Regulation of membrane peptides by the Pseudomonas plasmid alk regulon.

Pseudomonas putida strains carrying the plasmid alk genes will grow on n-alkanes. Induced alk+ strains contain membrane activities for alkane hydroxylation and dehydrogenation of aliphatic primary alcohols. P. putida cytoplasmic and outer membranes can be separated by sucrose gradient centrifugation after disruption of cells by either mild detergent lysis or passage through a French press. Both the membrane component of alkane hydroxylase and membrane alcohol dehydrogenase fractionated with the cytoplasmic membrane. Induction of the alk regulon resulted in the appearance of at least three new plasmid-determined cytoplasmic membrane peptides of about 59,000 (59K), 47,000 (47K), and 40,000 (40K) daltons as well as the disappearance of a pair of chromosomally encoded outer membrane peptides of about 43,000 daltons. The 40K peptide is the membrane component of alkane hydroxylase and the product of the plasmid alkB gene because the alkB1029 mutation altered the properties of alkane hydroxylase in whole cells, reduced its thermal stability in cell extracts, and led to increased electrophoretic mobility of the inducible 40K peptide. These results are consistent with a model for vectorial oxidation of n-alkanes in the cytoplasmic membrane of P. putida.     

Fractionation of inducible alkane hydroxylase activity in Pseudomonas putida and characterization of hydroxylase-negative plasmid mutations.

The plasmid-determined inducible alkane hydroxylase of Pseudomonas putida resolved into particulate and soluble fractions. Spinach reductase and spinach ferredoxin could replace the soluble hydroxylase component. Two alkane hydroxylase mutants show in vitro complementation (S. Benson and J. Shapiro, J. Bacteriol., 123: 759-760, 1975): one, alk-7, lacks an active soluble component and the other, alk-181, lacks an active particulate component. Together with previous results on a particulate alcohol dehydrogenase enzyme (Benson and Shapiro, J. Bacteriol., 126: 794-798, 1976), these results allowed us to assay three plasmid-determined inducible activities: soluble alkane hydroxylase (alkA+), particulate alkane hydroxylase (alkB+), and particulate alcohol dehydrogenase (alkC+). Growth tests and in vitro complementation assays revealed three groups of plasmid mutations that block expression of alkane hydroxylase activity: alkA, which so far includes only the alk-7 mutation; alkB, which includes alk-181 and 11 other mutations; and a pleiotropic-negative class, which includes nine mutations that lead to loss of alkA+, alkB+, and alkC+ activities. Thus, the alk+ gene cluster found on IncP-2 plasmids contains at least four cistrons. We believe it is significant that two of these determined the presence of membrane proteins. The accompanying paper shows that these loci are part of a single regulon.     

Distribution of mannosamine and mannosaminuronic acid among cell walls of Bacillus species

Some Escherichia coli K-12 lamB mutants, those producing reduced amounts of LamB protein (one-tenth the wild type amount), grow normally on dextrins but transport maltose when present at a concentration of 1 microM at about one-tenth the normal rate. lamB Dex- mutants were found as derivatives of these strains. These Dex- mutants are considerably impaired in the transport of maltose at low concentrations (below 10 microM), and they have a structurally altered LamB protein which is impaired in its interaction with phages lambda and K10 but still interacts with a lambda host range mutant lambda hh*. The Dex- mutants are double lamB mutants carrying one mutation, already present in the parental strains, that reduces LamB synthesis and a second that alters LamB structure. The secondary mutations, present in different independent Dex- mutants, are clustered in the same region of the lamB gene. Dex+ revertants were isolated and analyzed: when the altered LamB protein is made in wild-type amount, due to a reversion of the first mutation, the phenotype reverts to Dex+. However, these Dex+ revertants are still very significantly impaired in maltose transport at low concentrations (below 10 microM).     

