Pharmacogenetic allele nomenclature: International workgroup recommendations for test result reporting.

This article provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward.

Role of the periplasmic domain of the Escherichia coli NarX sensor-transmitter protein in nitrate-dependent signal transduction and gene regulation.

The narX, narQ and narL genes of Escherichia coli encode a nitrate-responsive two-component regulatory system that controls the expression of many anaerobic electron-transport- and fermentation-related genes. When nitrate is present, the NarX and NarQ sensor-transmitter proteins function to activate the response-regulator protein, NarL, which in turn binds to its DNA-recognition sites to modulate gene expression. The sensor-transmitter proteins are anchored in the cytoplasmic membrane by two transmembrane domains that are separated by a periplasmic region of approximately 115 amino acids. In this study we report the isolation and characterization of narX* (star) mutants that constitutively activate nitrate reductase (narGHJI) gene expression and repress fumarate reductase (frdABCD) gene expression when no nitrate is provided for the cell. An additional narX mutant was identified that has lost its ability to respond to environmental signals. Each narX defect was caused by a single amino acid substitution within a conserved 17 amino acid sequence, called the 'P-box', in the periplasmic exposed region of the NarX protein. As a result, DNA binding is then 'locked-on' or 'locked-off' to give the observed pattern of gene expression. Diploid analysis of these narX mutants showed that a NarX P-box mutant which conferred a 'locked-on' phenotype was trans dominant over wild-type NarX. Both were also trans dominant over the NarX P-box mutant which conferred a 'locked-off' phenotype. Certain narX P-box mutations, when combined with a narX 'linker' region mutation, were recessive to the NarX linker mutation. Finally, a truncated form of the NarX protein that lacked the periplasmic and membrane regions also showed a 'locked-on' phenotype in vivo. Thus, the periplasmic and membrane domains are essential for signal transduction to NarL. From these findings, we propose that nitrate is detected in the periplasmic space of the cell, and that a signal-transduction event through the cytoplasmic membrane into the interior of the cell modulates the NarX-dependent phosphorylation/dephosphorylation of NarL.

The dsg gene of Myxococcus xanthus encodes a protein similar to translation initiation factor IF3.

The dsg mutants of Myxococcus xanthus are defective in fruiting body development and sporulation, yet they grow normally. The deduced amino acid sequence of the dsg gene product is 50 and 51% identical to the amino acid sequence of translation initiation factor IF3 of both Escherichia coli and Bacillus stearothermophilus, respectively. However, the Dsg protein has a carboxy-terminal extension of 66 amino acids, which are absent from its E. coli and B. stearothermophilus homologs. The Shine-Dalgarno sequence GGAGG and 5 bases further upstream are identical in M. xanthus and several enteric bacteria, despite the wide phylogenetic gap between these species. The infC gene, which encodes IF3 in enteric bacteria, starts with the atypical translation initiation codon AUU, which is known to be important for regulating the cellular level of IF3 in E. coli. Translation of the Dsg protein overexpressed from the M. xanthus dsg gene in E. coli cells initiates at an AUC codon, an atypical initiation codon in the AUU class. The dsg mutants DK429 and DK439 carry the same missense mutation that changes Gly-134 to Glu in a region of amino acid identity.

The Myxococcus xanthus dsg gene product performs functions of translation initiation factor IF3 in vivo.

The amino acid sequence of the Dsg protein is 50% identical to that of translation initiation factor IF3 of Escherichia coli, the product of its infC gene. Anti-E. coli IF3 antibodies cross-react with the Dsg protein. Tn5 insertion mutations in dsg are lethal. When ample nutrients are available, however, certain dsg point mutant strains grow at the same rate as wild-type cells. Under the starvation conditions that induce fruiting body development, these dsg mutants begin to aggregate but fail to develop further. The level of Dsg antigen, as a fraction of total cell protein, does not change detectably during growth and development, as expected for a factor essential for protein synthesis. The amount of IF3 protein in E. coli is known to be autoregulated at the translational level. This autoregulation is lost in an E. coli infC362 missense mutant. The dsg+ gene from Myxococcus xanthus restores normal autoregulation to the infC362 mutant strain. Dsg is distinguished from IF3 of E. coli, other enteric bacteria, and Bacillus stearothermophilus by having a C-terminal tail of 66 amino acids. Partial and complete deletion of this tail showed that it is needed for certain vegetative and developmental functions but not for viability.

Nitrate- and molybdenum-independent signal transduction mutations in narX that alter regulation of anaerobic respiratory genes in Escherichia coli.

Escherichia coli can respire anaerobically by reducing nitrate, trimethylamine-N-oxide, dimethyl sulfoxide, or fumarate. When nitrate is present, expression of the genes for fumarate (frdABCD), trimethylamine-N-oxide, and dimethyl sulfoxide (dmsABC) is repressed while expression of the nitrate reductase (narGHJI) gene is induced. This regulation requires molybdate and is mediated by the narX and narL gene products, which together form a two-component regulatory system. We provide evidence that NarX is a nitrate and molybdenum sensor which activates NarL when nitrate is available to cells. Mutants generated by hydroxylamine mutagenesis were repressed for frdA-lacZ expression even when cells were grown in the absence of nitrate. The mutations responsible for three of these nitrate independence (NarX*) phenotypes were localized to narX and further characterized in vivo for their ability to repress frdA-lacZ expression. Two of the mutants (the narX64 and narX71 mutants) had a greatly reduced requirement for molybdenum to function but still responded to nitrate. In contrast, a third mutant (the narX32 mutant) required molybdenum but did not exhibit full repression of frdA-lacZ expression even when nitrate was present. These narX* alleles also caused the induction of nitrate reductase gene expression and the repression of a dmsA-lacZ fusion in the absence of nitrate. Each narX* mutation was determined to lie in an 11-amino-acid region of the NarX polypeptide that follows a proposed transmembrane domain. We suggest that the conformation of the narX* gene products is altered such that even in the absence of nitrate each of these gene products more closely resembles the wild-type NarX protein when nitrate is present. These data establish a clear role for the narX gene product in gene regulation and strongly suggest its role in sensing nitrate and molybdenum.

