Participation of lysine-sensitive aspartokinase in threonine production by S-2-aminoethyl cysteine-resistant mutants of Serratia marcescens.

S-2-Aminoethyl cysteine (AEC) reduced both growth rate and final growth level of Serratia marcescens Sr41. The growth inhibition was completely reversed by lysine. AEC inhibited the activity of lysine-sensitive aspartokinase to a lesser extent than lysine. The AEC addition to the medium lowered not only the level of lysine-sensite aspartokinase but also those of homoserine dehydrogenase and threonine deaminase, whereas lysine repressed the aspartokinase alone. To select mutations releasing lysine-sensitive aspartokinase from feedback controls, AEC-resistant colonies were isolated from strains HNr31 and HNr53, both of which were previously found to excrete threonine on the minimal plates but not on the plates containing excess lysine. Two of 280 resistant colonies excreted large amounts of threonine. Strains AECr174 and AECr301, derived from strains HNr31 and HNr53, respectively, lacked both feedback inhibition and repression of lysine-sensitive aspartokinase. These strains produced about 7 mg of threonine per ml in the medium containing glucose and urea.     

Threonine production by ethionine-resistant mutants of Serratia marcescens.

Ethionine reduced both the growth rate and the final growth level of Serratia marcescens Sr41. Growth inhibition was completely reversed by methionine. Strain D-315, defective in homoserine dehydrogenase I, was more sensitive to ethionine-mediated growth inhibition than was the wild-type strain. Ethionine-resistant mutants were isolated from cultures of strain D-316, which was derived from strain D-315 as a threonine deaminase-deficient mutant. Of 60 resistant colonies, 7 excreted threonine on minimal agar plates. One threonine-excreting strain, ETr17, was highly resistant to ethionine and, moreover, insensitive to methionine-mediated growth inhibition, whereas the parent strain was sensitive. When cultured in minimal medium with or without excess methionine, strain ETr17 had a higher homoserine dehydrogenase level than did strain D-316. The homoserine dehydrogenase activity was not inhibited by threonine or methionine. Transductional analysis revealed that the ethionine-resistant (etr-1) mutation carried by strain ETr17 was located in the metBM-argE region and caused the derepressed synthesis of homoserine dehydrogenase II. Strain ETr17 had a higher aspartokinase level than did the parent strain. By transductional cross with the argE+ marker, the etr-1 mutation was transferred into strain D-562 which was derived from D-505, a strain defective in aspartokinases I and III. The constructed strain had a higher aspartokinase level than did strain D-505 in medium with or without excess methionine, indicating that the etr-1 mutation led to the derepressed synthesis of aspartokinase II. Strain ETr17 produced about 8 mg of threonine per ml of medium containing sucrose and urea.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Proline production by regulatory mutants of Serratia marcescens

Summary Proline-producing strains of Serratia marcescens Sr41 were constructed by three rounds of mutagenesis. A strain SP103 which did not degrade l-proline carried the putA mutation leading to lack of proline oxidase. A 3,4-dehydroproline-resistant mutant SP105, derived from strain SP103, carried the dpr-1 mutation which resulted in desensitization of the feedback inhibition of glutamate kinase. Strain SP103 produced 5.5 mg of l-proline per ml of fermentation medium containing sucrose and urea. Growth inhibition by proline analogs was enhanced when succinate was used as a carbon source in the medium. A thiazolidine-4-carboxylate-resistant mutant SP126 derived from strain SP105 produced 20.5 mg of l-proline per ml of medium. The mutation carried by strain SP126 might be distant from dpr-1 and putA mutations on the chromosome. Pyrroline-5-carboxylate reductase was not repressed by proline in S. marcescens Sr41.     

Conformations of lysine-sensitive aspartokinase.

1. The technique of differential thermal and proteolytic inactivation has been employed as a conformational probe for the lysine-sensitive aspartokinase (EC 2.7.2.4) of Escherichia coli B. 2. L-Amino acid inhibitors of this enzyme each induce a characteristic enzyme conformation. This is evidenced by rates of thermal and proteolytic inactivation and Arrhenius activation energies for thermal inactivation which are characteristic of the amino acid present. 3. Phenylalanine and leucine binding are mutually exclusive as evidenced by competitive behavior in thermal inactivation experiments, suggesting a hydrophobic amino acid binding site with broad specificity. 4. The phenylalanine-dependent conformation and the leucine-dependent conformation differ considerably. In comparison with the native enzyme, the former is more labile to proteolysis by trypsin whereas the latter is more stable. First-order rate constants for thermal inactivation of the phenylalanine- and leucine-dependent conformations are, respectively, about one-half and one-tenth that of the native enzyme. 5. Items 3 and 4 taken together suggest that the conformations are ligand induced and do not arise via ligand stabilization of spontaneously arising conformers.     

L-Histidine production by histidase-less regulatory mutants of Serratia marcescens constructed by transduction.

2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.     

L-Histidine production by histidase-less regulatory mutants of Serratia marcescens constructed by transduction.

2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.     

Superlytic hemolysin mutants of Serratia marcescens.

