Mutants of Escherichia coli with altered level of beta-N-acetylglucosaminidase activities.

Five strains of Escherichia coli with reduced level of beta-N-acetylglucosaminidase activity were isolated. The mutations responsible for reduced activity of the enzyme have been localized in one of the strains between 25 and 27 minutes on the genetic map of E. coli. The mutants do not differ morphologically from the original strain. This suggest that beta-N-acetylglucosaminidase is of the secondary importance in the biosynthesis of murein.     

Mutants of Escherichia coli with an altered tyrosyl-transfer ribonucleic acid synthetase

We have isolated several mutants defective in the gene for tyrosyl-transfer ribonucleic acid (tRNA) synthetase (tyrS). One of these mutants is described in detail. It was isolated as a tyrosine auxotroph with defects both in the tyrosyl-tRNA synthetase and in the tyrosine biosynthetic enzyme, prephenate dehydrogenase. It also had derepressed levels of the tyrosine-specific 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase. The latter finding suggested that a wild-type tyrS gene was required for repression of the tyrosine biosynthetic enzymes. The following results demonstrated that this hypothesis was not correct. (i) When the defective tyrS gene was transferred to another strain, the tyrosine-specific DAHP synthetase in that strain was not derepressed, and (ii) two other mutants with defective tyrosyl-tRNA synthetases had repressed levels of the tyrosine biosynthetic enzymes. The tyrS gene was located near minute 32 on the Escherichia coli chromosome by interrupted mating experiments.     

Effect of redox potential on activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli

This report elucidates the distinctions of redox properties between two uptake hydrogenases in Escherichia coli. Hydrogen uptake in the presence of mediators with different redox potential was studied in cell-free extracts of E. coli mutants HDK103 and HDK203 synthesizing hydrogenase 2 or hydrogenase 1, respectively. Both hydrogenases mediated H(2) uptake in the presence of high-potential acceptors (ferricyanide and phenazine methosulfate). H(2) uptake in the presence of low-potential acceptors (methyl and benzyl viologen) was mediated mainly by hydrogenase 2. To explore the dependence of hydrogen consumption on redox potential of media in cell-free extracts, a chamber with hydrogen and redox ( E(h)) electrodes was used. The mutants HDK103 and HDK203 exhibited significant distinctions in their redox behavior. During the redox titration, maximal hydrogenase 2 activity was observed at the E(h) below -80 mV. Hydrogenase 1 had maximum activity in the E(h) range from +30 mV to +110 mV. Unlike hydrogenase 2, the activated hydrogenase 1 retained activity after a fast shift of redox potential up to +500 mV by ferricyanide titration and was more tolerant to O(2). Thus, two hydrogenases in E. coli are complementary in their redox properties, hydrogenase 1 functioning at higher redox potentials and/or at higher O(2) concentrations than hydrogenase 2.     

Metal binding activity of the Escherichia coli hydrogenase maturation factor HypB

The formation of the [NiFe] metallocenter of Escherichia coli hydrogenase 3 requires the participation of proteins encoded by the hydrogenase pleiotropy operon hypABCDEF. The insertion of Ni(II) into the precursor enzyme follows the incorporation of the iron center and is the function of HypA, a Zn(II)-binding protein, and HypB, a GTPase. The Ni(II) donor and the mechanism of transfer of Ni(II) into the hydrogenase precursor protein are not known. In this study, we demonstrate that HypB is a nickel-binding protein capable of binding 1 equiv of Ni(II) with a K(d) in the sub-picomolar range. In addition, HypB has a weaker metal-binding site that is not specific for Ni(II) over Zn(II). Examination of the isolated C-terminal GTPase domain revealed that the high-affinity metal binding capability was severely abrogated but the low-affinity site was intact. By mutating conserved cysteine and histidine residues in E. coli HypB, we have localized the high-affinity Ni(II)-binding site to an N-terminal CXXCGC motif and the low-affinity metal-binding site to the GTPase domain. A model for the function of HypB during the Ni(II) loading of hydrogenase is proposed.     

