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At least 1,711 records · Page 95Linked to original sources

PubMed1995

Mutational analysis of SpvR binding to DNA in the regulation of the Salmonella plasmid virulence operon.

The Salmonella plasmid-borne spvR gene encodes a 33-kDa regulatory protein that activates transcription of the spvABCD operon during the stationary phase of bacterial growth. We used gel mobility shift assays to demonstrate that SpvR recognizes a specific target DNA sequence within a 318-bp EcoRI-ApaI fragment upstream of spvA. The addition of unlabeled target DNA to the radioactive labeled DNA-SpvR complex resulted in competitive inhibition of band retardation confirming the specificity of SpvR binding. Introduction of target DNA on a high copy number plasmid into wild-type Salmonella dublin Lane resulted in a substantial decrease of SpvB synthesis, confirming the binding properties of this DNA segment in vivo. Three SpvR mutants were constructed and were shown to abolish the positive regulatory function of SpvR. By site-specific mutagenesis of spvR, three single amino acids within the putative SpvR N-terminal alpha-helix domains were substituted by prolines. This resulted in loss of binding to the spvA promoter sequence and in loss of activation of the spvABCD genes. This study demonstrates that the regulatory function of SpvR is mediated by specific binding to the promoter region of the spvABCD operon.

Amino Acid Sequence↗

PubMed2002

Influence of Tn917 insertion on transcription of the icaADBC operon in six biofilm-negative transposon mutants of Staphylococcus epidermidis.

Insertion of Tn917 into the icaADBC operon determines a biofilm-negative phenotype in biofilm-producing Staphylococcus epidermidis due to the inactivation of the genes responsible for the synthesis of the polysaccharide intercellular adhesin. We previously characterized six isogenic biofilm-negative transposon mutants of S. epidermidis 1457 with Tn917 insertions in either icaA or icaC. Northern blot analysis using ica- and Tn917-specific probes revealed that ica sequences located upstream and downstream of the transposon insertion site were still transcribed in five mutants in which Tn917 was inserted in the same transcriptional orientation. Outward-directed transcription initiating from within the transposon resulted in the complete expression of individual ica genes. Our results indicate that not only the inactivation of the entire operon but the isolated interruption of individual icaA and icaC genes led to a biofilm-negative phenotype in S. epidermidis. Tn917 mutagenesis may also result in the activation of genes located downstream of the insertion site.

Amino Acid Sequence↗

PubMed2001

The cell wall and cell division gene cluster in the Mra operon of Pseudomonas aeruginosa: cloning, production, and purification of active enzymes.

We have cloned the Pseudomonas aeruginosa cell wall biosynthesis and cell division gene cluster that corresponds to the mra operon in the 2-min region of the Escherichia coli chromosome. The organization of the two chromosomal regions in P. aeruginosa and E. coli is remarkably similar with the following gene order: pbp3/pbpB, murE, murF, mraY, murD, ftsW, murG, murC, ddlB, ftsQ, ftsA, ftsZ, and envA/LpxC. All of the above P. aeruginosa genes are transcribed from the same strand of DNA with very small, if any, intragenic regions, indicating that these genes may constitute a single operon. All five amino acid ligases, MurC, MurD, MurE, MurF, and DdlB, in addition to MurG and MraY were cloned in expression vectors. The four recombinant P. aeruginosa Mur ligases, MurC, MurD, MurE, and MurF were overproduced in E. coli and purified as active enzymes.

Amino Acid Sequence↗

PubMed1998

Regulation of the bacteriophage Mu gem operon.

The gem operon of bacteriophage Mu, responsible for the complex phenomenon of phage conversion, is included in the so called "semiessential early" region of phage DNA. Unlike the other early genes of the phage which are transcribed from the pe promoter, expression of the gem operon is driven by its own promoter, which escapes the control of the repressor. In fact, the transcript corresponding to gem was detected in immune lysogens by using a combined reverse transcription and a subsequent amplification of the resulting cDNA. The transcription initiation site from pgem was determined by primer extension mapping experiments and localized at 8217 bp from the left end of phage DNA. Two elements which could perform the negative control of gem were also identified. The first is a phage product, GemB, which presumably interferes with gem expression at a posttranscriptional level, whereas the second is a structural element, an inverted repeat immediately downstream of pgem, which acts as a terminator for the transcripts starting from pe. These transcripts could regulate gem expression by interfering with the initiation of transcription from pgem.

