of spinach BADH. ZBD1 was less tolerant to NaCl than spinach BADH. ZBD1 showed not only BADH activity but also aminoaldehyde dehydrogenase activity. The Km values of ZBD1 for betaine aldehyde, 4-aminobutyraldehyde (AB-ald), and 3-aminopropionaldehyde (AP-ald) were 291, 49, and 4.0 microM, respectively. ZBD1 showed higher specific activities for AB-ald and AP-ald than did spinach BADH.     

Properties of Escherichia coli mutants deficient in enzymes of glycolysis.

Physiological properties of mutants of Escherichia coli defective in glyceraldehyde 3-phosphate dehydrogenase, glycerate 3-phosphate kinase, or enolase are described. Introduction of a lesion in any one of the reversible steps catalyzed by these enzymes impaired both the glycolytic and gluconeogenic capabilities of the cell and generated an obligatory requirement for a source of carbon above the block (gluconeogenic) and one below (oxidative). A mixture of glycerol and succinate supported the growth of these mutants. Mutants lacking glyceraldehyde 3-phosphate dehydrogenase and glycerate 3-phosphate kinase could grow also on glycerol and glyceric acid, and enolase mutants could grow on glycerate and succinate, whereas double mutants lacking the kinase and enolase required l-serine in addition to glycerol and succinate. Titration of cell yield with limiting amounts of glycerol with Casamino Acids in excess, or vice versa, showed the gluconeogenic requirement of a growing culture of E. coli to be one-twentieth of its total catabolic and anabolic needs. Sugars and their derivatives inhibited growth of these mutants on otherwise permissive media. The mutants accumulated glycolytic intermediates above the blocked enzyme on addition of glucose or glycerol to resting cultures. Glucose inhibited growth and induced lysis. These effects could be substantially overcome by increasing the osmotic strength of the growth medium and, in addition, including 5 mM cyclic adenosine 3',5'-monophosphate therein. This substance countered to a large extent the severe repression of beta-galactosidase synthesis that glucose caused in these mutants.     

Selection, mapping, and characterization of osmoregulatory mutants of Escherichia coli blocked in the choline-glycine betaine pathway.

Osmotically stressed Escherichia coli cells synthesize the osmoprotectant glycine betaine by oxidation of choline through glycine betaine aldehyde (choline----glycine betaine aldehyde----glycine betaine; B. Landfald and A.R. Str?m, J. Bacteriol. 165:849-855, 1986. Mutants blocked at the level of choline dehydrogenase were isolated by selection of strains which did not grow at elevated osmotic strength in the presence of choline but grew when supplemented with glycine betaine. A gene governing the choline dehydrogenase activity was named betA. Mapping by P1 transduction, F' complementation, and deletion mutagenesis showed the betA gene to be located at 7.5 min in the argF-codAB region of the chromosome. Mutants carrying deletions of this region also lacked glycine betaine aldehyde dehydrogenase activity and high-affinity uptake activity for choline; these deletions did not influence the activities of glycine betaine uptake or low-affinity choline uptake, both of which were osmotically regulated.     

Expression of the ogt gene in wild-type and ada mutants of E. coli.

O6-alkylguanine (O6-AlkG) DNA alkyltransferase (ATase) and alkylphosphotriester (AlkP) ATase activity have been quantitated individually in extracts of various E. coli strains by means of ATase specific DNA substrates. O6-AlkG ATase activity was higher than AlkP ATase activity in the wild-type strains F26, AB1157 and SB229 and in the ada- mutants PJ1, PJ3, PJ5 and PJ6 indicating a 5-70 times higher level of expression of the ogt gene than the ada gene. The ada- mutant strains BS23, BS73 and GW5352 expressed O6-AlkG ATase but not AlkP ATase activity indicating expression only of the ogt gene. Southern analysis of DNA from F26, BS23, BS73, PJ1 and GW5352 showed a consistent pattern of hybridisation to an ogt probe but not to an ada probe. Exposure of E. coli to adaptive doses of N-methyl-N-nitro-N-nitroso-guanidine (MeNNG) caused an increase in AlkP ATase activity in F26, AB1156, SB229, PJ1, PJ3, PJ5 and PJ6. O6-AlkG ATase activity also increased in F26, AB1157 and SB229 but decreased to almost undetectable levels in all other strains examined except PJ3 where it remained constant. MeNNG increased ada mRNA abundance in F26 but no ada mRNA was detected in BS23, BS73 or GW5352: there was no evidence for increased ogt mRNA in any of the strains examined. In a limited survey, other bacterial strains have been shown to possess an ogt-like ATase activity.     

Comparison of active mutants and wild-type aspartate transcarbamoylase of Escherichia coli

Two active mutants of aspartate transcarbamoylase from Escherichia coli have been purified from strains which produce large quantities of enzyme. Each enzyme was isolated from a different spontaneous revertant of a pyrimidine auxotrophic strain produced by mutagenesis with nitrogen mustard. Both enzymes exhibit allosteric properties with one having significantly less and the other more cooperativity than wild-type enzyme. Isolated catalytic subunits had different values of Km and Vmax. Studies on hybrids constructed from mutant catalytic and wild-type regulatory subunits (and vice versa) indicate that catalytic chains encoded by pyrB and not the regulatory chains encoded by pyrI were affected by the mutations. Differential scanning calorimetry experiments support these conclusions. Both mutant enzymes undergo ligand-promoted conformational changes analogous to those exhibited by wild-type enzyme: a 3% decrease in the sedimentation coefficient and a 5-fold increase in the reactivity of the sulfhydryl groups of the regulatory chains. Interactions between catalytic and regulatory chains in the mutants are weaker than those in the wild-type enzyme. The gross conformational changes of the mutants upon adding the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate, in the presence of the substrate, carbamoylphosphate, and the activator, ATP, correlate with differences in cooperativity. The mutant with lower cooperativity is more readily converted from the low-affinity, compact, T-state to the high-affinity, swollen, R-state than is wild-type enzyme; this conversion for the more cooperative enzyme is energetically less favorable.     

Characterization of a functional recombinant rat liver aldehyde dehydrogenase: expression as a non-fusion protein in E. coli

A cDNA encoding a rat liver inducible aldehyde dehydrogenase carried in a pUC8 plasmid is expressed in E. coli as a dimeric enzyme molecule functionally and physically identical to the authentic rat enzyme. The cDNA appears to be transcribed using the lac promoter, but is translated from an initiator codon 174 base pairs from the 5' end of the cDNA. The aldehyde dehydrogenase polypeptide is not produced as a fusion protein. This is the first example of the production by E. coli of a catalytically active, multimeric eukaryotic protein which is not a fusion protein.