Biomedical subjects

J R Roth

Publications and source records attributed to J R Roth.

At least 55 records · Page 3Linked to original sources

PubMed1992

A single regulatory gene integrates control of vitamin B12 synthesis and propanediol degradation.

The cob operon of Salmonella typhimurium encodes enzymes required for synthesis of adenosyl-cobalamin (vitamin B12). The pdu operon encodes enzymes needed for use of propanediol as a carbon source, including an adenosyl-cobalamin-dependent enzyme, propanediol dehydratase. These two operons both map near min 41 of the S. typhimurium linkage map and are transcribed divergently. Here we report that the cob and pdu operons form a single regulon. Transcription of this regulon is induced by either glycerol or propanediol. The metabolism of these compounds is not required for induction. Propanediol induces the regulon either aerobically or anaerobically during growth on poor carbon sources. Aerobically glycerol induces only if its metabolism is prevented by a mutational block such as a glpK mutation. Under anaerobic conditions, glycerol induces in both glpK+ and glpK mutant strains during growth on poor carbon sources. A new class of mutations, pocR, prevents induction of the cob/pdu regulon by either propanediol or glycerol and causes a Cob- Pdu- phenotype. The pocR gene is located between the cob and pdu operons and appears to encode a trans-acting protein that acts as a positive regulator of both operons. Transcription of the pocR regulatory gene is induced, even without the PocR protein, during aerobic growth on poor carbon sources and during anaerobic respiration. With the functional PocR protein, transcription of the pocR gene is autoinduced by propanediol but not by glycerol. The growth conditions that increase pocR gene expression correlate with growth conditions that allow high induction of the cob/pdu regulon. A model for control of this regulon suggests that the PocR protein is a transcriptional activator of both the cob and pdu operons and that both glycerol and propanediol can individually serve as effectors of the PocR protein. We suggest that global control mechanisms cause variation in the level of the PocR protein; an increased level of the PocR protein permits higher induction by propanediol or glycerol.

Bacterial Proteins↗

PubMed1991

A genetic characterization of the nadC gene of Salmonella typhimurium.

The nadC gene of Salmonella encodes the pyridine biosynthetic enzyme PRPP-quinolinate phosphoribosyltransferase. Using a combination of genetic techniques, a deletion map for the Salmonella nadC gene has been generated which includes over 100 point mutants and 18 deletion intervals. The nadC alleles obtained by hydroxylamine mutagenesis include those suppressed by either amber, ochre, or UGA nonsense suppressors as well as alleles suppressed by the missense suppressor, sumA. Deletions were obtained by three separate protocols including spontaneous selection for loss of the nearby aroP gene, recombination between aroP::MudA and nadC::MudA insertion alleles, and selection for spontaneous loss of tetracycline resistance in a nearby guaC::Tn10dTc insertion mutant allele. The nadC mutants comprise one complementation group and the nadC+ allele is dominant to simple, nadC auxotrophic mutant alleles. Intragenic complementation of two nadC alleles, nadC493 and nadC494, mapping to deletion intervals 17 and 18, respectively, suggests that nadC encodes a multimeric enzyme. Both nadC and the nearby aroP locus are transcribed counterclockwise on the standard genetic map of Salmonella, in opposite orientation to the direction of chromosome replication.

Chromosome Mapping↗

PubMed1991

Ability of a bacterial chromosome segment to invert is dictated by included material rather than flanking sequence.

Homologous recombination between sequences present in inverse order within the same chromosome can result in inversion formation. We have previously shown that inverse order sequences at some sites (permissive) recombine to generate the expected inversion; no inversions are found when the same inverse order sequences flank other (nonpermissive) regions of the chromosome. In hopes of defining how permissive and nonpermissive intervals are determined, we have constructed a strain that carries a large chromosomal inversion. Using this inversion mutant as the parent strain, we have determined the "permissivity" of a series of chromosomal sites for secondary inversions. For the set of intervals tested, permissivity seems to be dictated by the nature of the genetic material present within the chromosomal interval being tested rather than the flanking sequences or orientation of this material in the chromosome. Almost all permissive intervals include the origin or terminus of replication. We suggest that the rules for recovery of inversions reflect mechanistic restrictions on the occurrence of inversions rather than lethal consequences of the completed rearrangement.