Biochemical characterization of the hamster thy mutator gene and its revertants

The thy- mutator phenotype of Chinese hamster ovary cells is distinguished by increased intracellular levels of dCTP, auxotrophy for thymidine, and elevated spontaneous mutational rates. To determine the biochemical lesion responsible for this complex phenotype, enzymes responsible for the synthesis of dCTP and dTTP were investigated. Levels of ribonucleotide reductase and dCMP deaminase were identical in mutant and wild type strains. In contrast, CTP synthetase activity in extracts from thy- strains was consistently altered in that 50% of enzyme activity was resistant to feedback inhibition by CTP. Additionally, thy- strains obtained by DNA transfection also had CTP-resistant CTP synthetase. Thy+ revertants lost the resistant enzyme, and total activity was reduced. CTP-resistant CTP synthetase was regained in thy- mutants reselected from thy+ revertants, but in these strains all activity was resistant. These experiments demonstrate that the thy- mutator phenotype is a consequence of a mutation of CTP synthetase and suggest that one pathway of reversion to the wild type state is by loss or inactivation of the mutant allele rendering the revertants hemizygous for the gene.     

Regulation of beta-glucuronidase synthesis in Escherichia coli K-12: pleiotropic constitutive mutations affecting uxu and uidA expression.

Among the beta-glucuronidase (UID)-constitutive mutants obtained by growth on methyl-beta-D-galacturonide, some strains are also derepressed for the two enzymes of the uxu operon: mannonate oxidoreductase (MOR) and mannonate hydrolyase (HLM). By conjugation and transduction experiments, two distinct constitutive mutations were separated in each pleiotropic mutant strain. One of them was specific for uidA gene expression and was characterized as affecting either uidO or uidR sites. The second type of mutation was mapped close to the uxu operon and was found to be responsible for the pleiotropic effect revealed in the primary mutants: after separation such a mutation still fully derepresses MOR and HLM synthesis but weakly derepresses UID synthesis. The pleiotropic effect of this mutation was maintained even though the activity of the structural genes was altered. This rules out the occurrence of an internal derepressing interaction between these enzymes. In merodiploid strains, uxu-linked constitutive mutations were recessive to the wild-type allele, suggesting that these mutations could affect a regulatory gene. The uxuR gene is probably a specific regulatory gene for a very close operon, uxu. Moreover, it has a weak effect on uidA expression. Thus, UID synthesis would be negatively controlled through the activity of two repressor molecules that are synthesized by two distinct regulatory genes, uidR and uxuR. These two repressing factors are antagonized, respectively, by phenyl-thio-beta-D-glucuronide and mannonic amide and could cooperate in a unique repression/induction control over uidA expression. Constitutive mutations affecting the control sites of uidA gene probably characterize two distinct attachment sites in the operator locus for each of the repressor molecules.     

Insertion mutations in the control region of the Gal operon of E. coli. I. Biological characterization of the mutations

Summary Galactose negative mutations are described which reduce the maximum expression of all three gal genes about 100-fold. The residual enzyme synthesis is not or only slightly inducible.These pleiotropic mutations map in the control region of the gal operon. No recombination is observed between these mutations. All mutants revert spontaneously to a Gal⁺ phenotype. In some mutations wildtype-like as well as constitutive revertants are obtained. The frequency of reversion can be increased by nitrosoguanidine (NG) in all mutants. The revertants, induced by this mutagen, are of a constitutive type.     

Regulation of Escherichia coli K-12 hexuronate system genes: exu regulon.

Two types of Escherichia coli K-12 regulatory mutants, partially or totally negative for the induction of the five catabolic enzymes (uronic isomerase, uxaC; altronate oxidized nicotinamide adenine dinucleotide: uxaB; mannonate hydrolyase, uxuA) and the transport system (exuT) of the hexuronate-inducible pathway, were isolated and analyzed enzymatically. Hexuronate-catabolizing revertants of the negative mutants showed a constitutive synthesis for some or all of these enzymes. Negative and constitutive mutations were localized in the same genetic locus, called exuR, and the following order for the markers situated between the min 65 and 68 was determined: argG--exuR--exuT--uxaC--uxaA--tolC. The enzymatic characterization of the pleiotropic negative and constitutive mutants of the exuR gene suggests that the exuR regulatory gene product exerts a specific and total control on the three exuT, uszB, and uxaC-uxaA operons of the galacturonate pathway and a partial control on the uxuA-uxuB operon of the glucuronate pathway. The analysis of diploid strains conatining both the wild type and a negative or constitutive allele of the exuR gene, as well as the analysis of thermosensitive mutants of the exuR gene, was in agreement with a negative regulatory mechanism for the control of the hexuronate system.     