Bacteriophage mu↗

PubMed1990

The cyanelle str operon from Cyanophora paradoxa: sequence analysis and phylogenetic implications.

The str operon containing the genes for the ribosomal proteins S12 (rps12) and S7 (rps7) and for the elongation factors G (fus) and Tu (tufA) has been characterized for some cyanobacteria and chloroplasts from algae and higher plants. In the case of plastids a stepwise reduction by one and two genes, respectively, has been observed due to gene transfer to the nuclear genome. The nucleotide sequence of the str operon on the cyanelle genome from Cyanophora paradoxa was determined as a first example for a chlorophyll b-less plastid. It comprises rps12, rps7 and tufA which are closely linked and not interrupted by introns. Transcript analysis revealed cotranscription of the two ribosomal protein genes whereas tufA gave rise to a monocistronic mRNA. Phylogenetic studies using these three different traits allowed an assessment of the position of Cyanophora paradoxa among oxygenic photoautotrophs.

Amino Acid Sequence↗

PubMed1993

Nucleotide sequence of maize chloroplast rpl32: completing the apparent set of plastid ribosomal protein genes and their tentative operon organization.

By sequencing the rpl32 gene, we have characterized the apparent complete set of the RP genes in Zea mays plastid genome. Key data for these 21 genes (total of 26 gene copies) and the proteins encoded by them are presented, and the operon organization is discussed on the basis of available transcription data. A nomenclature for the inferred 13 operons is suggested.

Amino Acid Sequence↗

PubMed1994

Synechocystis sp. PCC6803 fusB gene, located outside of the str operon, encodes a polypeptide related to protein synthesis factor EF-G.

Synechocystis sp. PCC6803, a cyanobacterium, possesses an unusual gene (fusB) which encodes a protein with strong homology to protein synthesis elongation factor G (EF-G), although it is not linked to the classical str operon. The fusB gene is redundant, since a Synechocystis gene similar to str operon-encoded fusA genes of other bacteria is also present (based on PCR and hybridization results). There is no evidence for the presence of a fusB homologue in other bacteria. The Synechocystis fusB gene encodes unusual amino acids at some positions that are highly conserved in fusA genes of other prokaryotes.

Amino Acid Sequence↗

PubMed1991

Evolution of the Rubisco operon from prokaryotes to algae: structure and analysis of the rbcS gene of the brown alga Pylaiella littoralis.

The rbcS gene coding for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the brown alga Pylaiella littoralis is located within the plastid genome and is transcribed as a single polycistronic mRNA with the gene for the large subunit of Rubisco, rbcL. The structure of the Rubisco operon from P. littoralis was determined. Molecular phylogenies for rbcS and rbcL with a wide range of prokaryotes and eukaryotes were constructed which are congruent with recent evidence for polyphyletic plastid origins. Both rbcL and rbcS of the beta-purple bacterium Alcaligenes eutrophus clearly cluster with the rhodophyte and chromophyte proteins. The data suggest that the Rubisco operons of red algal and chromophytic plastids derive from beta-purple eubacterial antecedents, rather than the cyanobacterial lineage of eubacteria from which other of their genes derive. This implies a lateral transfer of Rubisco genes from beta-purple eubacterial ancestors to the cyanobacterial ancestor of rhodophyte and chromophyte plastids.

Amino Acid Sequence↗

PubMed1992

Evolution of the plastid ribosomal RNA operon in a nongreen parasitic plant: accelerated sequence evolution, altered promoter structure, and tRNA pseudogenes.

The nucleotide sequence of a 7.4 kb region containing the entire plastid ribosomal RNA operon of the nongreen parasitic plant Epifagus virginiana has been determined. Analysis of the sequence indicates that all four rRNA genes are intact and almost certainly functional. In contrast, the split genes for tRNA(Ile) and tRNA(Ala) present in the 16S-23S rRNA spacer region have become pseudogenes, and deletion upstream of the 16S rRNA gene has removed a tRNA(Val) gene and most of the promoter region for the rRNA operon. The rate of nucleotide substitution in 16S and 23S rRNAs is several times higher in Epifagus than in tobacco, a related photosynthetic plant. Possible reasons for this, including relaxed translational constraints, are discussed.

Base Sequence↗

PubMed1993

Accumulation of adenine and thymine in a groE-homologous operon of an intracellular symbiont.