Chromosome Inversion↗

PubMed1991

Synthesis of thiamine in Salmonella typhimurium independent of the purF function.

In Salmonella typhimurium, the first five steps in purine biosynthesis also serve as the first steps in the biosynthesis of the pyrimidine moiety of thiamine (vitamin B1). Strains with null mutations of the first gene of purine-thiamine synthesis (purF) can, under some circumstances, grow without thiamine. This suggests the existence of an alternative pathway to thiamine that can function without the purF protein. To demonstrate the nature and map position of the purF mutations corrected, a fine-structure genetic map of the purF gene was made. The map allows identification of deletion mutations that remove virtually all of the purF gene, as defined by mutations. We describe conditions and mutations (panR) which allow B1 synthesis appears to require enzymes which act mutants lacking purF function. The alternative route of B1 synthesis appears to require enzymes which act subsequent to the purF enzyme in the purine pathway.

Adenine↗

PubMed1991

The nadI region of Salmonella typhimurium encodes a bifunctional regulatory protein.

Mutants of the nadI and pnuA genes were independently isolated on the basis of defects in repression of NAD biosynthetic genes and defects in transport nicotinamide mononucleotide (NMN). The mutations map at min 99 on the Salmonella chromosome, and the affected regions appear to be cotranscribed. Some pairs of nadI and pnuA mutations complement, suggesting the existence of independent functions. However, cis/trans tests with particular mutations provide evidence that both repressor and transport functions are actually performed by a single bifunctional protein. (This result confirms sequencing data of Foster and coworkers [J. W. Foster, Y. K. Park, T. Fenger, and M. P. Spector, J. Bacteriol. 172:4187-4196, 1990]). We have designated the gene for this bifunctional protein nadI and distinguish the regulatory and transport defects with phenotypic designations (R and T). When a nadI(R- T+) mutation (eliminating only repression function) is placed cis to a superrepressor mutation, nadI(Rs T-), the superrepression phenotype is lost. In contrast, placement of R- and Rs T- mutations in trans allows full superrepression. This result suggests that the transport function (eliminated by the Rs T- mutation) and the repression function are provided by the same protein. Insertion mutations in the promoter-proximal repressor region of the nadI gene eliminate transport function unless the inserted element can provide both for both transcription and translation start signals; this finding suggests that there is no transcriptional or translational start between the regions encoding repression and transport functions.

Bacterial Proteins↗

PubMed1991

Activity of the nicotinamide mononucleotide transport system is regulated in Salmonella typhimurium.

Transport of nicotinamide mononucleotide (NMN) requires two functions, NadI(T) and PnuC. The PnuC protein is membrane associated, as judged by isolation of active TnphoA gene fusions and demonstration that the fusion protein is membrane associated. The PnuC function appears to be the major component of the transport system, since mutant alleles of the pnuC gene permit NMN transport in the absence of NadI(T) function. We present evidence that the activity of the NMN transport system varies in response to internal pyridine levels (presumably NAD). This control mechanism requires NadI(T) function, which is provided by a bifunctional protein encoded by the nadI gene (called nadR by Foster and co-workers [J. W. Foster, Y. K. Park, T. Fenger, and M. P. Spector, J. Bacteriol. 172:4187-4196]). The nadI protein regulates transcription of the nadA and nadB biosynthetic genes and modulates activity of the NMN permease; both regulatory activities respond to the internal pyridine nucleotide level.

Bacterial Proteins↗

PubMed1990

cobA function is required for both de novo cobalamin biosynthesis and assimilation of exogenous corrinoids in Salmonella typhimurium.