Temperature-Sensitive Pleiotropic Revertants from a Mutant in the AROM Gene Cluster of NEUROSPORA CRASSA

Genetical and biochemical studies have been performed with revertants induced in a polyaromatic mutant (No. 58) in the arom gene cluster of Neurospora crassa. In addition to complete and partial revertants able to grow on minimal at both 25 degrees and 35 degrees , temperature-sensitive revertants capable of growth on minimal at 25 degrees but not at 35 degrees have been recovered. One of these revertants has been shown to lack biosynthetic dehydroquinase activity at both temperatures (utilizing the inducible catabolic isozyme for growth at 25 degrees ), to have dehydroshikimate reductase activity only at 25 degrees , and to form an arom aggregate having a molecular weight approximately one-half that of wild type. These results are interpreted as indicating that pleiotropic mutants in the arom gene cluster can result from missense mutations, as well as from nonsense mutations as indicated in previous studies.     

Determinants for overproduction of the Pseudomonas oleovorans cytoplasmic membrane protein alkane hydroxylase in alk+ Escherichia coli W3110.

The Pseudomonas oleovorans alkB gene is expressed in alk+ Escherichia coli W3110 to 10 to 15% of the total cell protein, which is exceptional for a (foreign) cytoplasmic membrane protein. In other E. coli recombinants such as alk+ HB101, AlkB constitutes 2 to 3% of the total protein. In this study, we have investigated which factors determine the expression level of alkB in alk+ W3110. In particular, we have investigated the role of AlkB-induced stimulation of phospholipid synthesis. Blocking phospholipid synthesis in alk+ W3110 did not specifically alter the expression of alkB, and we conclude that stimulation of phospholipid synthesis is not a prerequisite for high-level expression of the membrane protein. W3110 is able to produce exceptionally high levels of alkane monooxygenase, because the rate of alkB mRNA synthesis in W3110 is an order of magnitude higher than that in HB101. This may be due in part to the higher copy number of pGEc47 in W3110 in comparison with HB101.     

Positive selection for loss of RpoS function in Escherichia coli

Though RpoS, an alternative sigma factor, is required for survival and adaptation of Escherichia coli under stress conditions, many strains have acquired independent mutations in the rpoS gene. The reasons for this apparent selective loss and the nature of the selective agent are not well understood. In this study, we found that some wild type strains grow poorly in succinate minimal media compared with isogenic strains carrying defined RpoS null mutations. Using an rpoS+ strain harboring an operon lacZ fusion to the highly-RpoS dependent osmY promoter as an indicator strain, we tested if this differential growth characteristic could be used to selectively isolate mutants that have lost RpoS function. All isolated (Suc+) mutants exhibited attenuated beta-galactosidase expression on indicator media suggesting a loss in either RpoS or osmY promoter function. Because all Suc+ mutants were also defective in catalase activity, an OsmY-independent, RpoS-regulated function, it was likely that RpoS activity was affected. To confirm this, we sequenced PCR-amplified products containing the rpoS gene from 20 independent mutants using chromosomal DNA as a template. Sequencing and alignment analyses confirmed that all isolated mutants possessed mutated alleles of the rpoS gene. Types of mutations detected included single or multiple base deletions, insertions, and transversions. No transition mutations were identified. All identified point mutations could, under selection for restoration of beta-galactosidase, revert to rpoS+. Revertible mutation of the rpoS gene can thus function as a genetic switch that controls expression of the regulon at the population level. These results may also help to explain why independent laboratory strains have acquired mutations in this important regulatory gene.     

A new gene (alkB) of Escherichia coli that controls sensitivity to methyl methane sulfonate.

Seven mutants of Escherichia coli were isolated that are sensitive to methyl methane sulfonate but not to UV light. They exhibited decreased host cell reactivation capacity for methyl methane sulfonate-treated phage lambda. Five of the mutations were mapped in the same region as alkA (previously called alk) and may indeed be identical to known mutations. Another mutation was found near nalA, and the gene responsible was named alkB. Its phenotype was different from that of ada, since the alkB mutant exhibited a normal adaptive response to N-methyl-N'-nitro-N-nitrosoguanidine. A third type of mutation was mapped near polA, but this mutant contained an almost normal level of DNA polymerase I activity.     

