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PubMed2000

Prevalence of the multiple antibiotic resistance operon (marRAB) in the genus Salmonella.

The multiple antibiotic resistance operon (marRAB) is a member of the multidrug resistance (mdr) systems. Similar to other mdr systems, this operon when induced encodes resistance to structurally and functionally unrelated antibiotics. marRAB has been shown to be conserved in the family Enterobacteriaceae, but within the genus Salmonella certain species appeared to be lacking this operon. To investigate how conserved the marRAB operon was in Salmonella, 30 veterinary isolates were examined by PCR, Southern blot, and dot blot analysis. Using DNA primers based on the marRAB operon of S. typhimurium, a predicted 2.3-kb amplicon resulted after PCR in 16 of the 30 organisms. The 2.3-kb DNA band from S. enteritidis was cloned and sequenced and shown to possess 99% sequence homology to marRAB from S. typhimurium. Using a labeled marRAB gene probe from S. enteritidis, Southern blot and dot blot analysis confirmed the presence of the operon in all 30 Salmonella species examined. Furthermore, when these isolates were induced with low levels of either tetracycline or chloramphenicol, increased antimicrobial resistance was observed to structurally and functionally unrelated antibiotics. Thus, the widespread occurrence of the marRAB locus in this genus prescribes judicious use of antimicrobials to avoid induction of a mdr phenotype.

Animals↗

PubMed2001

Characterization of FruR as a putative activator of the fructose operon of Spiroplasma citri.

The role of fruR, the first gene of the Spiroplasma citri fructose operon, was investigated. In vivo transcription of the fructose operon is greatly enhanced by the presence of fructose in the growth medium while glucose has no effect. When fruR is not expressed, transcription of the fructose operon is not stimulated by fructose, and fructose fermentation is decreased, indicating that FruR is an activator of the fructose operon. The promoter of the fructose operon was localized by primer extension, and a direct T-rich repeat was found to overlap the -35 box. This repeat could be the binding site of FruR. The presence of fructose in the culture medium also decreases the toxicity of methyl alpha-glucoside, however FruR is not involved in this regulation. This is the first description of transcription regulation of a mollicute operon.

Bacterial Proteins↗

PubMed1997

Acquisition of a deliberately introduced phenol degradation operon, pheBA, by different indigenous Pseudomonas species.

Horizontal transfer of genes of selective value in an environment 6 years after their introduction into a watershed has been observed. Expression of the gene pheA, which encodes phenol monooxygenase and is linked to the pheBA operon (A. Nurk, L. Kasak, and M. Kivisaar, Gene 102:13-18, 1991), allows pseudomonads to use phenol as a growth substrate. Pseudomonas putida strains carrying this operon on a plasmid were used for bioremediation after an accidental fire in the Estonia oil shale mine in Estonia in 1988. The water samples used for studying the fate of the genes introduced were collected in 1994. The same gene cluster was also detected in Pseudomonas strains isolated from water samples of a nearby watershed which has been continuously polluted with phenols due to oil shale industry leachate. Together with the more frequently existing counterparts of the dmp genes (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), the pheA gene was also represented in the phenol-degrading strains. The area where the strains containing the pheA gene were found was restricted to the regular route of phenolic leachate to the Baltic Sea. Nine Pseudomonas strains belonging to four different species (P. corrugata, P. fragi, P. stutzeri, and P. fluorescens biotypes B, C, and F) and harboring horizontally transferred pheBA operons were investigated. The phe genes were clustered in the same manner in these nine phe operons and were connected to the same promoter as in the case of the original pheBA operon. One 10.6-kb plasmid carrying a pheBA gene cluster was sequenced, and the structure of the rearranged pheBA operon was described. This data indicates that introduced genetic material could, if it encodes a beneficial capability, enrich the natural genetic variety for biodegradation.

Base Sequence↗

PubMed1997

Phylogeny of mercury resistance (mer) operons of gram-negative bacteria isolated from the fecal flora of primates.