Salmonella typhimurium is able to synthesize cobalamin (B12) under anaerobic growth conditions. The previously described cobalamin biosynthetic mutations (phenotypic classes CobI, CobII, and CobIII) map in three operons located near the his locus (minute 41). A new class of mutant (CobIV) defective in B12 biosynthesis was isolated and characterized. These mutations map between the cysB and trp loci (minute 34) and define a new genetic locus, cobA. The anaerobic phenotype of cobA mutants suggests an early block in corrin ring formation; mutants failed to synthesize cobalamin de novo but did so when the corrin ring is provided as cobyric acid dicyanide or as cobinamide dicyanide. Under aerobic conditions, cobA mutants were unable to convert either cobyric acid dicyanide or cobinamide dicyanide to cobalamin but could use adenosylcobyric acid or adenosylcobinamide as a precursor; this suggests that the mutants are unable to adenosylate exogenous corrinoids. To explain the anaerobic CobI phenotype of a cobA mutant, we propose that the cobA gene product catalyzes adenosylation of an early intermediate in the de novo B12 pathway and also adenosylates exogenous corrinoids. Under anaerobic conditions, a substitute function, known to be encoded in the main Cob operons, is induced; this substitute function can adenosylate exogenous cobyric acid and cobinamide but not the early biosynthetic intermediate. The cobA gene of S. typhimurium appears to be functionally equivalent to the btuR gene of Escherichia coli.

Chromosome Mapping↗

PubMed1989

Transcriptional occlusion of transposon targets.

In Salmonella typhimurium, insertion of transposons Tn5, Tn10 and bacteriophage Mu is inhibited by transcription of some target sequences. The transcription effects on Tn5 are large when the lac operon is a target but are limited to a slight effect on the hisG gene of the his operon. The Tn10 element shows target occlusion in both operons. Phage Mu has been shown previously to be inhibited for insertion into the lac operon. In the his operon Mu is only inhibited for insertion into the hisG gene. The variability of the inhibition effect from one sequence to another suggests site or regional specificity for transcription effects. Reducing the probability of insertion into transcribed sequences may be of selective importance to transposons since it reduces the risk of killing the host while maintaining the ability to transpose.

DNA Transposable Elements↗

PubMed1989

Absence of insertions among spontaneous mutants of Salmonella typhimurium.

While insertion sequences (IS) in Escherichia coli transpose frequently to generate spontaneous insertion mutants, such mutations are rare in Salmonella typhimurium: the only documented insertion mutation is a hisD mutation caused by the Salmonella-specific IS element IS200. To obtain more examples of IS200 insertion mutations and to seek additional types of IS elements in Salmonella, we selected and characterized 422 independent, spontaneous His- mutants and some 2100 additional mutants that are not necessarily independent. None of the mutants showed the absolute polar effect characteristic of insertion mutations or the reversion properties characteristic of insertions (low spontaneous reversion frequency and no reversion induction by chemical mutagens). A few mutants, showing a high spontaneous reversion frequency, were screened physically. No insertion mutations were found. Thus insertion mutations appear to be rare in S. typhimurium, in strong contrast to E. coli and despite the possession in Salmonella of at least one type of insertion element (IS200). These results suggest that in Salmonella transposition of the endogenous elements has been controlled. The transposition ability of the elements may have been reduced or favored target sites removed from the host genome.

Anaerobiosis↗

PubMed1989

Heme-deficient mutants of Salmonella typhimurium: two genes required for ALA synthesis.

The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). We have isolated, mapped and characterized a large number of Salmonella typhimurium mutants auxotrophic for ALA. These mutants carry defects in either one of two genes, both required for ALA synthesis. The previously identified hemA gene maps at 35 min, and the hemL gene maps at 5 min on the S. typhimurium genetic map. Mutants in hemA and hemL are defective for aerobic and anaerobic respiration, and appear to be oxygen sensitive. The Hem- phenotype of hemL mutants is less severe than that of hemA mutants. Although hemA and hemL mutants are deficient in heme synthesis, genetic tests indicate that they still synthesize two minor products of the heme pathway, siroheme and cobalamin (vitamin B12), under anaerobic conditions. In contrast, hemB, hemC and cysG mutants, blocked after ALA synthesis, make neither siroheme nor vitamin B12. Double mutants defective in both hemA and hemL also make siroheme. We suggest that hemA and hemL are required for one route of ALA synthesis and that a second, minor route of ALA synthesis may operate in S. typhimurium; this second pathway would be independent of the hemA and hemL functions.

Aminolevulinic Acid↗

PubMed1989

Role of recBC function in formation of chromosomal rearrangements: a two-step model for recombination.