Transketolase Mutants of Escherichia coli

Transketolase mutants have been selected after ethyl methane sulfonate mutagenesis of Escherichia coli. These strains are unable to grow on any pentose and, in addition, require a supplement of aromatic amino acids or shikimic acid for normal growth on any other carbon source. Revertants are normal in both respects and also contain transketolase. Transketolase mutants do not require exogenous pentose for growth. Preliminary genetic mapping of the locus is presented.     

Nonchemotactic Mutants of Escherichia coli

We have isolated 40 mutants of Escherichia coli which are nonchemotactic as judged by their failure to swarm on semisolid tryptone plates or to make bands in capillary tubes containing tryptone broth. All the mutants have normal flagella, a fact shown by their shape and reaction with antiflagella serum. All are fully motile under the microscope and all are sensitive to the phage chi. Unlike its parent, one of the mutants, studied in greater detail, failed to show chemotaxis toward oxygen, glucose, serine, threonine, or aspartic acid. The failure to exhibit chemotaxis does not result from a failure to use the chemicals. The swimming of this mutant was shown to be random. The growth rate was normal under several conditions, and the growth requirements were unchanged.     

Porphyrin-Accumulating Mutants of Escherichia coli

Four mutants (pop-1, pop-6, pop-10, and pop-14) which accumulate a red water-insoluble pigment were obtained in Escherichia coli K-12 AB1621. For each mutant, the red pigment was shown to be protoporphyrin IX, a late precursor of heme. Mutagenic treatment of mutant pop-1 yielded a secondary mutant, pop-1 sec-20, which accumulated a brown water-soluble pigment. The brown pigment was shown to be coproporphyrin III. Mutant pop-1 resembled the parental strain in its cytochrome absorption spectrum, catalase activity, and ability to grow on nonfermentable carbon and energy sources; therefore, its ability to produce and utilize heme was unimpaired. Judged on the same criteria, the secondary mutant, pop-1 sec-20, was partially heme and respiratory deficient. Growth in anaerobic conditions decreased by 25% the accumulation of protoporphyrin by pop-1; under the same conditions, pop-1 sec-20 did not accumulate coproporphyrin or coproporphyrinogen. The mutations causing protoporphyrin accumulation in all four pop mutants were found to map in the lac to purE (10-13 min) region of the E. coli chromosome. In the case of mutant pop-1, the mutation was shown to be strongly linked to the tsx locus (12 min). In mutant pop-1 sec-20, the second mutation causing coproporphyrin accumulation was co-transducible with the gal locus at a frequency of 88 to 96%. The mechanism of porphyrin accumulation by the mutants is discussed.     

Reversible activation of hydrogenase from Escherichia coli

Hydrogenase from Escherichia coli exhibited low activity when assayed for hydrogen:methyl viologen reductase activity and no activity when assayed for hydrogen-uptake activity with acceptors of high redox potential (dichloroindophenol, methylene blue). Nor did the enzyme as isolated catalyse proton-tritium exchange activity. Incubation under hydrogen resulted in an increase in hydrogen-uptake activity with methyl viologen and the appearance of hydrogen-uptake activity with dichloroindophenol and methylene blue. Following such treatment, the enzyme also readily catalysed isotope exchange. This process is interpreted as the conversion of the hydrogenase from an inactive 'unready' state to an 'active' state. Oxidation of active hydrogenase with dichloroindophenol caused conversion to a state resembling that of the enzyme as isolated but capable of more rapid activation under reducing conditions. This form is termed the 'ready' state. Such interconversions have been reported for hydrogenases from Desulfovibrio gigas and D. desulfuricans, and the possibility that they constitute a regulatory mechanism suggested.     

