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PubMed1982

Amplification of maize ribulose bisphosphate carboxylase large subunit synthesis in E. coli by transcriptional fusion with the lambda N operon.

The maize chloroplast gene coding for the large subunit of ribulose bisphosphate carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) has been placed under the transcriptional control of the bacteriophage lambda promoter PL, by fusion with the lambda N operon located on a multicopy plasmid. Transcription from PL was repressed at 32 degrees C by the presence in the E. coli chromosome of a cIts gene that specifies a temperature-sensitive repressor. After inactivation of the repressor at 45 degrees C unmoderated transcription of the chloroplast gene occurred from the PL promoter. Translation was probably initiated from a chloroplast Shine-Dalgarno sequence located five nucleotides from the N-terminal methionine initiation codon to yield a polypeptide the same size as that synthesised in maize. This direct translation results in a level of expression of the chloroplast gene corresponding to approximately 2% of the total E. coli cell protein as ribulose bisphosphate carboxylase large subunits. Transcriptional fusions with the lambda N operon should provide a generally applicable, simple method for the amplification and regulation of chloroplast gene expression in E. coli.

Bacteriophage lambda↗
PubMed1989

Cotranscription of the S10- and spc-like operons in spinach chloroplasts and identification of three of their gene products.

The organisation and expression of the rpl22, rps3, rpl16 and rpl14 genes, which belong to the S10- and spc-like operons of spinach chloroplasts, have been studied. Northern experiments and nuclease S1 mapping show that the two operon-like groups of genes are cotranscribed. It is demonstrated that the intron-containing rpl16 gene is spliced in vivo. Based on amino acid composition and protein sequence data, the products of the rpl22, rpl16 and rpl14 genes are identified respectively as the spinach chloroplast ribosomal proteins CS-L13, CS-L24 and CS-L29. The rpl22 gene product is a 5S rRNA binding protein and therefore is distinguishable from the homologous Escherichia coli L22 ribosomal protein.

Amino Acid Sequence↗
PubMed1983

The lethal effect of a plasmid resulting from transcriptional readthrough of rplJ from the rplKA operon in Escherichia coli.

A high-copy plasmid, pGA217, which carries a deletion (lacking the carboxy-terminal 20 amino acids) of the structural gene for ribosomal protein L10 (rplJ) is lethal to the cell in the absence of the gene (rplL) for r-proteins L7/L12, but only if the upstream operon for r-proteins L11 (rplK) and L1 (rplA) is present on the same plasmid. Measurements of beta-galactosidase activity of a hybrid protein expressed by a rplL-lacZ fusion indicated that the L10 fragment peptide which lacks the carboxy-terminal 20 amino acids is capable of exerting feedback regulation. Double transformation experiments with two compatible plasmids showed that the detrimental effect of the rplJ deletion on pGA217 can be reversed by the addition of a second plasmid which carries a functional gene for L7/L12. These two pieces of evidence suggest that the lethal effect of pGA217 is due to its property of feeding back on L7/L12 production from the chromosomal rplK gene. The upstream rplKA operon was inferred to have a cis-acting, stimulating effect on rplJ expression from the following evidence: (1) donor plasmids carrying the genes for L11 and/or L1 fail to exert a trans-acting effect, (2) deletion mutants which removed portions of rplK and/or rplA, but maintained the rplKA promoter, rplKp, still retained a severe growth-inhibiting effect. We suggest that these results can be explained by assuming that there is transcription from the rplKA promoter through rplJ and perhaps beyond.

Chromosome Deletion↗
PubMed1986

The pyridine nucleotide cycle of Salmonella typhimurium: genetic characterization of the pncXA operon.

