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The chip and quantitative real-time PCR (qPCR) assays were optimized to study the expression of microcystin biosynthesis genes (mcy) with RNA samples extracted from cyanobacterial strains and environmental water samples. Both microcystin-producing Anabaena and Microcystis were identified in Lake Tuusulanjärvi samples. Microcystis transcribed the mcyE genes throughout the summer of 2006, while expression by Anabaena became evident later in August and September. Active mcyE gene expression was also detectable when microcystin concentrations were very low. Detection of Anabaena mcyE transcripts by qPCR, as well as certain cyanobacterial 16S rRNAs with the chip assay, showed slightly reduced sensitivity compared with the DNA analyses. In contrast, even groups undetectable or present in low quantities as determined by microscopy could be identified with the chip assay from DNA samples. The methods introduced add to the previously scarce repertoire of applications for mcy expression profiling in environmental samples and enable in situ studies of regulation of microcystin synthesis in response to environmental factors.

Pseudomonas syringae pv. tomato strain DC3000, a pathogen of tomato and Arabidopsis, occurs as an epiphyte. It produces N-acyl homoserine lactones (AHLs) which apparently function as quorum-sensing signals. A Tn5 insertion mutant of DC3000, designated PsrA(-) (Psr is for Pseudomonas sigma regulator), overexpresses psyR (a LuxR-type regulator of psyI) and psyI (the gene for AHL synthase), and it produces a ca. 8-fold-higher level of AHL than does DC3000. The mutant is impaired in its ability to elicit the hypersensitive reaction and is attenuated in its virulence in tomato. These phenotypes correlate with reduced expression of hrpL, the gene for an alternate sigma factor, as well as several hrp and hop genes during early stages of incubation in a Hrp-inducing medium. PsrA also positively controls rpoS, the gene for an alternate sigma factor known to control various stress responses. By contrast, PsrA negatively regulates rsmA1, an RNA-binding protein gene known to function as negative regulator, and aefR, a tetR-like gene known to control AHL production and epiphytic fitness in P. syringae pv. syringae. Gel mobility shift assays and other lines of evidence demonstrate a direct interaction of PsrA protein with rpoS promoter DNA and aefR operator DNA. In addition, PsrA negatively autoregulates and binds the psrA operator. In an AefR(-) mutant, the expression of psyR and psyI and AHL production are lower than those in DC3000, the AefR(+) parent. In an RpoS(-) mutant, on the other hand, the levels of AHL and transcripts of psyR and psyI are much higher than those in the RpoS(+) parent, DC3000. We present evidence, albeit indirect, that the RpoS effect occurs via psyR. Thus, AefR positively regulates AHL production, whereas RpoS has a strong negative effect. We show that AefR and RpoS do not regulate PsrA and that the PsrA effect on AHL production is exerted via its cumulative, but independent, effects on both AefR and RpoS.

A cryptic plasmid of Methylobacterium extorquens AM1 was found to encode tslI, a truncated luxI homolog. tslI was shown to be expressed and to control transcription of the acyl-homoserine lactone (HSL) synthase gene msaI and thus, indirectly, acyl-HSL production. In addition, tslI was found to positively regulate extracellular polysaccharide production.

The ability of rhizobia to symbiotically fix nitrogen from the atmosphere when forming nodules on their plant hosts requires various signal transduction pathways. LuxR-LuxI-type quorum-sensing systems have been shown to be one of the players in a number of rhizobium species. In this study, we found that Mesorhizobium tianshanense, a moderate-growth Rhizobium that forms nodules on a number of licorice plants, produces multiple N-acyl homoserine lactone (AHL)-like molecules. A simple screen for AHL synthase genes using an M. tianshanense genomic expression library in Escherichia coli, coupled with a sensitive AHL detector, uncovered a LuxI-type synthase, MrtI, and a LuxR-type regulator, MrtR, in M. tianshanense. Deletions of the mrtI or mrtR locus completely abolished AHL production in M. tianshanense. Using lacZ transcriptional fusions, we found that expression of the quorum-sensing regulators is autoinduced, as mrtI gene expression requires MrtR and cognate AHLs and mrtR expression is dependent on AHLs. Compared with the wild-type strains, quorum-sensing-deficient mutants showed a marked reduction in the efficiency of root hair adherence and, more importantly, were defective in nodule formation on their host plant, Glycyrrhiza uralensis. These data provide strong evidence that quorum sensing plays a critical role in the M. tianshanense symbiotic process.