Plasmid mutations affecting self-maintenance and host growth in Escherichia coli.

As reported in the accompanying paper, a number of mutants of the ColVBtrp plasmid that can not be maintained stably in the host cell of Escherichia coli have been isolated. Each of the mutated plasmids has been transferred to an isogenic Col minus strain, and the resulting Col+ strains were studied to examine the effects of plasmid mutations on some properties of the host bacteria. Many of the strains harboring a mutated plasmid were thus found to be temperature sensitive; they failed to grow and divide normally at high temperatures. Some of them formed "filaments" under these conditions. These abnormal growth characteristics were accompanied by an increased susceptibility to sodium deoxycholate and methylene blue, suggesting that the cytoplasmic membrane has been altered. Moreover, studies of temperature-independent revertants obtained from two of these temperature-sensitive Col+ strains suggested that a single mutation on the plasmid is responsible for the pleiotropic effects exerted on the host cell. The bearing of these findings on the mode of replication and segregated of stringent-type plasmids such as ColVBtrp in the host bacteria is discussed.     

Phenotypic reversion in some early blocked sporulation mutants of Bacillus subtilis. Genetic study of polymyxin resistant partial revertants

Summary A class of suppressor mutations restores, in pleiotropic sporulation mutants of B. subtilis (SPO mutants), the wild type level of resistance to Polymyxin, and, most often, other properties of the wild strain as well, but never the ability to sporulate. These suppressors, extracistronic, are active on mutations occurring in any one of the 5 genes in which SPO mutations have been found. The phenotype of the suppressed strains is dependent on both the suppressed (SPO) and the suppressive mutations. All these suppressors are located in a single locus and some of them are thermosensitive. The evidence suggests that a physiological compensation is at work in the partial revertants, so that the locus at which the suppressors are located was called cps X. Two hypotheses are discussed that might account for these observations.     

Isolation of super-repressor mutants in the histidine utilization system of Salmonella typhimurium.

Two super-repressor mutations in the histidine utilization (hut) operons of Salmonella typhimurium are described. Cells bearing either of these mutations have levels of hut enzymes that do not increase above the uninduced levels when growth is in the presence of either histidine or the gratuitous inducer imidazole propionate. Both mutations lie in the region of the gene for the hut repressor, hutC, and reverse mutations of both are to the constitutive (repressor-negative) rather than to the inducible (wild type) phenotype. In hybrid merodiploid strains the super-repressor mutations are dominant over either wild-type (hutC+) or repressor-negative (hutC-) alleles. Whereas both super-repressor mutations cause the uninducible synthesis of hut enzymes, the degree of repression is different. One mutation causes repression of enzyme synthesis in one of the two hut operons to a level below the basal, uninduced level of wild-type cells. The other mutation causes repression to a lesser degree than in wild-type cells, so that the hut enzymes are present at a level above the normal basal level; this partially constitutive synthesis is greater for the enzymes of one of the hut operons than for the enzymes of the other. Thus, both mutations apparently result in repressors with altered operator-binding properties, in addition to altered inducer-binding properties.     

Suppression of polarity of insertion mutations within the gal operon of E. coli.

Summary Phenotypic revertants of galOP::IS1 and galOP::IS2 mutations have been isolated after mutagenesis with nitrosoguanidine, they are probably caused by mutations in gene suA. The polarity suppressor mutations described in this study and a known mutation in gene suA isolated by D. Morse (Morse and Guertin, 1972) suppress polarity caused by IS1 more effectively than that caused by IS2 or IS4. Furthermore, suppressibility is influenced by the site and orientation of IS integration.The synthesis of the three enzymes in galOP::IS suA double mutants is constitutive and the ratio of the three enzymes is altered in comparison to the wild type. The reasons for constitutive synthesis of the galactose enzymes and for the altered ratio of enzyme synthesis are discussed.     

Revertants of v-fos-transformed rat fibroblasts: suppression of transformation is dominant.