A series of Mud1 and Tn10 insertions were identified in the pncA chromosome region of Salmonella typhimurium which is responsible for the production of nicotinamide deamidase. Both pncA (resulting in no nicotinamide deamidase activity) and pncX (resulting in lowered nicotinamide deamidase activity) insertions were constructed. In addition, mutants which could utilize nicotinamide as a sole source of nitrogen were isolated. These mutants, designated pncH, hyperproduce nicotinamide deamidase. Genetic studies utilizing pncX--lacZ and pncA--lacZ operon fusions indicate that pncX::Tn10 insertions reduce transcription of pncA--lac while pncH mutations increase the expression of both pncA--lacZ and pncX-lacZ. The gene order was determined as purB--pncA--pncX--gdh with transcription of both pncA and pncX occurring in the counterclockwise direction. Merodiploid studies suggest a model whereby pncX and pncA form an operon with the major promoter occurring upstream from pncX. A second, weaker promoter for pncA must be situated between pncX and pncA. The pncH mutations appear to occur in the pncX promoter (pncXp) increasing promoter activity.

Chromosome Mapping↗
PubMed1988

The rpoA341 allele of Escherichia coli specifically impairs the transcription of a group of positively-regulated operons.

The specificity of the transcription defect caused by the rpoA341(phs) allele has been investigated. Three apparently unlinked genetic systems have been found to be impaired in their transcription by this mutant allele of the alpha subunit of RNA polymerase. These three systems, the melAB operon, the cysA locus and the ara regulon, are apparently unrelated other than by their requirement for a regulon-specific positive regulator for the initiation of transcription. Expression of the gene for the positive regulator does not appear to be significantly affected in any of the three systems. However, mutations that render expression of the araBAD operon independent of the regulatory protein also confer insensitivity to the rpoA341 allele. The significance of these observations is discussed in the context of models of positive regulation.

Alleles↗
PubMed1985

The catabolite gene activator protein (CAP) is not required for indole-3-acetic acid to activate transcription of the araBAD operon of Escherichia coli K-12.

Kline et al. (1980) have reported that indole-3-acetic acid (IAA) and four other indole derivatives are able to substitute for cAMP in activating expression of the ara regulon of E. coli. We have examined this phenomenon in detail, utilizing fusions between the structural gene for beta-galactosidase and the promoters for the araBAD, araE, and araFG operons. We confirm that IAA potently stimulates transcription from the araBAD promoter. The effect is highly specific to araBAD, as IAA has no, or only slight, effects on the araE and araFG operons. However, contrary to the results of Kline et al., we find that the action of IAA does not require CAP. Thus, IAA fully stimulates the transcription of araBAD in a strain which bears a complete deletion of the crp gene.

Arabinose↗
PubMed1978

Restoration of polarity by N-deficiency in lambda phage containing a translocated trp operon segment.

When translation of trp mRNA is terminated by a nonsense codon or by antibiotics like chloramphenicol, the amount of the mRNA distal to the blocked ribosomes is found at much lower levels ("polarity"). Polarity is alleviated when the trp mRNA is formed as part of a long transcript from the phage lambda promoter PL (Segawa and Imamoto, 1974; Franklin, 1974); but the relief of polarity is itself largely dependent on the lambda protein N. In a phage that joins the trp operon segment (trpD, C, B A) to a point distal to the N gene, lacking the tL site, synthesis of trp mRNA starting at the PL promoter continues even when translation is generally inhibited by chloramphenicol, but in the absence of functional N gene product synthesis of the mRNA can be blocked by the antibiotic. Unexpectedly, in the absence of N function, even when translation is occurring, weak termination of transcription occurs at some sites in the translocated trp operon.

Chloramphenicol↗
PubMed1979

Constitutive mutation of cysJIH operon in a cysB deletion strain of Salmonella typhimurium.

In a cysB deletion strain a new mutation, denoted cys-2332 was isolated, which causes the constitutive expression of the cysJIH operon. cys-2332 is closely linked to cysJIH and presumably is located in the initiator region of this operon, rendering its expression independent of the cysB gene product and the internal inducer O-acetyl-L-serine. The presence of sulfite reductase (encoded by cysI and cysJ) activity in a cysB- cys-2332 double mutant indicates that cysG, which is not linked to cysJIH but is required for the synthesis of the sulfite reductase co-factor siroheme, is not controlled by cysB.