The stringent control response, which involves a rapid accumulation of ppGpp, is triggered if the marine Vibrio sp. strain S14 is subjected to carbon and energy starvation. By means of high-resolution two-dimensional gel electrophoresis analysis, we addressed the role of the major ppGpp-synthesizing enzyme (RelA) in the regulation of the carbon starvation response of Vibrio sp. strain S14. The finding that a large number of the carbon starvation-induced proteins were underexpressed in the Vibrio sp. S14 relA mutant strain after the onset of glucose starvation suggests that a rapid accumulation of ppGpp is required for induction of many of the carbon starvation-induced proteins. However, it was also found that a majority of the carbon starvation-induced proteins were significantly less induced if the stringent control response was provoked by amino acid starvation. We therefore also addressed the notion that a carbon starvation-specific signal transduction pathway, complementary to the stringent control, may exist in Vibrio sp. strain S14. It was found that a majority of the proteins that were underexpressed in the relA mutant strain were also underexpressed in the Vibrio sp. S14 spoT mutant strain (csrS1). Interestingly, a large proportion of these underexpressed proteins were found to belong to a group of proteins that are not, or significantly less, induced by starvation conditions that do not promote starvation survival. On the basis of these observations and the finding that the csrS1 strain survives poorly but accumulates ppGpp in a fashion similar to the wild type during carbon and energy source starvation, the gene product of the csrS gene is suggested to be responsible for the mediation of a signal which is complementary to ppGpp and essential for the successful development of the starvation- and stress-resistant cell. This conclusion was also supported by experiments in which changes in phenotypic characteristics known to be induced during carbon starvation were studied. The starvation induction of the high-affinity glucose uptake system was found to be dependent on the csrS gene but not relA, and the synthesis of carbon starvation-specific periplasmic space proteins was dependent, at different times of starvation, on both the relA and the csrS gene products.

Previous work of others reported an untranslated stretch of 12 nucleotides in the 5' coding sequence of carA from Pseudomonas aeruginosa. However, N-terminal protein sequencing of carA-lacZ translational fusions shows that these 12 nucleotides are normally translated in a continuous triplet manner, both in P. aeruginosa and in Escherichia coli.

The genetic organization of the Pseudomonas aeruginosa acetyl coenzyme A carboxylase (ACC) was investigated by cloning and characterizing a P. aeruginosa DNA fragment that complements an Escherichia coli strain with a conditional lethal mutation affecting the biotin carboxyl carrier protein (BCCP) subunit of ACC. DNA sequencing and RNA blot hybridization studies indicated that the P. aeruginosa accB (fabE) homolog, which encodes BCCP, is part of a 2-gene operon that includes accC (fabG), the structural gene for the biotin carboxylase subunit of ACC. P. aeruginosa homologs of the E. coli accA and accD, encoding the alpha and beta subunits of the ACC carboxyltransferase, were identified by hybridization of P. aeruginosa genomic DNA with the E. coli accA and accD. Data are presented which suggest that P. aeruginosa accA and accD homologs are not located either immediately upstream or downstream of the P. aeruginosa accBC operon. In contrast to E. coli, where BCCP is the only biotinylated protein, P. aeruginosa was found to contain at least three biotinylated proteins.

A gene designated purU has been identified and characterized. purU is adjacent to tyrT at min 27.7 on the Escherichia coli chromosome. The gene codes for a 280-amino-acid protein. The C-terminal segment of PurU from residues 84 to 280 exhibits 27% identity with 5'-phosphoribosylglycinamide (GAR) transformylase, the product of purN. Primer extension mapping and assays of lacZ in a promoter probe vector identified two promoters giving mono- and bi-cistronic purU mRNA. Neither mRNA was regulated by purines. Mutations in either of two pairs of genes are required to block synthesis of 5'-phosphoribosyl-N-formylglycinamide (FGAR) from GAR: purN purT (purT encodes an alternative formate-dependent GAR transformylase) or purN purU. On the basis of the growth of purU, purN, and purU purN mutants, it appears that PurU provides the major source of formate for the purT-dependent synthesis of FGAR.

Alpha-Dehydrobiotin, like biotin, represses coordinately the 7,8-diaminopelargonic acid aminotransferase and the dethiobiotin synthetase enzymes that are encoded on the l and r strands, respectively, of the bioA operon. The rate of synthesis for both enzymes is inhibited about 80% in the presence of alpha-dehydrobiotin. Homobiotin and alpha-methylbiotin are less effective than alpha-dehydrobiotin in repressing the synthesis of the two enzymes. The selective repression of transcription from l and by alpha-dehydrobiotin and homobiotin, previously reported in hybridization experiments, is not observed at the enzyme level. A combination of equal concentrations of biotin and alpha-dehydrobiotin which was reported to enhance selectively the level of messenger ribonucleic acid transcribed from the l strand does not increase the rate of synthesis of the aminotransferase enzyme. Instead, the enzymes encoded on both strands are essentially completely inhibited as with biotin alone. Strain differences have been ruled out to account for the different results obtained by the two methodologies. Our evidence would suggest that alpha-dehydrobiotin acts like biotin, presumably as the co-repressor, in the repression of the bioA operon. The low rates of enzyme synthesis observed in the presence of the biotin analogue is the result of incomplete repression due to a lower affinity of either the analogue for the repressor or of the co-repressor/repressor complex for the operator. While our evidence would support the concept of a two promoter/operator complex, both would have to respond equally to biotin and its analogues. The evidence, however, does not rule out other possible alternative models for the regulation of the biotin operon.