Nine polymorphic mer loci carried by 185 gram-negative fecal bacterial strains from humans and nonhuman primates are described. The loci were characterized with specific intragenic and intergenic PCR primers to amplify distinct regions covering approximately 80% of the typical gram-negative mer locus. These loci were grouped phylogenetically with respect to each other and with respect to seven previously sequenced mer operons from gram-negative bacteria (the latter designated loci 1, 2, 3, 6, 7, 8, and delta 8 by us here for the purpose of this analysis). Six of the mer loci recovered from primates are similar either to these previously sequenced mer loci or to another locus recently observed in environmental isolates (locus 4), and three are novel (loci 5, 9, and 10). We have observed merC, or a merC-like gene, or merF on the 5' side of merA in all of the loci except that of Tn501 (here designated mer locus 6). The merB gene was observed occasionally, always on the 3' side of merA. Unlike the initial example of a merB-containing mer locus carried by plasmid pDU1358 (locus 8), all the natural primate loci carrying merB also had large deletions of the central region of the operon (and were therefore designated locus delta 8). Four of the loci we describe (loci 2, 5, 9, and 10) have no region of homology to merB from pDU1358 and yet strains carrying them were phenylmercury resistant. Two of these loci (loci 5 and 10) also lacked merD, the putative secondary regulator of operon expression. Phylogenetic comparison of character states derived from PCR product data grouped those loci which have merC into one clade; these are locus 1 (including Tn21), locus 3, and locus 4. The mer loci which lack merC grouped into a second clade: locus 6 (including Tn501) and locus 2. Outlying groups lacked merD or possessed merB. While these mer operons are characterized by considerable polymorphism, our ability to discern coherent clades suggests that recombination is not entirely random and indeed may be focused on the immediate 5' and 3' proximal regions of merA. Our observations confirm and extend the idea that the mer operon is a genetic mosaic and has a predominance of insertions and/or deletions of functional genes immediately before and after the merA gene. chi sites are found in several of the sequenced operons and may be involved in the abundant reassortments we observe for mer genes.

Animals↗

PubMed2003

Interaction of Ler at the LEE5 (tir) operon of enteropathogenic Escherichia coli.

The genome of enteropathogenic Escherichia coli (EPEC) encodes a global regulator, Ler (locus of enterocyte effacement [LEE]-encoded regulator), which activates expression of several polycistronic operons within the 35.6-kb LEE pathogenicity island, including the LEE2-LEE3 divergent operon pair containing overlapping -10 regions and the LEE5 (tir) operon. Ler is a predicted 15-kDa protein that exhibits amino acid similarity with the nucleoid protein H-NS. In order to study Ler-mediated activation of virulence operons in EPEC, we used a molecular approach to characterize the interactions of purified Ler protein with the upstream regulatory sequences of the LEE5 operon. We determined the cis-acting DNA sequences necessary for Ler binding at LEE5 by mobility shift and DNase I protection assays, demonstrating that Ler acts directly at LEE5 by binding sequences between positions -190 and -73 in relation to the transcriptional start site. Based on the molecular weight of Ler, the similarity to H-NS, and the extended region of protection observed in a DNase I footprint at LEE5, we hypothesized that multiple Ler proteins bind upstream of the LEE5 promoter to increase transcriptional activity from a distance. Using an hns deletion strain, we demonstrated that like the LEE2-LEE3 operon pair, H-NS represses LEE5 transcription. We describe a model in which Ler activates transcription at both divergent overlapping paired and single promoters by displacing H-NS, which results in the disruption of a repressing nucleoprotein complex.

Bacterial Proteins↗

PubMed1974

Histidyl-transfer ribonucleic acid synthetase in positive control of the histidine operon in Salmonella typhimurium.