Chromosome Mapping↗
PubMed1985

Role of translation in the UTP-modulated attenuation at the pyrBI operon of Escherichia coli.

A 273 bp DNA fragment containing the attenuator of the pyrBI operon was inserted into a synthetic cloning site early in the lacZ gene on a plasmid. By this operation the first few codons of lacZ were joined through a linker to the last 39 codons of the open reading frame for the putative pyrB leader peptide. In addition a gene fusion encoding a hybrid protein with beta-galactosidase activity was formed between the pyrB start and the rest of lacZ. This gene fusion is expressed from the lac promoter and the transcript is subject to facultative termination at the pyrBI attenuator. Different variants of the lacZ start were used that either contained a stop codon or directed the translation toward the attenuator in any of the alternative reading frames. The following results were obtained. No significant read-through of transcription over the pyrB attenuator was seen when the leader translation ended 49 nucleotide residues, or more, upstream of the attenuator symmetry, but a UTP-modulated attenuation was established if the leader translation was allowed to proceed across the attenuator as for the putative leader peptide or in a frame-shifted version. The regulation, however, was not as great as for the native pyrB gene. This is probably because the substitution of the normal start of the leader peptide by the start of lacZ alters the coupling between transcription and translation and thereby the attenuation frequency. It cannot, however, be ruled out that the pyrBI operon is regulated at the promoters in addition to the control by attenuation.

Aspartate Carbamoyltransferase↗
PubMed1985

Effect of mutations in the RNA polymerase gene and that of the transcription termination factor rho on expression of the Escherichia coli galactose operon with an IS2 polar insertion.

We analysed the effects on the expression of the gal operon of six phenotypically different mutations in the Escherichia coli RNA polymerase genes in combination with wild-type, rho, and mutant, rho15, alleles of the gene for the transcription termination factor. RNA polymerase mutations can enhance (rpoB268) or reduce (rpoB255), rpoC3, rpoB265) termination by the rho15 factor at the IS2 terminator. The rpoC1 mutation enhances the transcription of the gal operon regardless of the IS2 insertion or the rho15 mutation. Thus RNA polymerase mutations can, independently, or in combination with the rho mutation, compensate for the IS-induced, specifically IS2-induced, termination, leading to a partial restoration of gene activity.

DNA Transposable Elements↗
PubMed1986

Cloning and complete nucleotide sequence of the Escherichia coli glutamine permease operon (glnHPQ).

The glutamine permease operon encoding the high-affinity transport system of glutamine in Escherichia coli could be cloned in one of the mini F plasmids, but not in pBR322 or pACYC184, by selection for restoration of the Gln+ phenotype, the ability to utilize glutamine as a sole carbon source. We determined the nucleotide sequence of the glutamine permease operon, which contains the structural gene of the periplasmic glutamine-binding protein (glnH), and indispensable component of the permease activity. The N-terminal amino acid sequence and the overall amino acid composition of the purified glutamine-binding protein were in good agreement with those predicted from the nucleotide sequence, if the N-terminal 22 amino acid residues were discounted. The latter comprised two Lys residues (nos. 2 and 6) followed by 16 hydrophobic amino acid residues and was assumed to be a signal peptide for transport into the periplasmic space. There were two additional reading frames (glnP and glnQ) downstream of glnH sharing a common promoter. It was concluded that the glnP and glnQ proteins as well as the glnH protein are essential for glutamine permease activity.

Amino Acid Transport Systems, Basic↗
PubMed1979

Do the tightly linked structural genes for nitrate and nitrite reductases in Aspergillus nidulans form an operon? Evidence from an insertional translocation which separates them.