Urea transport in Saccharomyces cerevisiae occurs by two pathways. The first mode of uptake is via an active transport system which: (i) has an apparent Km value of 14 muM, (ii) is absolutely dependent upon energy metabolism, (iii) requires pre-growth of the cultures in the presence of oxaluric acid, gratuitous inducer of the allantoin degradative enzymes, and (iv) is sensitive to nitrogen repression. The second mode of uptake which occurs at external urea concentrations in excess of 0.5 mM is via either passive or facilitated diffusion.

guaA and guaB mutants of Salmonella typhimurium were isolated utilizing the mutagen, nitrous acid. The guaB mutants were defective for inosine 5'-monophosphate (IMP) dehydrogenase activity and those mutants classified as guaA exhibited no xanthosine 5'-monophosphate aminase activity. In vivo complementation maps were determined for the mutants. The guaB map indicated that at least three complementation regions existed whereas five complementation regions were observed for the guaA mutants. The demonstration of in vitro complementation was also achieved for the guaB mutants by utilizing a process of denaturation and renaturation. All of the guaB mutants that exhibited in vivo complementation were found to exhibit in vitro complementation. No correlation was found between the degree of in vivo complementation exhibited by the various pairs of mutants and the specific enzyme activities of the same mutant pair that yielded in vitro complementation. The kinetic parameters for three of the most active guaB "complemented" enzymes and a renatured wild-type IMP dehydrogenase were then examined. No apparent differences were found in the K(m) values between any of the enzymes for substrate, IMP, activator ion, K(+), and coenzyme, oxidized nicotinamide adenine dinucleotide. Some differences were noted in the apparent V(max) values; the best "complemented" enzyme yielded only 20% of the velocity exhibited by renatured wild-type enzyme.

The synthesis and regulation of two of the enzymes of the biotin operon of Escherichia coli, 7,8-diaminopelargonic acid aminotransferase and dethiobiotin synthetase, were studied in vitro in a coupled transcription-translation system. These enzymes are encoded by genes located on opposite strands of the divergently transcribed operon (A. Guha, Y. Saturen, and W. Szybalski, J. Mol. Biol. 56:53-62, 1971). The kinetics of synthesis of both the enzymes were determined and the efficiency of the system was 0.3 to 0.4% that of the in vivo rate of synthesis in derepressed cells. Guanosine 3'-diphosphate 5'-diphosphate at 0.2 mM concentration stimulated the synthesis of 7,8-diaminopelargonic acid aminotransferase two- to threefold but had no effect on dethiobiotin synthetase synthesis. Biotin, which was most effective as the corepressor in vivo, also functioned in vitro at physiological concentrations in conjunction with a crude repressor protein isolated from a lysogen carrying the bioR gene. However, the two strands showed differential repression. At a repressor concentration where 7,8-diaminopelargonic acid aminotransferase synthesis was completely repressed, the repression of dethiobiotin synthetase was only 20% and did not exceed 50% with increasing repressor concentrations. Although the exact reason for the partial repression remains to be resolved, our data clearly suggest that the biotin operon is regulated from two separate operators.

Experiments were carried out to construct pseudomonad strains capable of the biodegradation of certain recalcitrant branched hydrocarbons via a combination of alkane and citronellol degradative pathways. To promote the metabolism of the recalcitrant hydrocarbon 2,6-dimethyl-2-octene we transferred the OCT plasmid to Pseudomonas citronellolis, a pseudomonad containing the citronellol pathway. This extended the n-alkane substrate range of the organism, but did not permit utilization of the branched hydrocarbon even in the presence of a gratuitous inducer of the OCT plasmid. In a separate approach n-decane-utilizing (Dec+) mutants of P. citronellolis were selected and found to be constitutive for the expression of medium- to long-chain alkane oxidation. The Dec+ mutants were capable of degradation of 2,6-dimethyl-2-octene via the citronellol pathway as shown by (i) conversion of the hydrocarbon to citronellol, determined by gas-liquid chromatography-mass spectrometry, (ii) induction of geranyl-coenzyme A carboxylase, a key enzyme of the citronellol pathway, and (iii) demonstration of beta-decarboxymethylation of the hydrocarbon by whole cells. The Dec+ mutants had also acquired the capacity to metabolize other recalcitrant branched hydrocarbons such as 3,6-dimethyloctane and 2,6-dimethyldecane. These studies demonstrate how enzyme recruitment can provide a pathway for the biodegradation of otherwise recalcitrant branched hydrocarbons.

The initiation of patulin biosynthesis in submerged batch cultures of Penicillium urticae NRRL 2159A was investigated at the enzyme level. In contrast to earlier studies, this study achieved a clear temporal separation of growing cells devoid of secondary metabolism-specific enzymes from nongrowing cells, which rapidly produce these enzymes. A spore inoculum, silicone-treated flasks, and two new media which supported a rapid, pellet-free, filamentous type of growth were used. In yeast extract-glucose-buffer medium, a marked drop in the specific growth rate (approximately equal to 0.26 h-1) coincided with the appearance of the first pathway-specific enzyme, 6-methylsalicylic acid synthetase, at about 19 h after inoculation. About 3 h later, when replicatory growth had ceased entirely, the sparsely branched mycelia (length, approximately equal to 550 microns) began the rapid synthesis of a later pathway enzyme, m-hydroxybenzyl alcohol dehydrogenase. A similar sequence of events occurred in a defined nitrate-glucose-buffer medium; 12 other strains or isolates of P. urticae, as well as some patulin-producing aspergilli, behaved in a similar manner. The age at which a culture produced m-hydroxybenzyl alcohol dehydrogenase was increased by increasing the nutrient nitrogen content of the medium or by decreasing the size of the spore inoculum. In each instance the appearance of enzyme was determined by the nutritional status of the culture and not by its age. A similar appearance of patulin pathway enzymes occurred when a growing culture was resuspended in a nitrogen-free 4% glucose solution with or without 0.1 M phosphate (pH 6.5). The appearance of both the synthetase and the dehydrogenase was arrested by the addition of cycloheximide (0.4 to 5 micrograms/ml) or actinomycin D (20 to 80 micrograms/ml). This requirement for de novo protein and ribonucleic acid syntheses was confirmed by the incorporation of labeled leucine into the dehydrogenase, and the possibility that latent or preformed proteins were being activated was eliminated.