As a result of the nucleotide sequence analysis of an aphid endosymbiont's operon homologous to the Escherichia coli groE, we noted that directional base substitutions tending toward an increase of A + T content represent an obvious evolutionary trend in this prokaryotic operon, housed for a long period by an eukaryotic cell. This result, when taken together with previous reports, raised the possibility that genomic DNA of prokaryotes residing in an eukaryotic cell is subject to A/T-biased directional mutation pressure and/or both negative and positive selection operating under conditions specific to the intracellular environments.

Adenine↗

PubMed1989

A regulatory element within a gene of a ribosomal protein operon of Escherichia coli negatively controls expression by decreasing the translational efficiency.

The trmD operon of Escherichia coli consists of the genes for the ribosomal protein (r-protein) S16, a 21 kDa protein (21K) of unknown function, the tRNA(m1G37)methyltransferase (TrmD), and r-protein L19, in this order. Previously we have shown that the steady-state amount of the two r-proteins exceeds that of the 21K and TrmD proteins 12- and 40-fold, respectively, and that this differential expression is solely explained by translational regulation. Here we have constructed translational gene fusions of the trmD operon and lacZ. The expression of a lacZ fusion containing the first 18 codons of the 21K protein gene is 15-fold higher than the expression of fusions containing 49 or 72 codons of the gene. This suggests that sequences between the 18th and the 49th codon may act as a negative element controlling the expression of the 21K protein gene. Evidence is presented which demonstrates that this regulation is achieved by reducing the efficiency of translation.

Amino Acid Sequence↗

PubMed1978

Re-initiation of tryptophan operon expression in a promoter deletion strain of Salmonella typhimurium.

A genetic and enzymological study was made of five spontaneous prototrophic revertants of a tryptophan auxotroph of Salmonella typhimurium which carries a deletion extending from the closely linked supX locus into the trp operator-promoter region. The revertants were found to have regained initiation of expression of all five trp genes. Recombinational tests showed that in each case the genetic change responsible for re-initiation is cotransducible with the trp-cysB region of the chromosome. Two different mechanisms leading to re-initiation of trp gene expression were established: (a) an extension of the limits of the original deletion resulting in the fusion of the trp structural genes with a nearby gene or gene set located outside the operator end of trp, and (b) translocation of a duplicate set of the trp structural genes to other chromosomal sites, located operator-distal to the normal trp operon, in such a manner that they are functionally fused to foreign genetic units. One revertant which arose by mechanism (a) was shown to have an extended deletion with one new terminus in trp and the other in the nearby cysB locus. All the revertants exhibit constitutive expression of the trp enzymes, with activities varying among strains from five to forty five times greater than the fully repressed wild type level. The protein product of trpA, the first structural gene of the operon, appears to have been partially damaged by the re-initiation event in at least two strains, while in the other strains, the enzyme appears in preliminary tests to be indistinguishable from that of wild type.

Amidophosphoribosyltransferase↗

PubMed1976

Expression of the guanine operon of Escherichia coli as analyzed by bacteriophage lambda induced mutations.

Studies were made on two guanine-requiring strains of Escherichia coli isolated independently as a result of insertion of prophage gamma into one of the structural genes of the guanine operon. These mutants do not exhibit any detectable guaB function but express the guaA function constitutively at a low level, presumably due to transcription from the pI promoter on the prophage. Various types of plaque-forming gua-transducing phages were generated from these lysogens. The approximate location and the mode of substitution of the gua genes in the phage genome were determined. These results clearly indicate that the gene guaB is located closer to the operator-promoter region of the gua operon than is guaA, and the gene order is "operator"-guaB-guaA.

Coliphages↗

PubMed1979

Structure of the malB region in Escherichia coli K12. I. Genetic map of the malK-lamB operon.

A series of deletions, Mu insertions and point mutations affecting the malK-lamB operon have been isolated. They were used to establish a deletion map of this operon, which could be divided in 27 intervals, with 16 in malK and 11 in lamB. One interesting feature of this map is the lack of randomness in the distribution of Mu insertions in the lamB gene; by using data published elsewhere on the physical length of the deletion intervals it can be concluded that about 25% of these Mu insertions are clustered in a segment representing 2 to 8% of the gene. This map is presently being used to study the biosynthesis, structure, and function of the lamB product, which is an outer membrane protein involved in the transport of maltose and maltodextrin, and which in addition constitutes the receptor for phage lambda.

Chromosome Mapping↗

PubMed1979

Structure of the malB region in Escherichia coli K12. II. Genetic map of the malE,F,G operon.