Histidyl-transfer ribonucleic acid (tRNA) synthetase (HRS), coded by the hisS gene, appears to play two roles in regulation of the histidine operon of Salmonella typhimurium: (i) in synthesis of a critical effector molecule, histidyl-tRNA, and (ii) a more direct effect elicited by the presence of the enzyme protein itself. The specific activity of HRS was elevated either by mutations in the strB locus or in hisS(+) merodiploids of Escherichia coli/S. typhimurium and S. abony/S. typhimurium. In each case, an increase in HRS was accompanied by an increase in histidine operon expression, indicating that HRS may be involved in positive control of the histidine operon. It is unlikely that HRS leads to increased histidine operon expression merely by acting as a "sponge" for charged tRNA. Rather, HRS appears to influence operon expression by interaction with some effector molecule other than charged tRNA or by a direct interaction with the histidine operator-promoter region. The functional level of histidine operon expression has no effect on HRS specific activity.

Alcohol Oxidoreductases↗

PubMed1979

Mutation spoT of Escherichia coli increases expression of the histidine operon deleted for the attenuator.

F'-episomes carrying the Salmonella typhimurium wild-type or attenuator-deleted histidine (his) operons were introduced into Escherichia coli strains containing relA or spoT single and double mutations known to affect guanosine 3'-diphosphate 5'-diphosphate (ppGpp) and guanosine 3'-triphosphate 5'-diphosphate (pppGpp) levels. Expression of the his operon and expression of the gene for 6-phosphogluconate dehydrogenase (gnd) were measured during balanced growth in amino acid-rich and minimal media. The data were consistent with the interpretation that ppGpp is a positive effector of his operon expression, whereas pppGpp is not an essential effector. The conclusion that his operon expression is maximally stimulated at a lower than maximum intracellular ppGpp concentration was further confirmed. Neither ppGpp nor pppGpp appeared to influence gnd gene expression. The metabolic regulation of the E. coli his operon was found to be similar to the ppGpp-meidated metabolic regulation of the S. typhimurium his operon.

Escherichia coli↗

PubMed1982

Ribosomal protein S4 acts in trans as a translational repressor to regulate expression of the alpha operon in Escherichia coli.

Ribosomal protein (r-protein) S4 is the translational repressor which regulates the synthesis rates of r-proteins whose genes are in the alpha operon: r-proteins S13, S11, S4, and L17. In a strain having a mutation in the gene for r-protein S4 (rpsD), the mutant S4 fails to regulate expression of the alpha operon, resulting in specific and significant overproduction of r-proteins S13, S11, and S4. This confirms and extends similar observations made with rpsD mutants (M. O. Olsson and L. A. Isaksson, Mol. Gen. Genet. 169:271-278, 1979) before post-transcriptional regulation of r-protein synthesis was proposed and is consistent with the established regulatory role of r-protein S4. The rpsD mutant has been used to study the question of whether regulatory r-proteins function in trans or strictly in cis as translational repressors. The mutant strain was lysogenized with one or two specialized transducing phages carrying a wild-type S4 gene to obtain strains which were diploid or triploid with respect to the alpha operon. The wild-type and mutant forms of S4 were separated by two-dimensional polyacrylamide gel electrophoresis, which allowed accurate measurement of the relative contributions of r-proteins from different alpha operons within a single cell. We found that expression of r-proteins from the chromosomal alpha operon containing the rpsD allele was reduced when the wild-type S4 was present, with the effect being greater in the triploid strain than in the diploid strain. We conclude that the wild-type S4 acts in trans as a translational repressor to regulate expression from the chromosomal alpha operon.

Bacteriophage lambda↗

PubMed1984

Level of rRNA, not tRNA, synthesis controls transcription of rRNA and tRNA operons in Escherichia coli.