Saccharomyces cerevisiae degrades allantoin in five steps to ammonia, CO(2), and glyoxylate. Previously we demonstrated that allophanic acid, the last intermediate of the pathway, was required for induction of all five degradative enzymes. The data presented here indicate that oxaluric acid, an allophanate analogue, is capable of serving as a non-metabolizable inducer. Oxaluric acid brings about a high level of induction even in strains lacking urea carboxylase. Induction observed with parabanic acid was found to result from its spontaneous breakdown to oxaluric acid.

A temperature-sensitive mutation in Escherichia coli K-12 was shown to affect acetyl coenzyme A carboxylase and to map at min 63. This site is designated fabE.

We have shown that allantoin degradation in Saccharomyces cerevisiae proceeds exclusively through the intermediate formation of allantoic acid, urea, and allophanic acid. The number of reactions between allantoic acid and urea, however, remains obscure owing to our inability to isolate a mutant defective in ureidoglycolate hydrolase. Structural genes for the enzymes, allantoinase (dal1) and allantoicase (dal2) are located on chromosome IX promixal to the centromere in the order dal1-dal2-lysl.

At least two pathways exist in Klebsiella aerogenes for glutamate synthesis. A mutant blocked in one pathway due to the loss of glutamate dehydrogenase (gltD) does not require glutamate and has the same growth characteristics as the parent strain in most media; however, its growth is inhibited by the analogues methionine sulfoximine and methionine sulfone. Wild-type Klebsiella is resistant to 0.1 M methionine sulfoximine or methionine sulfone, whereas the gltD mutant is sensitive to 1 mM concentrations. Either glutamate or glutamine is effective in overcoming this inhibition. Activities of both glutamine synthetase and glutamate synthetase, two enzymes involved in the second pathway of glutamate synthesis, are inhibited by methionine sulfoximine and methionine sulfone. The primary effect of methionine sulfoximine appears to be the prevention of glutamine production necessary for subsequent glutamate synthesis via glutamate synthetase enzyme.

Nester, E. W. (University of Washington, Seattle), and R. A. Jensen. Control of aromatic acid biosynthesis in Bacillus subtilis: sequential feedback inhibition. J. Bacteriol. 91:1594-1598. 1966.-The three major end products of aromatic acid synthesis, tyrosine, phenylalanine, and tryptophan, were tested for their ability to inhibit the first enzymes of the three terminal branches of the pathway as well as the enzyme common to both tyrosine and phenylalanine synthesis. Tyrosine inhibits the activity of prephenate dehydrogenase and also prephenate dehydratase to a limited extent. Phenylalanine inhibits the activity of prephenate dehydratase and, at much higher concentrations, prephenate dehydrogenase. Tryptophan inhibits the activity of anthranilate synthetase and, to some extent, prephenate dehydrogenase and prephenate dehydratase. Chorismate mutase is not inhibited by either 1 mm tyrosine or 1 mm phenylalanine when these are present singly or together in the reaction mixture. The significance of the feedback control of the terminal branches to the feedback control of that part of the pathway common to the synthesis of all three amino acids is discussed.

The kinetic properties of Escherichia coli glutamine synthetase are markedly influenced by the manner in which the organism is grown. Enzyme obtained from stationary-phase cells grown on glycerol and glutamate is strongely inhibited by each of the eight feedback effectors known to influence this enzyme; however, the enzyme from log-phase cells grown on glucose and growth-limiting concentrations of NH(4)Cl is stimulated by some of these effectors. Of the growth variables examined, nitrogen source and time of harvest were the most important; carbon source and aeration seemed to have no effect. Two purified enzyme preparations have been obtained from cells grown under two different conditions, designated enzymes I and II for convenience. Enzyme I is stimulated by adenosine 5'-monophosphate, histidine, and tryptophan in the transfer assay, whereas enzyme II is strongly inhibited by all effectors tested. Enzyme I has a higher specific activity in the forward assay in the presence of Mg(++) or Co(++), whereas enzyme II is more active in the presence of Mn(++).

