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PubMed2006

The acid-induced operon Rv3083-Rv3089 is required for growth of Mycobacterium tuberculosis in macrophages.

The Rv3083-Rv3089 operon of Mycobacterium tuberculosis has been shown to be induced 17-33-fold when tubercle bacilli were exposed in vitro to acidic conditions which may mimic those that the bacilli encounter early during the infection and it is induced during growth in macrophages. To understand the role of this operon in intracellular survival, we constructed a knockout of the operon in the M. tuberculosis H37Rv strain. No differences were observed in the growth of mutant and wild-type mycobacteria on axenic media. Though the uptake of mutant and wild-type bacteria by eukaryotic cells was similar, the mutant failed to grow subsequently. By 192h post-infection, the fold differences between the wild-type and mutant bacteria were significant thus leading to the conclusion that the mutant is defective for intracellular growth in these cell lines. Complementation of the knockout restored intracellular growth to wild-type levels. During the first 24-48h post-infection, mutant bacteria also stimulated production of significantly less IL-1beta, IL-6, IL-8, RANTES, and MCP-1 by THP-1 cells than wild-type bacteria. Overall, the data indicate that the operon plays an important role in the ability of M. tuberculosis to grow inside host cells.

Animals↗

PubMed2005

The two major subspecies of Fusobacterium necrophorum have distinct leukotoxin operon promoter regions.

Fusobacterium necrophorum, a gram-negative, non-spore-forming anaerobe, is a normal inhabitant of the alimentary tract of animals and humans. Two types of F. necrophorum, subspecies necrophorum (biotype A) and funduliforme (biotype B), have been recognized, which differ morphologically, biochemically and biologically. The organism is an opportunistic pathogen that causes numerous necrotic conditions (necrobacillosis) such as bovine hepatic abscesses and ruminant foot abscesses. Subspecies necrophorum strains are considered to be more virulent for cattle and have been shown to produce greater amounts of leukotoxin than subspecies funduliforme strains. The leukotoxin operon of F. necrophorum consists of three genes (lktBAC) of which the leukotoxin structural gene (lktA) is the second gene in the operon. In this study, the promoter regions of the leukotoxin operons from the two subspecies were identified and their nucleotide sequence compared. The promoter regions were found to differ in sequence, in length of the sequence between the upstream determinant (oppF) and the first gene of the leukotoxin operon (lktB), and in promoter strength as assayed in Escherichia coli host cells.

Animals↗

PubMed1990

Escherichia coli ribosomal protein L4 stimulates transcription termination at a specific site in the leader of the S10 operon independent of L4-mediated inhibition of translation.

Transcription of the 11-gene S10 operon of Escherichia coli is inhibited by excess ribosomal protein L4, the product of the third gene of the operon. Previous studies suggested that L4 regulates transcription by modulating the level of readthrough at an attenuator in the S10 leader. To understand better the molecular details of the transcriptional regulation, we have determined the site of L4-induced termination of transcription using a method that allows us to map the 5' and 3' ends of newly synthesized RNA. Our results indicate that L4 stimulates termination about 140 bases from the transcription start site. Thus, the termination point is more than 30 bases upstream from the most proximal structural gene of the S10 operon, and coincides with a string of U residues on the descending side of a terminator-like hairpin structure. Since L4 is also known to inhibit translation of the S10 operon, we have analyzed the role of translation control in the protein's regulation of transcription by deleting sequences downstream from the termination site, including bases involved in translation initiation of the proximal structural gene. We find that the first 150 bases of the S10 leader contain the information sufficient for L4-mediated attenuation control and, therefore, that L4 regulates transcription by a mechanisms that is independent of the protein's inhibition of translation.

Base Sequence↗

PubMed2002

Restriction fragment length polymorphism of rRNA operons for discrimination and intergenic spacer sequences for cataloging of Bacillus subtilis sub-groups.