We have recently proposed a model for the negative feedback control of rRNA and tRNA synthesis in Escherichia coli by products of rRNA operons or their derivatives (e.g., nontranslating ribosomes) (S. Jinks-Robertson, R.L. Gourse, and M. Nomura, Cell 33:865-876, 1983). In this paper, we examined the following questions. (i) Are the spacer tRNAs carried within rRNA operons the products responsible for the regulation of rRNA and tRNA transcription? (ii) Are tRNAs capable of regulating their own syntheses? We measured tRNA accumulations in cells containing plasmids with intact or defective rRNA operons or with tRNA operons. From the results obtained, we conclude that neither the tRNAs encoded within rRNA operons nor the tRNAs encoded in non-rRNA operons are capable of controlling rRNA or tRNA transcription. Therefore, the products responsible for the initial step leading to rRNA and tRNA regulation are rRNAs (or their derivatives).

Aspartate-tRNA Ligase↗

PubMed1986

Novel form of transcription attenuation regulates expression the Bacillus subtilis tryptophan operon.

Transcription of the trp operon of Bacillus subtilis is regulated in response to the availability of tryptophan. The first structural gene of the operon is preceded by a 204-base-pair transcribed leader region that contains a segment with the features of a procaryotic termination site. Transcription of the leader region was analyzed in vivo and in vitro to determine whether this putative termination site was used to regulate operon expression. When RNA was isolated from wild-type cells grown in the presence of excess tryptophan, transcripts of the operon ended at the putative termination site. In contrast, RNA isolated from cells grown in the absence of tryptophan or from a mutant strain which is constitutive for trp operon expression contained trp transcripts that extended beyond the termination site into the structural genes. To assess termination quantitatively in vivo, a trpE-lacZ fusion was constructed in which the trp promoter and leader region controls hybrid beta-galactosidase formation. The effects on hybrid beta-galactosidase levels of point mutations and deletions introduced into this leader region were determined. The results obtained establish that transcription of the trp operon structural genes is regulated in the leader region. This regulation appears to be mediated by the formation of alternative secondary structures of the leader transcript. In vitro transcription studies with wild-type and mutant templates provided additional evidence that the identified alternative RNA secondary structures regulate transcription termination. We hypothesize that binding of a tryptophan-activated regulatory protein to a specific segment of the nascent leader transcript prevents formation of one of the alternative secondary structures, thereby directing RNA polymerase to terminate transcription.

Amino Acid Sequence↗

PubMed1987

Cloning and characterization of the repressor gene of the Staphylococcus aureus lactose operon.

The genes responsible for utilization of lactose in Staphylococcus aureus are organized as an inducible operon, with galactose 6-phosphate being the intracellular inducer. To clone the repressor gene of this operon, we constructed an integration vehicle carrying 1.9 kilobases (kb) of DNA sequences from a region upstream of the structural genes of the operon. Through integration and subsequent rescue of this plasmid, we were able to clone approximately 7 kb of staphylococcal chromosomal DNA. We have shown that the plasmid insert complemented lac constitutive mutants. This repressor activity was localized to a 1.8-kb DNA fragment and, through maxicell analysis, was shown to correlate with the presence of a polypeptide with an apparent molecular weight of 32,000. Furthermore, a region between the repressor gene and the other genes of the operon was identified which, when carried on multicopy plasmids, resulted in expression of the operon in the absence of any exogenous induction. This region may represent an operator-type element capable of titrating repressor molecules away from chromosomal operator, allowing transcription of the operon in the absence of induction.

Cloning, Molecular↗

PubMed1987

Positive and negative regulation of the bgl operon in Escherichia coli.

We have analyzed the functions encoded by the bgl operon in Escherichia coli K-12. Based on the ability of cloned regions of the operon to complement a series of Bgl- point mutations, we show that the three bgl structural genes, bglC, bglS, and bglB, are located downstream of the regulatory locus bglR in the order indicated. Using a bgl-lacZ transcriptional fusion, we show that bglC and bglS are involved in regulating operon expression. The presence of the bglC gene in trans is absolutely required for the expression of the fusion, which is constitutive when only the bglC gene is present. When the bglC and the bglS genes are both present in the cell, expression of the fusion requires a beta-glucoside inducer. From these observations, we conclude that (i) the bglC gene encodes a positive regulatory of bgl operon expression and (ii) the bglS gene encodes a negative regulator of operon expression, causing the requirement for a beta-glucoside inducer. These conclusions are supported by our observations that (i) a majority of bglC mutants exhibits a Bgl- phenotype, whereas rare trans-dominant mutations in bglC result in constitutive expression of the bgl operon and the fusion, and (ii) mutations in the bglS gene lead to constitutive expression of the fusion. Based on several lines of evidence presented, we propose that the bglS gene product has an additional role as a component of the beta-glucoside transport system.