The role of recBC functions has been tested for three types of chromosomal recombination events: (1) recombination between direct repeats to generate a deletion, (2) recombination between a small circular fragment and the chromosome, and (3) recombination between inversely oriented repeats to form an inversion. Deletion formation by recombination between direct repeats, which does not require a fully reciprocal exchange, is independent of recBC function. Circle integration and inversion formation are both stimulated by the recBC function; these events require full reciprocality. The results suggest that half-reciprocal exchanges can occur without recBC, but recBC functions greatly stimulate completion of a fully reciprocal exchange. We propose that chromosomal recombination is a two-step process, and recBC functions are primarily required for the second step.

Chromosome Deletion↗

PubMed1989

Recombination between homologies in direct and inverse orientation in the chromosome of Salmonella: intervals which are nonpermissive for inversion formation.

Sequences placed in inverse order at particular chromosome sites (permissive) recombine to generate an inversion; the same sequences, placed at other sites (nonpermissive) interact recombinationally but do not form the expected inversion recombinants. We have investigated the events that occur between sequences at nonpermissive sites. Genetically marked lac operons in inverse order were placed at nonpermissive sites in a single chromosome and Lac+ recombinants were selected. No inversions were formed. The Lac+ recombinants recovered include double-recombinant types in which information appears to have undergone a nonreciprocal information exchange; one mutant copy is repaired with no alteration of the other copy. Recombination within the lac operon is stimulated more than 100-fold by the presence of extensive homology (antenna sequences) outside of the region for which recombination is selected. Sequences placed in direct order at the ends of the same noninvertible chromosome segment recombine to form all the expected recombinant types including those in which a reciprocal exchange has generated a duplication. All the detected recombinant types can be accounted for by recombination between sister chromosomes. These results are discussed in terms of two alternative models. One explanation of the failure to detect inversion of some intervals is that particular inversions are lethal, despite the fact that no essential sequences are disrupted. Another explanation is that chromosome topology prevents sequences at nonpermissive sites in a single chromosome from engaging in the direct interaction required for inversion formation, but allows the sister strand exchanges that can generate the recombinant observed.

Base Sequence↗

PubMed1989

Role of gene duplications in the adaptation of Salmonella typhimurium to growth on limiting carbon sources.

Duplication-containing cells are selected when growth of Salmonella typhimurium is limited by the availability of any one of several carbon and energy sources. Under conditions of extreme starvation, growth occurs almost exclusively in the duplication-containing fraction of the population. Cells with duplications of one large segment of the chromosome are repeatedly selected regardless of which of these carbon sources limits growth. The duplicated chromosomal segment encodes the transport systems for all of these carbon sources. This duplication is not selected during growth on a carbon source for which the permease is not included within the duplication segment. This suggests that the growth advantage conferred by the duplication may be due to increased transport of the limiting carbon source. Inclusion of the permease alone is not sufficient to explain the growth advantage of the duplications, since other common duplications that include the permease are not selected.

Adaptation, Physiological↗

PubMed1989

recB and recC genes of Salmonella typhimurium.

We have investigated the genetic organization of the recB (exonuclease V) and recC (exonuclease V) genes of Salmonella typhimurium. A detailed genetic map is constructed that includes the relative order in the chromosome, P22 cotransduction frequencies, and the orientation of transcription of the recB and recC genes. In addition, the isolation and characterization of Mu dJ insertion mutations in recB and recC are discussed.

Chromosome Mapping↗

PubMed1989

Mutations affecting regulation of cobinamide biosynthesis in Salmonella typhimurium.

Transcription of the genes for cobalamin biosynthesis is reduced during aerobic growth. We isolated and characterized mutants that showed a 2- to 90-fold increase in aerobic expression of the cobinamide biosynthesis (CobI) genes, depending on the particular mutation and growth conditions. Four different classes of mutations were characterized. All mutations (CobRI through CobRIV) were cis-acting, dominant mutations that mapped near the promoter end of the CobI operon. Two of these classes of mutations (III and IV) caused an increase in anaerobic as well as aerobic transcription of the CobII and CobIII operons; this led to increased biosynthesis of cobalamin under anaerobic growth conditions. A recessive mutation (cobF) mapping far from the CobI operon increased anaerobic CobI operon expression by about fourfold.

Chromosome Mapping↗

PubMed1989

Redox regulation of the genes for cobinamide biosynthesis in Salmonella typhimurium.