Previous work (Rand and Arst, 1977) led to the proposal that the nis-5 mutation results in a new low activity promoter for niiA, the structural gene for nitrite reductase in Aspergillus nidulans. Expression of niiA via this promoter differs from expression of niiA via its normal promoter/initiator in that expression by the new promoter is not subject to nitrate induction or ammonium repression. nis-5 reduces but does not abolish niiA expression mediated by the normal promoter/initiator. In this work we show that nis-5 is associated with and is probably identical to a non-reciprocal translocation in which a considerable portion of the centromere proximal region of the right arm of linkage group II is inserted into linkage group VIII between niiA and niaD, the tightly linked, probably contiguous structural genes for nitrate reductase. This implies that niiA, along with its normal promots yet unidentified by its normal role. Further, it indicates that niiA is transcribed from the niaD-proximal side. As niiA and niaD are separated by a large number of unrelated genes in nis-5 strains, we can safely conclude that expression of niiA does not occur solely by synthesis of a messenger which carries a niaD as well as a niiA transcript. Clearly, niiA and niaD do not form an operon for which a di- (or poly-) cistronic messenger by the only transcript. This is consistent with other experimental evidence which shows that the syntheses of nitrate and nitrite reductases are not coordinately regulated. Nevertheless, all of these data would also be consistent with a model in which niiA and niaD form an operon-type structure having overlapping transcripts, one being di- (or poly-) cistronic and including both niiA and niaD and another being monocistronic for niiA. The reduced niiA expression mediated by the normal promoter/initiator in nis-5 strains could be a consequence of the functioning or positioning of the new linkage group II niiA promoter. An alternative, but not mutually exclusive, explanation would be that the insertional translocation prevents synthesis of a niiA niaD dicistronic transcript so that only that component of niiA expression which is due to a monocistronic niiA messenger can be induced by nitrate (and nitrite) in nis-5 strains. The apparently low activity of the new linkage group II promoter in comparison to the normal niiA promoter/initiator might betoken considerable efficiency of the latter rather than any particular lack of efficiency of the former. In addition, this work has involved extensive new mapping in linkage group II, including both mitotic mapping of the centromere and meiotic mapping of previously unlocated markers. A series of crosses in cluding genotype combinations both heterozygous and homozygous for nis-5 has been used to map the break-points and orientation of the translocation. As one break-point is closer to the centromere of linkage group II than the most centromere proximal identified gene on the same (i.e...

Aspergillus nidulans↗
PubMed1984

The hisD-hisC gene border of the Salmonella typhimurium histidine operon.

We have sequenced the hisD-hisC gene border of the Salmonella typhimurium histidine operon. The translation termination codon of the hisD gene overlaps with the translation initiation codon of the hisC gene in the manner AUGA. The Shine-Dalgarno sequence of the hisC gene is contained entirely within hisD and there is no intercistronic space since all of the bases are utilized in coding. Two mutations that alter the hisD-hisC gene border are analyzed. Both mutations simultaneously abolish the termination codon of hisD and modify the initiation codon of hisC. One of the mutations changes the hisC initiation codon from AUG to AUU. The AUU codon is 10 to 20% as efficient as AUG for initiation of translation of the hisC gene. The mutant hisC ribosome binding site is compared to the ribosome binding site of the Escherichia coli infC gene which has been reported to contain an AUU initiation codon. The role of overlapping termination/initiation codons in regulating translation of polycistronic mRNAs in bacterial operons is discussed.

Amino Acid Sequence↗
PubMed1996

Regulation of the operon responsible for broad-spectrum mercury resistance in Streptomyces lividans 1326.