Phenotypic revertants of Finkel-Biskis-Riley (FBR)-murine sarcoma virus-transformed rat fibroblasts were isolated on the basis of their adherence to plastic tissue culture dishes in the absence of divalent cations. Some revertants had sustained deletions or inactivating mutations of the v-fos gene. However, two revertants expressed a functional v-fos gene at levels equal to that in the transformed parental cells, and therefore phenotypic reversion was due to mutations in nonviral genes. These revertants were considered nontransformed according to four criteria: (i) they were flat and had a nontransformed morphology, (ii) they were contact inhibited when grown to confluence, (iii) they did not display anchorage-independent growth in soft agar, and (iv) they did not form tumors in nude mice. Somatic-cell hybrids between the revertants and the transformed parental cells were nontransformed, suggesting that the revertants had sustained an activating mutation of a gene capable of suppressing transformation. The expression of c-jun, junB, and junD was not altered in the revertants, and they could not be transformed by transfection with a c-jun expression vector. The revertants were resistant to transformation by an activated c-Ha-ras gene but were susceptible to transformation by simian virus 40. Our results demonstrate the existence of a class of revertants that harbor genes capable of suppressing transformation by v-fos and some other oncogenes. This contrasts with previously described revertants of transformation by v-fos that contain recessive mutations.     

Ammonia regulation of amino acid permeases in Saccharomyces cerevisiae.

The activities of the proline-specific permease (PUT4) and the general amino acid permease (GAP1) of Saccharomyces cerevisiae vary 70- to 140-fold in response to the nitrogen source of the growth medium. The PUT4 and GAP1 permease activities are regulated by control of synthesis and control of activity. These permeases are irreversibly inactivated by addition of ammonia or glutamine, lowering the activity to that found during steady-state growth on these nitrogen sources. Mutants altered in the regulation of the PUT4 permease (Per-) have been isolated. The mutations in these strains are pleiotropic and affect many other permeases, but have no direct effect on various cytoplasmic enzymes involved in nitrogen assimilation. In strains having one class of mutations (per1), ammonia inactivation of the PUT4 and GAP1 permeases did not occur, whereas glutamate and glutamine inactivation did. Thus, there appear to be two independent inactivation systems, one responding to ammonia and one responding to glutamate (or a metabolite of glutamate). The mutations were found to be nuclear and recessive. The inactivation systems are constitutive and do not require transport of the effector molecules per se, apparently operating on the inside of the cytoplasmic membrane. The ammonia inactivation was found not to require a functional glutamate dehydrogenase (NADP). These mutants were used to show that ammonia exerts control of arginase synthesis largely by inducer exclusion. This may be the primary mode of nitrogen regulation for most nitrogen-regulated enzymes of S. cerevisiae.     

Physiological changes and alk gene instability in Pseudomonas oleovorans during induction and expression of alk genes.

The alk genes of Pseudomonas oleovorans, which is able to metabolize alkanes and alkenes, are organized in alkST and alkBFGHJKL clusters, in which the expression of alkBFGHJKL is positively regulated by AlkS. Growth of the wild-type strain GPo1 and P. oleovorans GPo12 alk recombinants on octane resulted in changes of cellular physiology and morphology. These changes, which included lower growth rates and a reduction of the number of CFU due to filamentation, were also seen when the cells were grown on aqueous medium, and the alk genes were induced with dicyclopropylketone, a gratuitous inducer of the alk genes. These effects were seen only for recombinants carrying both alkST and alkBFGHJKL operons. Deletion of parts of either alkB or alkJ, which encode two major Alk proteins located in the cytoplasmic membrane, modified but did not eliminate the effects described above, suggesting that they were due to induction and expression of several alk genes. Continuous growth of the cells in the presence of dicyclopropylketone for about 10 generations led to inactivation, but not elimination, of the alk genes. This resulted in a return of the recombinants to normal physiology and growth.     

Enhanced UV sensitivity of Escherichia coli strain uvrA crp]

UV-sensitivity and UV-induced mutability to tryptophan independence has been studied in isogenic crp, cya, crp+, uvrA crp and uvrA crp+ strains of Escherichia coli. crp and cya strains are found to have the same UV-sensitivity as an isogenic wild type strain. UV-sensitivity of uvrA crp strain seems to be one-two orders increased as compared with the sensitivity exhibited by the uvrA - crp+ strain. The yield of UV-induced revertants is slightly higher in crp, cya and uvrA crp strains than in the wild type cells. The existence of cap-dependent inducible error-free repair pathway is supposed due to the data obtained.