Starting with a strain containing a malK-lacZ fusion, a series of lambda plaque-forming phages which carry varying amounts of the malE,F operon have been isolated. We have used these phages to construct a deletion map of the malE,F operon. The construction of this deletion map has led to the identification of a new gene, malG. The malG gene is located distal to malF. The malG gene product is a protein required for the active transport of maltose and maltodextrins.

Biological Transport↗

PubMed1981

Expression of the L11-L1 operon in mutants of Escherichia coli lacking the ribosomal proteins L1 or L11.

Using a variety of immunological techniques, the supernatant levels of ribosomal proteins were measured in mutants lacking the ribosomal proteins L1 or L11, and in wild-type strains. There was a 2.5--5-fold elevation of protein L11 level in the supernatant of strains lacking protein L1, compared to wild-type. In contrast, there was no elevation, but rather a diminution, in the corresponding L1 level in strains lacking protein L11, compared to wild-type. These results are consistent with a model for the control of expression of the L11-L1 operon in which protein L1 is an inhibitor of expression of that operon, but protein L11 is not. The supernatant concentrations of other proteins were indistinguishable in all strains.

Bacterial Proteins↗

PubMed1978

A mutation to 5-methyltryptophan dependence in the tryptophan (trp) operon of Salmonella typhimurium. II. Studies of 5-methyltryptophan-dependent mutants and their revertants.

Mutants of S. typhimurium with a defect in the first structural gene of the trp operon can utilize anthranilic acid (AA) as a growth factor. Among a group of 5-methyltryptophan (MT) resistant derivatives of trpA mutants we encountered several with a novel phenotype: they actually grew better in the presence of MT than in its absence. Normally MT inhibits growth of S. typhimurium at the concentration we employed due to its ability to act as co-repressor of the trp operon and as a feedback inhibitor of anthranilate synthetase (AS) the first enzyme for tryptophan biosynthesis. Mutations to MT-dependence were only found in strains carrying extremely polar trpA mutations. In all cases analyzed, mutations causing MT-dependence mapped at the extreme operator distal end of trpA. The mutation trpA515 responsible for MT-dependence in strain SO61 (genotype trpA49trpA515) was recombined away from the polar mutation. The strain thus obtained, SO495 was totally dependent on MT for growth on AA supplement. Strain SO495 lacks AS and under repressing growth conditions synthesizes the trp enzymes constitutively at 2--3 times the basal level. Under derepression, while the levels of the distal enzymes, as represented by tryptophan synthetase--beta subunit (TSbeta), did not increase there was a marked drop in the activity of anthranilate-PRPP phosphoribosyltransferase, (PRT) the enzyme catalyzing the second step of tryptophan biosynthesis. trpA515 was found to revert to prototrophy at a low frequency (about 10(-8)) which was not increased by chemical mutagens or ultraviolet radiation. In contrast, it was found to revert to MT-independence (growth on AA in the absence of MT) at a fairly high spontaneous frequency (about 10(-6)) and this frequency could be increased approximately tenfold by mutagens causing base substitutions or deletions but not by frameshift mutagens. About one hundred MT-independent revertants of trpA515 were mapped and found to fall into three general classes: (A) mutations at or near the trpA515 site (B) secondary mutations located upstream from trpA515, (C) deletions of various sizes. Based on a detailed genetic and physiological study of twelve representative MT-independent revertants, it appears that trpA515 may be caused by the insertion of a piece of DNA with some of the properties described for the IS elements found in Escherichia coli. The trpA515 insertion should contain (in this order), a transcription terminator, a low efficiency promoter and, probably, a translation start signal.

Chromosome Mapping↗

PubMed1979

Expression of Escherichia coli tryptophan operon in Rhizobium leguminosarum.

RP4-trp hybrid plasmid containing Escherichia coli whole tryptophan operon was conjugatively transferred from E. coli to Rhizobium leguminosarum strains carrying mutations in different trp genes, converting their Trp- phenotype to Trp+. That the phenotype change of the R. leguminosarum cells was due to the presence of the E. coli tryptophan operon was verified by the isolation of RP4-trp hybrid plasmid from the R. leguminosarum conjugant cells, and by re-transfer of RP4-trp plasmid by conjugation back to E. coli trp and Pseudomonas putida trp strains. Enzymatic activities of anthranilate synthetase and beta subunit of tryptophan synthetase in crude extracts of R. leguminosarum cells containing RP4-trp plasmid were much higher than that of the wild-type cells and were not repressed by the presence of tryptophan in the culture medium.

Anthranilate Synthase↗