Restriction fragment length polymorphism of rRNA operons (RFLP) and 16S-23S rRNA intergenic region (ISR) sequences of Bacillus subtilis subsp. subtilis, B. subtilis subsp. spizizenii, and B. atrophaeus were compared. ISR sequences of the B. subtilis subspecies were extremely similar (W23 versus 168 rrn H, J, G,W; 96.8%; rrn D, E; 98.4%; rrnB; 97.9%) and, therefore, not useful for their differentiation. However, RFLP of rRNA operons of the B. subtilis subspecies were distinct in terms of numbers and organization within the genome (e.g. the 168 sub-group generally contained 8.3- and 8.0-kb fragments absent in the W23 sub-group). The more distantly related B. atrophaeus was distinct from both B. subtilis subspecies in terms of ISR sequence and rRNA operon number and organization. RFLP of rRNA operons discriminates the two sub-groups of Bacillus subtilis that are indistinguishable by ISR sequence. However, ISR sequence defines the relatedness of B. subtilis to other species (e.g. B. atrophaeus) within the genus Bacillus.

Bacillus subtilis↗

PubMed1996

Structure and regulation of the Salmonella typhimurium rnc-era-recO operon.

The Escherichia coli rnc-era-recO operon encodes ribonuclease III (RNase III; a dsRNA endonuclease involved in rRNA and mRNA processing and decay), Era (an essential G-protein of unknown functions and RecO (involved in the RecF homologous recombination pathway). Expression of the rnc and era genes is negatively autoregulated: RNase III cleaves the rncO 'operator' in the untranslated leader, destabilizing the operon mRNA. As part of a larger effort to understand RNase III and Era structure and function, we characterized rnc operon structure, function and regulation in the closely related bacterium Salmonella typhimurium. Construction of a S typhimurium strain conditionally defective for RNase III and Era expression showed that Era is essential for cell growth. This mutant strain also enabled selection of recombinant clones containing the intact S typhimurium rnc-era-recO operon, whose nucleotide sequence, predicted protein sequence, and predicted rncO RNA secondary structure were all highly conserved with those of E coli. Furthermore, genetic and biochemical analysis revealed that S typhimurium rnc gene expression is negatively autoregulated by a mechanism very similar or identical to that in E coli, and that the cleavage specificities of RNase IIIs.t. and RNase IIIE.c. are indistinguishable with regard to rncO cleavage and S typhimurium 23S rRNA fragmentation in vivo.

Bacterial Proteins↗

PubMed2003

Transcriptional starts for cytadherence-related operons of Mycoplasma genitalium.

One mechanism of mycoplasma cytadherence possessed by several mycoplasmas, including Mycoplasma genitalium, necessitates coordination of multiple adhesins and adherence-associated proteins. The genes encoding these adherence-related proteins are located in three different regions of the M. genitalium genome and exhibit an operon-like organization with surrounding genes. To understand whether genes encoding adherence-related proteins in M. genitalium are regulated as operons, we performed transcriptional and reverse transcription-polymerase chain reaction (RT-PCR) analyses on the loci mg191 (encoding major cytadhesin P140 localized at the specialized tip organelle) and mg218 (encoding high molecular mass cytadherence-related protein MG218 required for tip-mediated adherence). Primer extension suggested that transcription of mg191 was under the control of two transcriptional starts, one immediately upstream of mg191 (Prm(MG191)) and the other upstream of mg190 (Prm(MG190)). In contrast, mg218 appeared to be transcribed by a single transcriptional start, located upstream of mg217. RT-PCR indicated that transcription was continuous from mg190 to mg192 and mg217 to mg219, suggesting that these loci constitute true operons. Additional data revealed heretofore undetected similarities between adherence-related operons of M. genitalium and Mycoplasma pneumoniae.

Adhesins, Bacterial↗

PubMed2001

Cell toxicity caused by products of the p(L) operon of bacteriophage lambda.

Induction of a lambda prophage causes the death of the host cell even in the absence of phage replication and lytic functions due to expression of functions from the lambda p(L) operon. We genetically modified the lambda prophage to determine which lambda p(L) operon functions were involved in cell killing. Viability assays and flow cytometry were used to monitor cell death and filamentation. The kil gene was shown to cause cell death and filamentation as described previously. Another killing activity was mapped within the p(L) operon to the gam gene. Inspection of the DNA sequence showed that there are two possible translation start points for both kil and gam. In both cases, the shorter of the two possible products could cause cell killing. The shorter products were also sufficient for the known filamentation and recombination activities of the respective Kil and Gam functions. The expression level of the p(L) operon is down-regulated by Cro repressor. In the absence of Cro, higher p(L) expression levels allow either Kil or Gam to be lethal or growth inhibitory, whereas at lowered expression in Cro-repressed conditions, only Kil is lethal. The filamentation function of Kil and recombination activity of Gam are unaffected at Cro-repressed levels of expression.