Detailed study of methionine-mediated repression of enzymes involved in methionine biosynthesis in Saccharomyces cerevisiae led to classification of these enzymes into two distinct regulatory groups. Group I comprises four enzymes specifically involved in different parts of methionine biosynthesis, namely, homoserine-O-transacetylase, homocysteine synthetase, adenosine triphosphate sulfurylase, and sulfite reductase. Repressibility of these enzymes is greatly decreased in strains carrying a genetically impaired methionyl-transfer ribonucleic acid (tRNA) synthetase (mutation ts(-) 296). Conditions leading to absence of repression in the mutant strain have been correlated with a sharp decrease in bulk tRNA(met) charging, whereas conditions which restore repressibility of group I enzymes also restore tRNA(met) charging. These findings implicate methionyl-tRNA in the regulatory process. However, the absence of a correlation in the wild type between methionyl-tRNA charging and the levels of methionine group I enzymes suggests that only a minor iso accepting species of tRNA(met) may be devoted with a regulatory function. Repressibility of the same four enzymes (group I) was also decreased in strains carrying the regulatory mutation eth2(r). Although structural genes coding for two of these enzymes, as well as mutations ts(-) 296 and eth2(r) segregate independently to each other, synthesis of group I enzymes is coordinated. The pleiotropic regulatory system involved seems then to comprise beside a "regulatory methionyl tRNA(met)," another element, product of gene eth2, which might correspond either to an aporepressor protein or to the "regulatory tRNA(met)" itself. Regulation of group II enzymes is defined by response to exogenous methionine, absence of response to either mutations ts(-) 296 and eth2(r), and absence of coordinacy with group I enzymes. However, the two enzymes which belong to this group and are both involved in threonine and methionine biosynthesis undergo distinct regulatory patterns. One, aspartokinase, is subject to a bivalent repression exerted by threonine and methionine, and the other, homoserine dehydrogenase, is subject only to methionine-mediated repression. Participation of at least another aporepressor and another corepressor, different from the ones involved in regulation of group I enzymes, is discussed.

The accumulation of imidazoleglycerol phosphate during growth of Neurospora crassa in the presence of 3-amino-1,2,4-triazole was found to cause derepression of tryptophan synthetase and to inhibit the induction of kynureninase. Accumulation of indoleglycerol phosphate in response to growth in the presence of indole acrylic acid or anthranilic acid was also accompanied by derepressed synthesis of tryptophan synthetase. Enzyme synthesis in mutants (his-7 and trp-4) unable to form these intermediates was not altered under similar conditions. The rate of formation of tryptophan synthetase and kynureninase was found to differ in the presence of tryptophan and indole.

Gaudy, Elizabeth T. (University of Illinois, Urbana), R. Bojanowski, R. C. Valentine, and R. S. Wolfe. Ureidoglycolate synthetase of Streptococcus allantoicus. I. Measurement of glyoxylate and enzyme purification. J. Bacteriol. 90:1525-1530. 1965.-A new spectrophotometric method for the determination of glyoxylate is described. The technique is based on measurement of the initial rate of formation of glyoxylic acid phenylhydrazone in neutral solution. Its advantages include rapidity and convenience, suitability for use with mixtures containing acid-labile substrates, and elimination of possibly inhibitory reagents from the enzyme incubation mixture. Ureidoglycolate synthetase, which cleaves ureidoglycolate to glyoxylate and urea, was purified from crude extracts of Streptococcus allantoicus grown on allantoin-containing medium. The purification procedures include treatment with MnCl(2), fractionation on calcium phosphate gel, fractional precipitation with ammonium sulfate, and column chromatography on diethylaminoethyl cellulose. The final enzyme preparation was purified 77-fold and contained 35% of the total activity of the extract.

Inactivation of bacteriophage T4 by ethyl methanesulfonate (EMS) is a complex process which depends critically upon the conditions of treatment and upon both the viral and the host genotypes. EMS-inactivated particles are capable of multiplicity and cross-reactivation, indicating the need for caution in using EMS in certain types of mutation studies. The pyrimidine dimer excision systems of the phage and the host do not affect the EMS sensitivity of T4, but the T4x(+)y(+) system does. Mutational defects in the deoxyribonucleic acid (DNA) ligase and the DNA polymerase systems both of the virus and of its host also affect viral EMS sensitivity.

Burns, R. O. (Cold Spring Harbor Laboratories of Quantitative Biology, Cold Spring Harbor, N.Y.), J. Calvo, P. Margolin, and H. E. Umbarger. Expression of the leucine operon. J. Bacteriol. 91:1570-1576. 1966.-The four genes which specify the structure of the three enzymes specifically involved in the biosynthesis of leucine in Salmonella typhimurium constitute a single operon. Three types of control mutants have been delineated on the basis of their location on the Salmonella chromosome and the manner in which they coordinately affect the rates of synthesis of the pertinent enzymes. The three types of mutants correspond to operator-negative, operator-constitutive, and regulator-negative. The rate of synthesis of the enzymes can also be altered by varying the amount of leucine made available to the cell. Leucine can be effectively limited by limiting the supply of alpha-ketoisovalerate, but in doing so two of the three enzymes, alpha-isopropylmalate synthetase and isopropylmalate isomerase, are labilized. This observation was correlated with an in vivo diminution of the levels of the substrates of these enzymes and the fact that alpha-ketoisovalerate and alpha-isoporpylmalate protect the respective enzymes against thermal inactivation in vitro. The functional association of the structural genes is also illustrated by the presence of polarity mutations; that is, certain structural gene mutations lower the rates of synthesis of the enzymes specified by genes located distally to the mutated gene and the operator segment of the operon.