Arbutin↗

PubMed1987

Physical and genetic characterization of the glucitol operon in Escherichia coli.

The glucitol (gut) operon has been identified in the colony bank of Clark and Carbon (A. Sancar and W. D. Rupp, Proc. Natl. Acad. Sci. USA 76:3144-3148, 1979). We subcloned the gut operon by using paCYC184, pACYC177, and pBR322. The operon, which is encoded in a 3.3-kilobase nucleotide fragment, consists of the gutC, gutA, gutB, and gutD genes. The repressor of the gut operon seemed to be encoded in the region downstream from the operon. The gene products of the gut operon were identified by using maxicells. The apparent molecular weights of the glucitol-specific enzyme II (product of the gutA gene), enzyme III (product of the gutB gene), and glucitol-6-phosphate dehydrogenase (product of the gutD gene) were about 46,000, 13,500, and 27,000, respectively, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Chromosome Mapping↗

PubMed1988

Flagellar transcriptional activators FlbB and FlaI: gene sequences and 5' consensus sequences of operons under FlbB and FlaI control.

The regulation of the expression of the operons in the flagella-chemotaxis regulon in Escherichia coli has been shown to be a highly ordered cascade which closely parallels the assembly of the flagellar structure and the chemotaxis machinery (T. Iino, Annu. Rev. Genet. 11:161-182, 1977; Y. Komeda, J. Bacteriol. 168: 1315-1318). The master operon, flbB, has been sequenced, and one of its gene products (FlaI) has been identified. On the basis of the deduced amino acid sequence, the FlbB protein has similarity to an alternate sigma factor which is responsible for expression of flagella in Bacillus subtilis. In addition, we have sequenced the 5' regions of a number of flagellar operons and compared these sequences with the 5' region of flagellar operons directly and indirectly under FlbB and FlaI control. We found both a consensus sequence which has been shown to be in all other flagellar operons (J. D. Helmann and M. J. Chamberlin, Proc. Natl. Acad. Sci. USA 84:6422-6424) and a derivative consensus sequence, which is found only in the 5' region of operons directly under FlbB and FlaI control.

Amino Acid Sequence↗

PubMed1989

Evolution of a tRNA operon in gamma purple bacteria.

Genomic DNA from eubacteria belonging to the gamma-3 subdivision of purple bacteria, as classified by Woese (C.R. Woese, Microbiol. Rev. 51:221-271, 1987), were probed with the argT operon of Escherichia coli encoding 5'-tRNA(Arg)-tRNA(His)-tRNA(Leu)-tRNA(Pro)-3'. The homologous operon from Vibrio harveyi was isolated and sequenced. Comparison of the five available sequences of this tRNA cluster from members of the families Enterobacteriaceae, Aeromonadaceae, and Vibrionaceae led to the conclusion that variations in different versions of this operon arose not only by point mutations but also by duplication and addition-deletion of entire tRNA genes. This data base permitted the formulation of a proposal dealing with the evolutionary history of this operon and suggested that DNA regions containing tRNA genes are active centers (hot spots) of recombination. Finally, since the operon from V. harveyi was not highly repetitive and did not contain tRNA pseudogenes, as in the Photobacterium phosphoreum operon, hybridization of genomic DNAs from different photobacterial strains with probes specific for the repeated pseudogene element was performed. We conclude that the phylogenetic distribution of the repetitive DNA is restricted to strains of P. phosphoreum.

