Search

A temperature-sensitive mutant (dna-11) with the phenotype of a mutant defective in the initiation of DNA replication, was isolated from an Hfr-like FP2 donor of Pseudomonas aeruginosa. Reversion of its temperature-sensitive character was achieved by integrative suppression rather than by backmutation or an additional suppressor mutation. The dna-11 mutant proved to be helpful in stabilizing the Hfr status of the original host.

The kinetics of isoleucine, leucine, and valine transport in Escherichia coli K-12 has been analyzed as a function of substrate concentration. Such analysis permits an operational definition of several transport systems having different affinities for their substrates. The identification of these transport systems was made possible by experiments on specific mutants whose isolation and characterization is described elsewhere. The transport process with highest affinity was called the "very-high-affinity"process. Isoleucine, leucine, and valine are substrates of this transport process and their apparent K(m) values are either 10(-8), 2 x 10(-8), or 10(-7) M, respectively. Methionine, threonine, and alanine inhibit this transport process, probably because they are also substrates. The very-high-affinity transport process is absent when bacteria are grown in the presence of methionine, and this is due to a specific repression. Methionine and alanine were also found to affect the pool size of isoleucine and valine. Another transport process is the "high-affinity" process. Isoleucine, leucine, and valine are substrates of this transport process, and their apparent K(m) value is 2 x 10(-6) M for all three. Methionine and alanine cause very little or no inhibition, whereas threonine appears to be a weak inhibitor. Several structural analogues of the branched-chain amino acids inhibit the very-high-affinity or the high-affinity transport process in a specific way, and this confirms their existence as two separate entities. Three different "low-affinity" transport processes, each specific for either isoleucine or leucine or valine, show apparent K(m) values of 0.5 x 10(-4) M. These transport processes show a very high substrate specificity since no inhibitor was found among other amino acids or among many branched-chain amino acid precursors or analogues tried. The evolutionary significance of the observed redundancy of transport systems is discussed.

Three mutations (ilvH611, ilvH612, and ilvH613) are described which make Escherichia coli K-12 resistant to valine inhibition and are located near leu. The expression of the ilv genes appears to be normal in these mutants since the isoleucine-valine biosynthetic enzymes are not derepressed relative to the wild type. The intracellular concentration of valine is, however, higher in the mutants than in the isogenic ilvH(+) strain. These mutants also excrete valine, probably because of the high intracellular concentration of this amino acid. The pool size of valine is regulated independently from that of isoleucine and leucine. The increased intracellular concentration of valine is due to a decreased feedback inhibition that valine exerts on its own biosynthetic pathway. In fact, acetolactate synthase activity assayed in extracts of ilvH612 and ilvH613 mutants is more resistant to valine inhibition than the activity assayed in the ilvH(+) isogenic strain. Two forms of acetolactate synthase activity can be separated from these extracts by adsorption and elution on hydroxylapatite. One of them is as sensitive to valine inhibition as that of the wild type, the other is more resistant to valine inhibition.

A method for the positive selection of dadA mutants defective in D-amino acid dehydrogenase has been devised. It consists in isolating mutants resistant to beta-chloro-D-alanine and screening for mutant colony color on a special agar medium. All 70 Escherichia coli K12 dadA mutants isolated either by this method or by other selection procedures map at a locus which is near to hemA and closely linked with dadR. Since some of the dadA mutants are thermosensitive in D-methionine utilization in vivo and have thermolabile D-amino acid dehydrogenase in vitro, it is proposed that the dadA gene codes for the enzyme structure. The broad substrate specificity, apparent membrane localization, inducibility by alanine, and repressibility by glucose strongly suggest that the D-amino acid dehydrogenase coded by the dadA gene is a species variant of the enzyme described under the same name in Salmonella typhimurium. It may be identical or homologous with the enzymes described under the names alaninase, D-alanine oxidase or D-alanine dehydrogenase in E. coli K12 or B.

Uptake of isoleucine, leucine, and valine in Escherichia coli K-12 is due to several transport processes for which kinetic evidence has been reported elsewhere. A very-high-affinity transport process, a high-affinity transport process, and three different low-affinity transport processes were described. In this paper the existence of these transport processes is confirmed by the isolation and preliminary characterization of mutants altered in one or more of them. The very-high-affinity transport process is missing either in strains carrying the brnR6(am) mutation or in strains carrying the brn-8 mutation. This appears to be a pleiotropic effect since other transport systems are also missing. Mutant analysis shows that more than one transport system with high affinity is present. One of them, high-affinity 1, which needs the activity of a protein produced by the brnQ gene, transports isoleucine, leucine, and valine and is unaffected by threonine. The other, high-affinity 2, which needs the activity of a protein produced by the brnS gene, transports isoleucine, leucine, and valine; this uptake is inhibited by threonine which probably is a substrate. Another protein, produced by the brnR gene, is required for uptake through both high-affinity 1 and high-affinity 2 transport systems. The two systems therefore appear to work in parallel, brnR being a branching point. The brnQ gene is located close to phoA at 9.5 min on the chromosome of E. coli, the brnR gene is located close to lac at 9.0 min, and the brnS gene is close to pdxA at 1 min. A mutant lacking the low-affinity transport system for isoleucine was isolated from a strain in which the high-affinity system was missing because of a brnR mutation. This strain also required isoleucine for growth because of an ilvA mutation. The mutant lacking the low-affinity transport system was unable to grow on isoleucine but could grow on glycylisoleucine. This mutant had lost the low-affinity transport for isoleucine, whereas those for leucine and valine were unaffected. A pleiotropic consequence of this mutation (brn-8) was a complete absence of the very-high-affinity transport system due either to the alteration of a common gene product or to any kind of secondary interference which inhibits it. Mutants altered in isoleucine-leucine-valine transport were isolated by taking advantage of the inhibition that valine exerts on the K-12 strain of E. coli. Mutants resistant both to valine inhibition (Val(r)) and to glycylvaline inhibition are regulatory mutants. Val(r) mutants that are sensitive to glycylvaline inhibition are transport mutants. When the very-high-affinity transport process is repressed (for example by methionine) the frequency of transport mutants among Val(r) mutants is higher, and it is even higher if the high-affinity transport process is partially inhibited by leucine.

About 600 compounds were screened as possible carbon or nitrogen sources for Salmonella typhimurium LT-2. About 100 utilizable compounds were found.

Secondary mutants able to utilize d-histidine, dhu, were isolated in histidine auxotrophs of Salmonella typhimurium. Mutations of one class (dhuA) are closely linked with the hisP locus which codes for a component of histidine permease. The specific activity of l-histidine permeation was estimated as increased two- to seven-fold in dhuA mutants. The dhuB mutants which have not been mapped also had elevated specific activity of l-histidine permeation. The uptake of d-histidine, barely detectable in the parental strains, was prominent in dhuA mutants and showed an apparent Michaelis constant about 1,000-fold higher than that observed with l-histidine. No change was detected in the kinetics of l-histidine permeation. d- and l-histidine competed in the uptake process. Tertiary mutants which lost the ability to grow on d-histidine were isolated by ampicillin counter-selection in dhuA his(-) strains. All of them mapped in the dhuA hisP region. Most of them had all known properties of hisP mutants. It is inferred from these data that the dhuA mutations increase synthesis of components critical to d- and l-histidine permeation.