Mutants of Escherichia coli K-12 with altered excision efficiency of transposons Tn5 and Tn9

HFETn5, HFETn9 and LFETn9 mutants of Escherichia coli K-12 have been isolated. The frequency of Tn5 precise excision from the chromosomal lac operon is increased 3-660-fold in nine HFETn5 mutants. The majority of these mutations have no influence on the efficiency of precise excision of transposon Tn9, though hfeTn5-04 and hfeTn5-06 mutations decrease excision efficiency 2-13-fold. The Tn9 transposon is excised in HFETn9 mutant about 20-fold more efficiently than in the wild type strain. This mutation does not stimulate excision of Tn5 and Tn10. LfeTn9 mutation decreases excision frequency of Tn9 11-17-fold, but has no effect on Tn5 excision and increases that of Tn10 about 20-fold. The differences in genetic control and mechanisms of excision of the transposons with long and short inverted repeats are discussed.     

Mutants of Escherichia coli specifically deficient in respiratory formate dehydrogenase activity

Escherichia coli K12 mutants lacking phenazine-methosulphate-linked formate dehydrogenase (FDH-PMS) activity, but still capable of producing normal levels of benzyl-viologen-linked formate dehydrogenase (FDH-BV) and nitrate reductase activities, have been isolated following P1 localized mutagenesis. The relevant mutations mapped with the same cotransduction frequency close to the rhaD gene, at 88 min on the E. coli chromosome. They were further subdivided into two classes. Class I consisted of six fdhD mutants which synthesized an inactive FDH-PMS protein with the same subunit composition as the wild-type enzyme. In contrast, class II contained four fdhE mutants totally devoid of this antigen. Construction of merodiploid strains harbouring various combinations of the mutated alleles, fdhE on the episome and fdhD on the chromosome, led to the restoration of FDH-PMS activity by complementation of the products encoded by the respective wild-type alleles. Difference spectroscopy suggested that both fdhD and fdhE mutants contained normal amounts of the cytochrome b559 associated with FDH-PMS although the cytochrome had lost its capacity for formate-dependent reduction.     

Partial characterization of an electrophoretically labile hydrogenase activity of Escherichia coli K-12

A mutant of Escherichia coli K-12 is described that is specifically impaired in only one hydrogenase isoenzyme. By means of Tn5-mediated insertional mutagenesis, a class of mutants was isolated (class I) that had retained 20% of the overall hydrogenase activity. As determined by neutral polyacrylamide gel electrophoresis, the mutant contained normal amounts of the hydrogenase isoenzymes 1 and 2. Therefore, the hydrogenase activity affected seemed to be electrophoretically labile and was called hydrogenase L. The presence of such an activity was recently suggested in various papers and was called isoenzyme 3. Hydrogenase L might be identical or part of the latter isoenzyme. By DEAE ion-exchange chromatography it could be separated from hydrogenases 1 and 2. Hydrogenase activity in the parent strain HB101, determined manometrically with cell-free preparations and methylviologen as the electron acceptor, immediately showed maximal activity. However, class I mutants showed a lag phase which was dependent on the protein concentration utilized in the assay. This suggested that the fast initial activity of HB101 was due to hydrogenase L. The enzyme or enzyme complex showed an Mr around 300,000 and a pH optimum between 7 and 8. Strong indications about its physiological role were provided by the finding that in class I mutants H2 production by the formate-hydrogen lyase pathway was unimpaired, whereas fumarate-dependent H2 uptake was essentially zero. Complementation with F-prime factor F'116 but not with F'143 and coconjugation and cotransduction experiments localized the mutation (hydL) close to metC at approximately 64.8 min.     