Bacterial Proteins↗

PubMed2002

Genetic and transcriptional analysis of a novel plasmid-encoded copper resistance operon from Lactococcus lactis.

A plasmid-borne copper resistance operon (lco) was identified from Lactococcus lactis subsp. lactis LL58-1. The lco operon consists of three structural genes lcoABC. The predicted products of lcoA and lcoB were homologous to chromosomally encoded prolipoprotein diacylglyceral transferases and two uncharacterized proteins respectively, and the product of lcoC is similar to several multicopper oxidases, which are generally plasmid-encoded. This genetic organization represents a new combination of genes for copper resistance in bacteria. The three genes are co-transcribed from a copper-inducible promoter, which is controlled by lcoRS encoding a response regulator and a kinase sensor. The five genes are flanked by two insertion sequences, almost identical to IS-LL6 from L. lactis. Transposon mutagenesis and subcloning analysis indicated that the three structural genes were all required for copper resistance. Copper assay results showed that the extracellular concentration of copper of L. lactis LM0230 containing the lco operon was significantly higher than that of the host strain when copper was added at concentrations from 2 to 3 mM. The results suggest that the lco operon conferred copper resistance by reducing the intracellular accumulation of copper ions in L. lactis.

Bacterial Proteins↗

PubMed1996

Cloning and nucleotide sequence analysis of the Streptococcus mutans membrane-bound, proton-translocating ATPase operon.

The function of the membrane-bound ATPase in S. mutans is to regulate cytoplasmic pH values for the purpose of maintaining delta pH. Previous studies have shown that as part of its acid-adaptive ability, S. mutans is able to increase H(+)-ATPase levels in response to acidification. As part of the study of ATPase regulation in S. mutans, we have cloned the ATPase operon and determined its genetic organization. The structural genes from S. mutans were found to be in the order: c, a, b, delta, alpha, gamma, beta, and epsilon; where c and a were reversed from the more typical bacterial organization. The operon contained no I gene homologue but was preceded by a 239-bp intergenic space. Deduced aa sequences from open reading frames indicated that genes encoding homologues of glycogen phosphorylase and nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase flank the H(+)-ATPase operon, 5' and 3' respectively. Sequence analysis indicated the presence of three inverted-repeat nt sequences in the glgP-uncE intergenic space. Primer extension analysis of mRNAs prepared from batch-grown or steady-state cultures demonstrated that the transcriptional start site did not change as a function of culture pH value. The data suggest that potential stem-and-loop structures in the promoter region of the operon do not function to alter the starting position of ATPase-specific mRNA transcription.

Amino Acid Sequence↗

PubMed1999

Transcriptional analysis of levU operon encoding saccharolytic enzymes and two apparent genes involved in amino acid biosynthesis in Zymomonas mobilis.

Extracellular levansucrase (LevU) and sucrase (InvB) are two of the three saccharolytic enzymes involved in the sucrose metabolism of Zymomonas mobilis. The levU and invB genes were clustered with a 155bp interval on the chromosome. Both genes were transcribed constitutively at the basal level and the transcription of both genes was induced significantly when sucrose was added to the medium. These genes were transcribed as a bicistronic mRNA and the expression was modulated by a single promoter, which is located upstream of the levU gene. The transcriptional initiation site was mapped to -64bp from the translation start site of levU gene. These results indicated that two genes are most likely to constitute an operon. The glk operon, which encodes four glycolytic enzymes, was located close to the levU operon on the chromosome. Two apparent ORFs (ORF3 and 4) were found at the intervening sequence located between the glk and levU operons. These ORFs were transcribed divergently and showed high homology at the amino acid level with the bacterial global regulatory protein (Lrp) and aspartate racemase.

Amino Acid Isomerases↗

PubMed2003

An RTX operon in hemolytic Moraxella bovis is absent from nonhemolytic strains.