Transcription of the cobinamide biosynthetic genes (the CobI operon) was induced under three different physiological conditions: anaerobiosis (anaerobic respiration or fermentation), aerobic respiration at low oxygen levels, and aerobic respiration with a partial block of the electron transport chain. After a shift to inducing conditions, there was a time lag of approximately 50 min before the onset of CobI induction. Under conditions of anaerobic respiration, the level of CobI transcription was dependent on the nature of both the electron donor (carbon and energy source) and the acceptor. Cells grown with electron acceptors with a lower midpoint potential showed higher CobI expression levels. The highest level of CobI transcription observed was obtained with glycerol as the carbon source and fumarate as the electron acceptor. The high induction seen with glycerol was reduced by mutational blocks in the glycerol catabolic pathway, suggesting that glycerol does not serve as a gratuitous inducer but must be metabolized to stimulate CobI transcription. In the presence of oxygen, CobI operon expression was induced 6- to 20-fold by the following: inhibition of cytochrome o oxidase with cyanide, mutational blockage of ubiquinone biosynthesis, and starvation of mutant cells for heme. We suggest that the CobI operon is induced in response to a reducing environment within the cell and not by the absence of oxygen per se.

Aerobiosis↗

PubMed1989

DNA ligase and the pyridine nucleotide cycle in Salmonella typhimurium.

Bacterial DNA ligases use NAD as an energy source. In this study we addressed two questions about these enzymes. First, what is the physiological consequence of completely removing the NAD-dependent enzyme and replacing it with an ATP-dependent DNA ligase? We constructed Salmonella typhimurium strains in which the endogenous NAD-dependent DNA ligase activity was inactivated by an insertion mutation and the ATP-dependent enzyme from bacteriophage T4 was provided by a cloned phage gene. Such strains were physiologically indistinguishable from the wild type, even under conditions of UV irradiation or treatment with alkylating agents. These results suggest that specific functional interactions between DNA ligase and other replication and repair enzymes may be unimportant under the conditions tested. Second, the importance of DNA ligation as the initiating event of the bacterial pyridine nucleotide cycle was critically assessed in these mutant strains. Surprisingly, our results indicate that DNA ligation makes a minimal contribution to the pyridine nucleotide cycle; the Salmonella strains with only an ATP-dependent ligase had the same NAD turnover rates as the wild-type strain with an NAD-dependent ligase. However, we found that NAD turnover was significantly decreased under anaerobic conditions. We suggest that most intracellular pyridine nucleotide breakdown occurs in a process that protects the cell against oxygen damage but involves a biochemical mechanism other than DNA ligation.

Adenosine Triphosphate↗

PubMed1989

Functions required for vitamin B12-dependent ethanolamine utilization in Salmonella typhimurium.

When B12 is available, Salmonella typhimurium can degrade ethanolamine to provide a source of carbon and nitrogen. B12 is essential since it is a cofactor for ethanolamine ammonia-lyase, the first enzyme in ethanolamine breakdown. S. typhimurium makes B12 only under anaerobic conditions; in the presence of oxygen, exogenous B12 must be provided to permit ethanolamine utilization. Genes required for ethanolamine utilization are encoded in the eut operon. For complementation testing, an F' plasmid containing the eut genes was constructed by transposition of the eut operon (flanked by two Tn10 elements) to an existing F plasmid. Complementation tests defined six genes in the eut operon. Three of these genes encode enzymes known to be involved in degradation of ethanolamine: ethanolamine ammonia-lyase (eutB and eutC) and acetaldehyde dehydrogenase (eutE). One gene (eutR) seems to encode a positive regulatory protein required for induction of transcription of eut. The function of one of the remaining two genes (eutA) was shown to be required for ethanolamine utilization only when cyano-B12 or hydroxy-B12 were the precursors of the adenosyl-B12 cofactor of ethanolamine ammonia-lyase; eutA mutants could use ethanolamine as the nitrogen source only when adenosyl-B12 was provided. No function has been assigned to the eutD gene, which is required for use of ethanolamine as a carbon source. Ethanolamine uptake assays of eut mutants suggest that no ethanolamine permease is encoded in the eut operon.

Biological Transport↗