The broad-spectrum mercury resistance of Streptomyces lividans 1326 is mediated by six open reading frames (orf). These are arranged in two divergently transcribed operons. The orfs mer A (mercuric reductase) and mer B (organolyase) form one of the two operons. These genes and their regulation were further studied by deletion analysis and transcriptional fusion to the reporter gene xylE in the plasmid pXE4. An increase in XylE activity in response to the presence of mercuric ions was observed. The function of ORF2 (MerT) and ORF3 (MerP) as mercury-specific transport proteins, previously postulated based on the structural features of the predicted proteins, was confirmed. Transcription of the mer genes starts within the intercistronic region and two divergent promoters were identified by S1 nuclease mapping. Expression of the genes was negatively regulated by the product of orf1, now called merR. The repressor function was confirmed by gel retardation assays. MerR, produced in Escherichia coli, bound to two sites (operators) in the fragment containing the promoter region between merA and merR. Addition of mercuric ions and phenylmercuric acetate prevented the binding of MerR.

Amino Acid Sequence↗
PubMed1996

The str gene cluster for the biosynthesis of 5'-hydroxystreptomycin in Streptomyces glaucescens GLA.0 (ETH 22794): new operons and evidence for pathway-specific regulation by StrR.

Two divergently oriented operons, strXU and strVW, located within the gene cluster for 5'-hydroxystreptomycin (5'-OH-Sm) biosynthesis in Streptomyces glaucescens strain GAL.0 (ETH 22794), were analysed by DNA sequencing and transcription/regulation studies. Three genes, strU and strVW, are conserved in a similar arrangement but in a different location within the str/sts gene cluster of the Sm-producing strain S. griseus N2-3-11. The four putative products resemble NDP-4-ketohexose 3,5-epimerases (StrX, M(r) 20.2 kDa), NAD(P)-dependent oxidoreductases (StrU, 45.6 kDa), and ABC-transporters (StrV, 61.8 kDa; StrW, 63.4 kDa). These genes are apparently involved in the biosynthesis of 5'-OH-Sm because the promoters of both operons are activated in trans by the activator StrR of S. griseus N2-3-11, when cloned in S. lividans 66 TK23. A sequence motif resembling the consensus sequence GTTCGActG(N)11CagTcGAAc for binding of StrR was identified within the intergenic region of strX and strV. Specific binding of StrR to this site was demonstrated by gel retardation assays using purified His*Tag-StrR.

ATP-Binding Cassette Transporters↗
PubMed1996

Sequence and characterisation of a ribosomal RNA operon from Agrobacterium vitis.

One of the four ribosomal RNA operons (rrnA) from the Agrobacterium vitis vitopine strain S4 was sequenced, rrnA is most closely related to the rrn operons of Bradyrhizobium japonicum and Rhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case of R. sphaeroides. The 16S rRNA sequence of S4 differs from the A. vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids. The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem. The 3' ends of the three other rrn copies of S4 were also cloned and sequenced. Sequence comparison delimits the 3' ends of the four repeats and defines two groups: rrnA/rrnB and rrnC/rrnD.

Base Sequence↗
PubMed1991

Phylogenetic depth of Thermotoga maritima inferred from analysis of the fus gene: amino acid sequence of elongation factor G and organization of the Thermotoga str operon.

The gene (fus) coding for elongation factor G (EF-G) of the extremely thermophilic eubacterium Thermotoga maritima was identified and sequenced. The EF-G coding sequence (2046 bp) was found to lie in an operon-like structure between the ribosomal protein S7 gene (rpsG) and the elongation factor Tu (EF-Tu) gene (tuf). The rpsG, fus, and tuf genes follow each other immediately in that order, which corresponds to the order of the homologous genes in the str operon of Escherichia coli. The derived amino acid sequence of the EF-G protein (682 residues) was aligned with the homologous sequences of other eubacteria, eukaryotes (hamster), and archaebacteria (Methanococcus vannielii). Unrooted phylogenetic dendrograms, obtained both from the amino acid and the nucleotide sequence alignments, using a variety of methods, lend further support to the notion that the (present) root of the (eu)bacterial tree lies between Thermotoga and the other bacterial lineages.

Amino Acid Sequence↗