Mutant strains of Escherichia coli K-12 have been isolated in which the synthesis of 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase (trp) is partially constitutive. The mutation causing derepression is closely linked to aroH [the structural gene for DAHP synthetase (trp)] and occurs in a locus designated aroJ. The aroJ mutation is not recessive in an aroJ(+)/aroJ(-) diploid strain, as the synthesis of DAHP synthetase (trp) is still derepressed in this strain. On the basis of its close linkage to aroH and its continued expression in an aroJ(+)/aroJ(-) diploid, it is postulated that aroJ is an operator locus controlling the expression of the structural gene aroH. In support of this conclusion, the synthesis of anthranilate synthetase is still normally repressible in aroJ(-) strains, whereas, in trpR(-) strains, both DAHP synthetase (trp) and anthranilate synthetase are synthesized constitutively. The synthesis of DAHP synthetase (trp) remains repressible in an operator-constitutive mutant of the tryptophan operon. In two trpS mutants which possess defective tryptophanyl transfer ribonucleic acid synthetase enzymes, neither DAHP synthetase (trp) nor anthranilate synthetase derepress under conditions in which the defective synthetase causes a decrease in growth rate. On the other hand, an effect of the trpS mutant alleles on the level of anthranilate synthetase has been observed in strains which are derepressed for the synthesis of this enzyme, because of a mutation in the gene trpR. Possible explanations for this effect are presented.

Methionine biosynthesis and regulation of four enzymatic steps involved in this pathway were studied in Saccharomyces cerevisiae, in relation to genes concerned with resistance to ethionine (eth(1) and eth(2)). Data presented in this paper and others favor a scheme which excludes cystathionine as an obligatory intermediate. Kinetic data are presented for homocysteine synthetase [K(m)(O-acetyl-l-homoserine) = 7 x 10(-3)m; K(i) (l-methionine) = 1.9 x 10(-3)m]. Enzymes catalyzing steps 3, 4, 5, and 9 were repressible by methionine. Enzyme 4 (homoserine-O-transacetylase) and enzyme 9 (homocysteine synthetase) were simultaneously derepressed in strains carrying the mutant allele eth(2) (r). Studies on diploid strains confirmed the dominance of the eth(2) (s) allele over eth(2) (r). Regulation of enzyme 3 (homoserine dehydrogenase) and enzyme 5 (adenosine triphosphate sulfurylase) is not modified by the allele eth(2) (r). The other gene eth(1) did not appear to participate in regulation of these four steps. Gene enzyme relationship was determined for three of the four steps studied (steps 3, 4, and 9). The structural genes concerned with the steps which are under the control of eth(2) (met(8): enzyme 9 and met(a): enzyme 4) segregate independently, and are unlinked to eth(2). These results are compatible with the idea that the gene eth(2) is responsible for the synthesis of a pleiotropic methionine repressor and suggest the existence of at least two different methionine repressors in S. cerevisiae. Implications of these findings in general regulatory mechanisms have been discussed.

Temperature-sensitive mutations in bacteriophage T4 gene 30 (polynucleotide ligase) were examined for their effects on spontaneous and proflavine-induced frameshift mutagenesis in the rII and ac (acridine resistance) cistrons. Only small (fourfold or less) effects on mutation rates were observed, even when selection artifacts involving suppression of gene 30 mutations by rII mutations were taken into account. The deoxyribonucleic acid ligase gene of T4 therefore appears to be only a minor determinant of frameshift mutation rates. This result is consistent with the particular nature of frameshift mutagenesis in bacteriophage T4.

Guanine auxotrophs of Escherichia coli K-12 were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methane sulfonate, or the acridine mustard ICR 372. guaA (xanthosine 5'-monophosphate [XMP] aminase-less) mutants were distinguished from guaB (inosine 5'-monophosphate [IMP] dehydrogenase-less) mutants by their growth response to xanthine and by enzyme assay. Mutations were classified as base substitutions or frameshift on the basis of mutagen-induced reversion patterns. All guaA strains, including three frameshift mutants, produced derepressed levels of IMP dehydrogenase when cultured with a growth-limiting concentration of guanine. The guaB strains were of two types: (i) those producing derepressed levels of XMP aminase, and (ii) those producing basal levels of XMP aminase when grown under conditions of guanine starvation. In the guaB strains of the second type, the expression of the adjacent guaA gene is reduced. It is proposed that this pleiotropic effect of some guaB mutations is a result of polarity. The orientation of polarity suggests the gene order "operator"-guaB-guaA. Gel diffusion studies with IMP dehydrogenase antiserum showed that strains carrying polar guaB mutations do not produce cross-reacting material (CRM). The remaining guaB mutants were either CRM(+) or CRM(-). Mapping the mutations by three-factor crosses showed that polar and nonpolar guaB sites are clustered in a small genetic region cotransducible with guaA. The relative positions of the guaB mutational sites established that the polar mutations lie within the structural gene for IMP dehydrogenase.