Base Sequence↗

PubMed1989

Mercury operon regulation by the merR gene of the organomercurial resistance system of plasmid pDU1358.

The structural basis for induction of the mercury resistance operon with inorganic mercury and with the organomercurial compound phenylmercuric acetate was addressed by DNA sequencing analysis and by lac fusion transcription experiments regulated by merR in trans from broad-spectrum-resistance plasmid pDU1358 (Hg2+ and phenylmercury responding). The lac fusion results were compared with those from a narrow-spectrum-resistance (Hg2+ responding but not phenylmercuric responding) operon and the pDU1358 merR deleted at the 3' end. The nucleotide sequence of the beginning region of the broad-spectrum mer operon of plasmid pDU1358 was determined, including that of the merR gene, the operator-promoter region, the merT and merP genes, and the first 60% of the merA gene. Comparison of this sequence with DNA sequences of narrow-spectrum mer operons from transposon Tn501 and plasmid R100 showed that a major difference occurred in the 3' 29 base pairs of the merR gene, resulting in unrelated C-terminal 10 amino acids. A hybrid mer operon consisting of the merR gene from pDU1358, a hybrid merA gene (determining mercuric reductase enzyme), and lacking the merB gene (determining phenylmercury lyase activity) was inducible by both phenylmercury and inorganic Hg2+. This shows that organomercurial lyase is not needed for induction by organomercurial compounds. A mutant form of pDU1358 merR missing the C-terminal 17 amino acids responded to inorganic Hg2+ but not to phenylmercury. Thus, the C-terminal region of the MerR protein of the pDU1358 mer operon is involved in the recognition of phenylmercury.

Amino Acid Sequence↗

PubMed1989

Spermidine biosynthesis in Escherichia coli: promoter and termination regions of the speED operon.

Two enzymes, S-adenosylmethionine decarboxylase and spermidine synthase, are essential for the biosynthesis of spermidine in Escherichia coli. We have previously shown that the genes encoding these enzymes (speD and speE) form an operon and that the area immediately upstream from the speE gene is necessary for the expression of both the speE and speD genes. We have now studied the upstream promoter and the downstream terminator regions of this operon more completely. We have shown that the major mRNA initiation site (Ia) of the operon is located 475 base pairs (bp) upstream from the speE gene and that there is an open reading frame that encodes for a polypeptide of 115 amino acids between the Ia site and the ATG start codon for the speE gene. Downstream from the stop codon for the speD gene is a potential hairpin structure immediately followed by an mRNA termination site, t. An additional mRNA termination site, t', is present about 110 bp downstream from t and is stronger than t. By comparing our DNA fragments with those prepared from this region of the E. coli chromosome by Kohara et al., we have located the speED operon on the physical map of the E. coli chromosome. We have shown that the orientation of the speED operon is counterclockwise and that the operon is located 137.5 to 140 kbp (2.9 minutes) clockwise from the zero position of the E. coli chromosomal map.

Adenosylmethionine Decarboxylase↗

PubMed1989

Characterization of five genes in the upper-pathway operon of TOL plasmid pWW0 from Pseudomonas putida and identification of the gene products.

The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes which transform toluene and xylenes to benzoate and toluates. The genetic organization of the operon was characterized by cloning of the upper operon genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the upper operon contains at least five genes in the order of xylC-xylM-xylA-xylB-xylN. Between the promoter of the operon and xylC, there is a 1.7-kilobase-long space of DNA in which no gene function was identified. In contrast, most of the DNA between xylC and xylN consists of coding sequences. The xylC gene encodes the 57-kilodalton benzaldehyde dehydrogenase. The xylM and xylA genes encode 35- and 40-kilodalton polypeptides, respectively, which were shown by genetic complementation tests to be subunits of xylene oxygenase. The structural gene for benzyl alcohol dehydrogenase, xylB, encodes a 40-kilodalton polypeptide. The last gene of this operon is xylN, which synthesizes a 52-kilodalton polypeptide of unknown function.

DNA Transposable Elements↗