Hemolysis by Serratia marcescens is caused by two proteins, ShlA and ShlB. ShlA is the hemolysin proper, and ShlB transports ShlA through the outer membrane, whereby ShlA is converted into a hemolysin. Superhemolytic ShlA derivatives that displayed 7- to 20-fold higher activities than wild-type ShlA were isolated. ShlA80 carried the single amino acid replacement of G to D at position 326 (G326D), ShlA87 carried S386N, and ShlA80III carried G326D and N236D. Superhemolysis was attributed to the greater stability of the mutant ShlA derivatives because they aggregated less than the wild-type hemolysin, which lost activity within 3 min at 20 degrees C. In contrast to the highly hemolytic wild-type ShlA at 0 degrees C, the hyperlytic hemolysins were nonhemolytic at 0 degrees C, suggesting that the hyperlytic derivatives differed from wild-type ShlA in adsorption to and insertion into the erythrocyte membrane. However, the size of the pores formed at 20 degrees C by superhemolytic hemolysins could not be distinguished from that of wild-type ShlA. In addition to the N-terminal sequence up to residue 238, previously identified to be important for activation and secretion, sites 326 and 386 contribute to hemolysin activity since they are contained in regions that participate in hemolysin inactivation through aggregation.     

Valine accumulation by alpha-aminobutyric acid-resistant mutants of Serratia marcescens

alpha-Aminobutyric acid, norvaline, and norleucine, which are analogues of branched-chain amino acids, inhibited the growth of Serratia marcescens. The inhibitory effect of these three analogues was counteracted by branched-chain amino acids. A number of mutants resistant to these analogues were isolated. alpha-Aminobutyric acid-resistant (abu-r) mutants markedly accumulated l-valine in the culture medium, but the other analogue-resistant mutants did not. Acetohydroxy acid synthetase, which seems to be rate-limiting for the biosynthesis of l-valine, was derepressed in abu-r mutants. One of the abu-r mutants, no. 140, which accumulated over 8 mg of l-valine per ml, had about a 20-fold increase in the enzyme level. Most of the abu-r mutants had acetohydroxy acid synthetase activity which was sensitive to feedback inhibition by l-valine to the same extent as in the parent strain. However, the enzyme of two of abu-r mutants was less sensitive to l-valine, and one of the two was the best valine accumulator.     

Nucleotide sequence of the Serratia marcescens threonine operon and analysis of the threonine operon mutations which alter feedback inhibition of both aspartokinase I and homoserine dehydrogenase I.

The nucleotide sequence of the Serratia marcescens threonine operon (thrA1A2BC) was determined. Three long open reading frames were identified; these open reading frames code for aspartokinase I (AKI)-homoserine dehydrogenase I (HDI), homoserine kinase, and threonine synthase, in that order. The predicted amino acid sequences of these enzymes were similar to the amino acid sequences of the corresponding enzymes in Escherichia coli. The AKI-HDI protein is apparently a tetramer composed of monomer polypeptides that are 819 amino acids long. A deletion analysis revealed that the central and C-terminal region was responsible for threonine-resistant HDI activity, a monomeric fragment extending from the N terminus to residue 306 was responsible for threonine-resistant AKI activity, and an N-terminal portion containing 468 residues was responsible for threonine-sensitive AKI activity. The thrA(1)1A(2)1 and thrA(1)5A(2)5 mutations of threonine-excreting strains HNr21 and TLr156, which result in the loss of threonine-mediated feedback inhibition of both AKI activity and HDI activity, cause single amino acid substitutions (Gly to Asp at position 330 and Ser to Phe at position 352, respectively) in the central region of the AKI-HDI protein. The thrA1+A(2)2 mutation of strain HNr59, which results in a threonine-sensitive AKI and a threonine-resistant HDI, also causes a single amino acid substitution (Ala to Thr at position 479).     

Lysine excretion by S-(2-aminoethyl)L-cysteine resistant mutants of Bacillus subtilis

S-(2-Aminoethyl)L-cysteine (AEC) at 2 X 10(-1) mM concentration completely inhibited the growth of Bacillus subtilis. This inhibitory effect was readily reversed by 2 X 10(-2) mM L-lysine. Besides L-lysine, L-aspartic acid was only effective of all the natural amino acids tested in reversing the AEC-mediated growth inhibition. AEC resistant mutants of B. subtilis were isolated and found to excrete L-lysine in high yields.     

Culture conditions affect cytotoxin production by Serratia marcescens

Abstract Cytotoxins have been implicated in the pathogenesis of bacterial infections. In this study, the influence of different culture conditions was evaluated on cytotoxin production by Serratia marcescens . Parameters such as culture media, incubation temperature, starting pH of culture medium, aeration, anaerobiosis, carbon sources, iron concentration in the culture media, and release of cell-bound toxin by polymyxin B were investigated. The data suggest that this cytotoxin is predominantly extracellular and is not induced by iron limitation. Aerobic culture with shaking resulted in higher cytotoxicity than static aerobic or anaerobic culture. Bacteria grown in glucose, sucrose or galactose were more cytotoxic than those grown in inositol or maltose. The culture conditions that were identified as optimal for cytotoxin production by Serratia marcescens were incubation temperature ranging from 30 to 37°C, in medium adjusted to pH 8.5, with shaking. This work will contribute to further studies on the identification of this cytotoxic activity.     

Threonine degradation by Serratia marcescens.

The wild strain of Serratia marcescens rapidly degraded threonine and formed aminoacetone in a medium containing glucose and urea. Extracts of this strain showed high threonine dehydrogenase and "biosynthetic" threonine deaminase activities, but no threonine aldolase activity. Threonine dehydrogenase-deficient strain Mu-910 was selected among mutants unable to grow on threonine as the carbon source. This strain did not form aminoacetone from threonine, but it slowly degraded threonine. Strain D-60, deficient in both threonine dehydrogenase and threonine deaminase, was derived from strain Mu-910 and barely degraded threonine. A glycine-requiring strain derived from the wild strain grew in minimal medium containing threonine as the glycine source, whereas a glycine-requiring strain derived from strain Mu-910 did not grow. This indicates that threonine dehydrogenase participates in glycine formation from threonine (via alpha-amino-beta-ketobutyrate) as well as in threonine degradation to aminoacetone.