Derepression of some methionine biosynthetic enzymes (methionine group I enzymes) obtained in methionine limitation has been found to be accompanied by a significant lack of in vivo charging of bulk methionine transfer ribonucleic acid (tRNA(Met)) and in addition by a decreased rate of synthesis of all tRNAs. Under the same conditions, methionyl-tRNA synthetase (MTS) was derepressed rather than repressed. These results are in agreement with those previously published based on studies of a mutant with an impaired MTS (5) and reinforce the idea that the rate of synthesis of methionine group I enzymes can be related to the total content of methionyl (Met)-tRNA (Met) per cell. They also render unlikely that MTS could be a constituent of the regulatory signal.

It has been reported that the level of d-biotin in the growth medium of Lactobacillus plantarum regulates the synthesis of apoacetyl-coenzyme A (CoA) carboxylase; high levels cause repression, and deficient levels effect derepression. In this study, evidence has been obtained which suggests that coenzyme repression by biotin is an indirect effect; i.e., biotin regulates the synthesis of unsaturated fatty acids which are the true repressors of the acetyl-CoA carboxylase. This was observed in an experiment in which long-chain unsaturated fatty acids were added to media containing deficient, sufficient, or excess levels of d-biotin. In every case, independently of the biotin concentration for growth, the unsaturated fatty acids caused a severe repression of the carboxylase. Saturated fatty acids were without effect. The level of oleic acid required to give maximal repression was 50 mug/ml. The free fatty acids had no adverse effect on the activity of the cell-free extracts nor on the permeation of d-biotin into the cell. Saturated and unsaturated fatty acids decreased the rate of holocarboxylase formation from d-biotin and the apoacetyl-CoA carboxylase in the extracts. It is concluded that there are at least three mechanisms that control the acetyl-CoA carboxylase in this organism: (i) indirect coenzyme repression by d-biotin, (ii) repression by unsaturated fatty acids, and (iii) regulation of the activity of the holocarboxylase synthetase by both saturated and unsaturated fatty acids.

The formation of the five tryptophan biosynthetic enzymes of Neurospora crassa was shown to be derepressed in histidine-starved cells. This histidine-mediated derepression was not due to a lowered intracellular concentration of tryptophan in these cells. Furthermore, histidine-mediated derepression of tryptophan enzymes was found to be coordinate and not subject to reversal by tryptophan of either exogenous or biosynthetic origin. The synthesis of tryptophan enzymes also was found to be coordinate in cells which were not histidine-starved. Although histidine is clearly involved in regulating the synthesis of tryptophan enzymes, it did not prevent either tryptophan-mediated derepression of tryptophan enzymes or indole-3-glycerol phosphate-mediated derepression of tryptophan synthetase.

The timing of the suppression of gene 30 (deoxyribonucleic acid ligase) mutations by rII mutations was studied by temperature shift-down experiments with a temperature-sensitive rII mutation. The rII function must remain inactivated for about 5 to 8 min at 37 C for suppression to occur, thus making suppression an early function. This result is in agreement with the timing of expression of other rII functions. A gene 30 defect can also be overcome by replacing the Na(+) cation in the growth medium with the Mg(2+) cation, a result similar to the relief of the lethality of rII mutations in lambda lysogens. Prior infection with bacteriophages T3 or T7, which produce their own deoxyribonucleic acid ligases, can also partially overcome the lethality of gene 30 mutations.

The control of the synthesis of certain key enzymes of aromatic amino acid biosynthesis was studied. Tyrosine represses the first enzyme of the 3-deoxy-d-arabino heptulosonic acid 7-phosphate pathway, DAHP synthetase, as well as shikimate kinase and chorismate mutase about fivefold in cultures grown under conditions limiting the synthesis of the aromatic amino acids. A mixture of tyrosine and phenylalanine represses twofold further. Tryptophan does not appear to be involved in the control of these enzymes. The specific activity of at least one early enzyme, dehydroquinase, remains essentially constant under a variety of nutritional supplementations. Two enzymes in the terminal branches are repressed by the amino acids they help to synthesize: prephenate dehydrogenase can be repressed fourfold by tyrosine, and anthranilate synthetase can be repressed over 200-fold by tryptophan. There is no evidence that phenylalanine represses prephenate dehydratase. Regulatory mutants have been isolated in which various enzymes of the pathway are no longer repressible. One class is derepressed for several of the prechorismate enzymes, as well as chorismate mutase and prephenate dehydrogenase. In another mutant, several enzymes of tryptophan biosynthesis are no longer repressible. Thus, the rate of synthesis of enzymes at every stage of the pathway is under control of various aromatic amino acids. Tyrosine and phenylalanine control the synthesis of enzymes involved in the synthesis of the three aromatic amino acids. Each terminal branch is under the control of its end product.

A study of the regulation of the synthesis of the enzyme glutamine synthase in Bacillus subtilis was initiated. An assay, based on the measurement of glutamo-hydroxamate, was used to characterize the enzyme in crude preparations and in toluene-treated cells. Determinations were made of the Michaelis constants for adenosine triphosphate, hydroxylamine, and glutamate (9 x 10(-3), 4 x 10(-3), and 2.2 x 10(-2)m, respectively), the pH optimum (7.6 to 7.7), and the stability. The differential rate of synthesis was determined under various growth conditions. The enzyme was found to be relatively insensitive to regulation. Partial repression was caused by glutamine, arginine, asparagine, and glutamate, or by carbon limitation in a chemostat. Derepression was caused by exhaustion of externally added amino acids or by nitrogen limitation in a chemostat.