Genetic and physiological characterization of new Escherichia coli mutants impaired in hydrogenase activity

The Mu dl (ApR lac) bacteriophage was used to generate mutants of Escherichia coli which were defective in formate hydrogenlyase. Three mutants were chosen for further analysis: they lacked hydrogenase (hydrogen: benzyl viologen oxidoreductase) activity, but produced normal levels of fumarate reductase activity and two- to three-fold reduced levels of benzyl viologen (BV)-dependent formate dehydrogenase activity. Two of them (hydC) were shown to contain about 4-fold reduced amounts of formate hydrogenlyase and fumarate-dependent H2 uptake activities. The third one (hydD) was totally devoid of both activities. Their insertion sites were located at 77 min on the E. coli map. Subdivision of these mutants into two classes was subsequently based on the restoration capacity of hydrogenase activity with high concentration of nickel in the growth media. Addition of 500 microM NiCl2 led to a complete recovery of hydrogenase activity, and to the concomitant restoration of normal BV-linked formate dehydrogenase, formate hydrogenlyase and fumarate-dependent H2 uptake activities in the hydC mutants. The hydD mutant was insensitive to the effect of nickel. Expression of the lac operon in hydC and hydD mutants was induced by anaerobiosis. It was not increased by the addition of formate under anaerobic conditions. The presence of nitrate resulted in slightly reduced beta-galactosidase activities in the hydC mutants, whereas those found in the hydD mutant reached only one third of the level obtained in its absence. Fumarate had no effect on both classes. Moreover, in contrast to the hydD locus, the hydC::Mu dl fusions were found to be dependent upon the positive control exerted by the nirR gene product and were totally repressed by an excess of nickel. In addition, the low levels of overall hydrogenase-dependent activities found in a nirR strain were also relieved by the presence of nickel. Our results strongly suggest that the pleiotropic regulatory gene nirR is essential for the expression of a gene (hydC) involved in either transport or processing of nickel in the cell, whose alteration leads to a loss of hydrogenase activity.     

Mutants of Escherichia coli with altered deoxyribonucleases. II. Isolation and characterization of mutants for exonuclease I

Mutants of Escherichia coli K12 deficient in exonuclease I (xon^?)³ were identified by enzymic assay of randomly selected, heavily mutagenized clones. From one of the six mutants of independent origin a thermolabile variant of exonuclease I was partially purified and identified, indicating that the mutation is probably in a structural gene for the enzyme. Transduction of this mutation into a recB^? recC^? strain did not result in the suppression of any of the phenotypic traits of the recipient. Although the five other mutants also appear to have temperature-sensitive exonuclease I activities in crude extracts, these enzymes were not sufficiently stable to permit purification. These latter mutations were of the xonA^? type; they produced a temperature-dependent suppression of the sensitivity to ultraviolet light and to mitomycin C manifested by a recB^? recC^? strain. None of the six mutations were of the sbcB^? type; that is, they did not suppress the recombination deficiency of a recB^? recC^? strain.In experiments with bacteriophage Plke, the six mutations were 41 to 62% cotransducible with the his region of E. coli. Heterozygous F′-merodiploids were constructed and studied for possible complementation of exonuclease I activity. All six mutations and an sbcB^? mutation were recessive to the wild-type alleles, and all were found to belong to a single complementation group. The results suggest that alterations of a structural gene for exonuclease I may result in the indirect suppression of the ultraviolet and mitomycin sensitivity manifested by recB^? recC^? strains.     

Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity

Anaerobic growth in the presence of 0.6 mM NiCl2 was able to restore hydrogenase and benzyl-viologen-linked formate dehydrogenase activities to a mutant (FD12), which is normally defective in these activities. This mutant carries a mutation located near minute 58 in the genome. Hydrogenase isoenzyme I and II activities were restored along with the hydrogenase activity that forms part of the formate hydrogen lyase system. A plasmid (pRW1) was constructed, containing a 4.8 kb chromosomal DNA insert, which was able to complement the lesion in mutant FD12. Further mutants with mutations near 58 minutes on the chromosome, and which lacked hydrogenase and formate dehydrogenase activities were isolated. These mutants were divided into three groups. Class I mutants were restored to the wild-type phenotype either by growth with 0.6 mM NiCl2 or following transformation with pRW1. Class II mutants were also complemented by pRW1 but were unaffected by growth with NiCl2. Class III mutants were unaffected by both pRW1 and growth with NiCl2. The cloned 4.8 kb fragment of chromosomal DNA therefore encodes two genes essential for hydrogenase activity. Restriction analysis indicates that the cloned DNA is the same as a fragment that has previously been cloned and which complements the hydB locus (Sankar et al. (1985) J. Bacteriol., 162, 353-360). None of the three classes of mutants possess mutations in hydrogenase structural genes.     