Identification of a second gene involved in global regulation of fumarate reductase and other nitrate-controlled genes for anaerobic respiration in Escherichia coli.

Fumarate reductase catalyzes the final step of anaerobic electron transport in Escherichia coli when fumarate is used as a terminal electron acceptor. Transcription of the fumarate reductase operon (frdABCD) was repressed when cells were grown in the presence of either of the preferred terminal electron acceptors, oxygen or nitrate, and was stimulated modestly by fumarate. We have previously identified a locus called frdR which pleiotropically affects nitrate repression of fumarate reductase, trimethylamine N-oxide reductase, and alcohol dehydrogenase gene expression and nitrate induction of nitrate reductase expression (L. V. Kalman and R. P. Gunsalus, J. Bacteriol. 170:623-629, 1988). Transformation of various frdR mutants with plasmids identified two complementation groups, indicating that the frdR locus is composed of two genes. One class of mutants was not completely restored to wild-type frdA-lacZ expression or nitrate reductase induction when complemented with multicopy narX+ plasmids, whereas low-copy narX+ plasmid-containing strains were. A second class of frdR mutants was identified and shown to correspond to a previously described gene, narL (frdR2). Complementation of these strains with multicopy narL+ plasmids resulted in superrepression of frdA-lacZ expression and moderate elevation of nitrate reductase expression. Multicopy plasmids containing both narL+ and narX+ or only narL+ were able to complement narL mutants, whereas narX+ plasmids complemented narX mutants only when present in a copy number approximately equal to that of narL. Both narL and narX mutants retained normal oxygen control of frdA-lacZ expression. Both types of mutants are pleiotropic, as evidenced by derepressed levels of the fumarate reductase and trimethylamine N-oxide reductase enzymes and by defective induction of nitrate reductase when cells were grown in the presence of nitrate. These results indicate that both the narL and narX gene products must be present in a defined ratio in the cell. We conclude that these proteins interact to effect normal nitrate control of the anaerobic electron transport-associated operons. From these studies, we propose that narX encodes a nitrate sensor protein while narL encodes a DNA-binding regulatory protein which together function in a manner analogous to other two-component regulatory systems.

Nucleotide sequence of the narL gene that is involved in global regulation of nitrate controlled respiratory genes of Escherichia coli.

The DNA sequence was determined for the narL gene of Escherichia coli. This gene is involved in global regulation of a number of nitrate controlled genes including frdABCD, tor, narGHJI, and adhE which are associated with bacterial respiration and fermentation. Comparison of the deduced amino acid sequence of narL to that of other bacterial genes revealed significant homologies to the phoB, ompR, and virG gene products based on the presence of similar protein domains. These DNA binding proteins are members of two-component regulatory systems. The similarities suggest that narL may also participate in such a two-component regulatory system and that the narR gene, which lies upstream of narL, may encode a second component required for nitrate control of gene regulation. In vitro protein synthesis experiments using a narL plasmid identified a putative NarL protein of 29 kDa in size consistent with the DNA sequence analysis. Primer extension experiments revealed the presence of two 5' termini for narL mRNA, and indicates that transcription may be complex.

The frdR gene of Escherichia coli globally regulates several operons involved in anaerobic growth in response to nitrate.

Fumarate reductase catalyzes the terminal step of anaerobic electron transport with fumarate as a terminal electron acceptor. Transcription of the fumarate reductase (frdABCD) operon in Escherichia coli is repressed in the presence of the preferred terminal electron acceptors, oxygen and nitrate. To identify trans-acting genes involved in regulation by nitrate, a number of E. coli mutants were generated in which expression of a frdA'-'lacZ protein fusion was no longer fully repressed by nitrate. One of these mutants, strain LK23R35, exhibited 17-fold higher beta-galactosidase activity than the wild-type strain when grown anaerobically in the presence of nitrate. When grown aerobically in the presence of nitrate, it contained three- to fourfold more beta-galactosidase activity than the wild-type strain did. Oxygen regulation of frd expression, however, was unaffected by the mutation, since the level of beta-galactosidase activity in both strains was nearly identical when they were grown in the absence of nitrate either aerobically or anaerobically. To confirm that the mutation acts in trans to frdABCD, we measured fumarate reductase levels and found them to parallel FrdA'-beta-galactosidase activity under all growth conditions tested. The effect of the mutation is pleiotropic, since the levels of nitrate reductase in LK23R35 were not induced by the addition of nitrate. The frdR mutant was also derepressed for nitrate control of the trimethylamine-N-oxide reductase and alcohol dehydrogenase enzymes. The mutation maps in a region between trp and hemA at 27 min on the E. coli chromosome. This gene, where we call frdR, is involved in both positive and negative regulation of electron transport and fermentation associated genes. A cloned 4.9-kilobase fragment of chromosomal DNA was found to complement the frdR mutation; both repression of fumarate reductase gene expression and activation of nitrate reductase gene expression were restored.