Megraw, Robert E. (Albert Einstein Medical Center, Philadelphia, Pa.), Henry C. Reeves, and Samuel J. Ajl. Formation of lactyl-coenzyme A and pyruvyl-coenzyme A from lactic acid by Escherichia coli. J. Bacteriol. 90:984-988. 1965.-Cell extracts of propionate-adapted Escherichia coli were found to contain a lactyl-coenzyme A (CoA) synthetase which catalyzes the formation of the CoA thiolester from lactate, CoA, and cofactors. The extracts also catalyzed the nicotinamide adenine dinucleotide-dependent oxidation of lactyl-CoA. The product of this oxidation was identified chromatographically as pyruvyl-CoA.

The antibiotic polyoxin D was shown to inhibit the incorporation of (14)C-glucosamine into cell wall chitin in Neurospora crassa at levels which were comparable with those required for inhibition of fungal growth. At the same time, the antibiotic increased the accumulation of a nucleotide, which was identified as uridine diphosphate (UDP)-N-acetylglucosamine, indicating inhibition of chitin synthesis. Chitin synthetase (UDP-N-acetylglucosamine: chitin N-acetylglucosaminyl transferase, EC 2.4.1.16) of N. crassa was found to be strongly inhibited by polyoxin D, as determined by the transfer of (14)C-N-acetylglucosamine from (14)C-UDP-N-acetylglucosamine to the particulate fraction. The inhibition was competitive with respect to UDP-N-acetylglucosamine and specific for chitin synthetase. The K(i) for polyoxin D in the reaction was 1.40 x 10(-6)m, and the K(m) for UDP-N-acetylglucosamine was 1.43 x 10(-3)m. The formation of osmotically sensitive, protoplast-like structures, when the fungus Cochliobolus miyabeanus was grown in the presence of polyoxin D, also suggested that the primary site of action of polyoxin D was in the formation of cell wall structures.

A prototrophic, thermophilic bacillus is in a state of biotin insufficiency when grown in medium consisting of inorganic salts and a carbon source. The effect of this biotin deficiency on the growth rate is severe only if the functioning of pyruvate carboxylase is essential for the utilization of the particular growth substrate. A mutant, PC2, of the thermophile devoid of active pyruvate carboxylase has been isolated. The properties of this mutant confirm the anaplerotic role of this enzyme in the utilization for growth of compounds like glucose and lactate which are catabolized via pyruvate. This conclusion is supported by the finding that revertants isolated from strain PC2 have regained simultaneously the ability to synthesize active pyruvate carboxylase and the ability to utilize glucose or lactate for growth. The growth of mutant PC2 on acetate, unlike that of the parent wild type, is inhibited when glucose or lactate is added to the medium. Secondary mutants obtained from PC2, which are resistant to such inhibition, still carry the original pyruvate carboxylase lesion but are derepressed for isocitrate lyase. This suggests that the inhibition of the growth of mutant PC2 is due to a block in the functioning of the glyoxylate cycle, produced by the glucose or lactate supplement.

Deoxyribonucleic acid (DNA) from several "DNA-deficient" amber mutants was observed to be either nicked (amber 22, 82, 122, and wild type) or cut (amber 453) after injection into a nonpermissive host. This effect was inhibited by chloramphenicol (CM), indicating that it is due to phage-induced enzymes. Although most of the mutants tested for replication in a density-label system were in fact DNA-deficient (amber 22, 82, 122), one (amber 81) was found to replicate almost identically to the wild type, and another (amber 453) was found to assume a hybrid density only. The hybrid moiety was less than, or equal to, one phage equivalent length, and was more efficiently extracted from infected bacteria than was similarly replicated DNA from wild-type phage. Interparental recombination between heavy and light parental DNA was observed for amber 82, 122, and wild type, but was not observed for amber 453; it was inhibited by CM. In contrast to amber 82 and wild type, the amber 453 intracellular DNA does not have single-strand regions, Because amber 453, unlike amber 82 and wild type T4, does not recombine, nicking and exposure of single-strand regions is postulated to be a prerequisite for recombination.

The following compounds or treatments have been shown to inhibit the oxidation of ammonia, but not the oxidation of hydroxylamine in cells of Nitrosomonas: (i) metal-binding agents such as allylthiourea or potassium cyanide; (ii) compounds such as SKF 525 which interact with cytochrome P-450 of mammalian microsomes; (iii) carbon monoxide; (iv) inhibitors of catalase, peroxidase, and amine oxidases such as thiosemicarbazide, ethylxanthate, and iproniazid, respectively; (v) uncouplers of oxidative phosphorylation such as m-chlorocarbonylcyanidephenylhydrazone; (vi) electron acceptors such as phenazine methosulfate; (vii) compounds such as methanol or N(2)O which react with free radicals; and (viii) illumination with 420 lux (5,000 foot candles) of light.