Mutants of Escherichia coli with Cold-Sensitive Deoxyribonucleic Acid Synthesis

Ten cold-sensitive mutants defective in deoxyribonucleic acid (DNA) synthesis at 20 C have been identified among 218 cold-sensitive mutants isolated from a mutagenized population of Escherichia coli K-12. Four of the ten mutant alleles, dna-339 dna-340, dna-341, and dna-342, cotransduce with serB(+) and hence may be dnaC mutants. Two of these, dna-340 and dna-341, are recessive to their wild-type allele. The gene product of their wild-type allele is trans acting. Complementation tests have demonstrated that dna-340 and dna-341 are in the same cistron. The mapping of the remaining six mutations is in progress. In an attempt to determine whether LW4 and LW21 were initiator mutants, cultures of these strains were starved of an essential amino acid at 37 C and then incubated at 15 C with the essential amino acid. The amount of DNA synthesis observed under these circumstances was insignificant. These data are consistent with the idea that LW4 and LW21 are initiator mutants. However, attempts to integratively suppress LW4 and LW21 with F' factors were unsuccessful. To resolve the question of whether or not LW4 and LW21 are initiator mutants, more specific tests and criteria are required. Cultures of LW4 and LW21 were toluene treated and used to measure in vitro DNA synthesis. If the cells were incubated either at 15 or 20 C before toluene treatment, they were capable of markedly less DNA synthesis than if preincubation had not occurred. The amount of in vitro DNA synthesis is directly proportional to the amount of DNA synthesis occurring during preincubation in vivo; i.e., more DNA synthesis is observed at 20 than at 15 C. The fact that the cold-sensitive mutants are unable to synthesize DNA when supplied with deoxyribonucleoside triphosphates, DNA precursors, is evidence they are not defective in precursor synthesis.     

The glucose transporter of Escherichia coli. Mutants with impaired translocation activity that retain phosphorylation activity.

The glucose transporter of the bacterial phosphotransferase system couples translocation with phosphorylation of the substrate in a 1:1 stoichiometry. It is a complex consisting of a transmembrane subunit (IIGlc) and a hydrophilic subunit (IIIGlc). Both subunits are transiently phosphorylated. IIIGlc is phosphorylated at a histidyl residue by the cytoplasmic phosphoryl carrier protein phospho-heat-stable phosphoryl carrier protein; IIGlc is phosphorylated at a cysteinyl residue by phospho-IIIGlc. The IIGlc subunit consists of two domains. The N-terminal hydrophobic domain is presumed to span the membrane several times; the C-terminal cytoplasmic domain includes the phosphorylation site. IIGlc phosphorylates glucose and methyl-alpha-D-glucopyranoside in transit across the inner membrane but can also phosphorylate intracellular glucose. Ten mutants resistant against extracellular toxic methyl-alpha-D-glucopyranoside yet capable of phosphorylating intracellular glucose were isolated. Strong impairment of transport activity in these mutants was accompanied by only a slight decrease of phosphorylation activity. Amino acid substitutions occurred at six sites that are clustered in three presumably hydrophilic loops in the transmembrane domain of IIGlc: M17T, M17I, G149S, K150E, S157F, H339Y, and D343G. We presume that the three polypeptide segments are directly involved in sugar translocation and/or binding but are of little importance for phosphorylation activity, folding, and membrane localization of IIGlc.