Pathogenic isolates of Moraxella bovis express a calcium-dependent transmembrane pore forming cytotoxin that is an RTX toxin encoded by mbxA. The DNA flanking mbxA was cloned and sequenced to determine if M. bovis contained a classical RTX operon. Open reading frames (ORFs) with deduced amino acid sequence homology to putative activation (RTX C) and transport (RTX B and D) proteins were identified and have been designated MbxC, MbxB, and MbxD, respectively. Thus, hemolytic M. bovis contains a typical RTX operon comprised of four genes arranged (5'-3') mbxCABD. In addition, the deduced amino acid sequences of DNA flanking mbxCABD revealed ORFs with amino acid sequence similarity to transposases (5'). At the 3' end of the mbx gene cluster, an ORF with homology to bacterial tolC genes was identified. Thus, as with the cya RTX operon of Bordetella pertussis, M. bovis appears to have a secretion accessory protein linked to RTX genes. Analysis of genomic DNA isolated from 5 nonhemolytic M. bovis strains by PCR and Southern blotting revealed the absence of mbxCABD. These strains did, however, amplify with primers specific for the 5' region flanking mbxC. M. bovis harbors a classical RTX operon that is absent in nonhemolytic strains.

Amino Acid Sequence↗

PubMed1999

Selfish operons: the evolutionary impact of gene clustering in prokaryotes and eukaryotes.

The Selfish Operon Model postulates that the organization of bacterial genes into operons is beneficial to the constituent genes in that proximity allows horizontal cotransfer of all genes required for a selectable phenotype; eukaryotic operons formed for very different reasons. Horizontal transfer of selfish operons most probably promotes bacterial diversification.

Animals↗

PubMed2000

Gene context conservation of a higher order than operons.

Operons, co-transcribed and co-regulated contiguous sets of genes, are poorly conserved over short periods of evolutionary time. The gene order, gene content and regulatory mechanisms of operons can be very different, even in closely related species. Here, we present several lines of evidence which suggest that, although an operon and its individual genes and regulatory structures are rearranged when comparing the genomes of different species, this rearrangement is a conservative process. Genomic rearrangements invariably maintain individual genes in very specific functional and regulatory contexts. We call this conserved context an uber-operon.

Base Sequence↗

PubMed1989

Clonal analysis of the lac operons from Klebsiella M5al and the Lac plasmid (pRE2) from Klebsiella V9A.

The chromosomal lac region of the coliform bacterium Klebsiella M5al was cloned into the multicopy plasmid pBR322 to give pHE7 and pHE8. pHE8 contains 12.6 kb of M5al DNA, including its complete lac operon, and pHE7 contains 2.5 kb of M5al DNA and includes the complete lac Y gene and a small segment of lacZ. The M5al operon has the same gene order as the Escherichia coli lac operon. The lac genes of the Lac plasmid of Klebsiella V9A were cloned into pBR322 to give pHE1 and pHE2, of approximately 39 and 43 kb. Both plasmids were unstable in an E. coli RecA-strain, in contrast to the stability of pHE8. Polyacrylamide gel electrophoresis tests suggested that the M5al beta-galactosidase monomer is about 5% longer, i.e. has about 50 more amino acids, than that of the E. coli Z gene. Tests made on the enzymes coded by the lac operons of M5al, another Klebsiella strain (V9A) and its resident Lac plasmid, and several Lac+ Enterobacteria, led to the conclusion that only Escherichia coli among the Enterobacteria contains an active lacA gene.

Acetyltransferases↗

PubMed1991

Nucleotide sequence of the promoter and fadB gene of the fadBA operon and primary structure of the multifunctional fatty acid oxidation protein from Escherichia coli.

The primary structure of a multifunctional protein, the large alpha-subunit of the Escherichia coli fatty acid oxidation complex, was determined by sequencing the fadB region of the fadBA operon. The amino-terminal sequence of this protein had been established by Edman degradation. The transcription start site of the fadBA operon was located 42 nucleotides upstream of the initiator codon of the fadB gene by primer extension analysis. Sequences of -10 and -35 regions of the promoter responsible for interaction with RNA polymerase were found to be CACACT and TTTGCA, respectively. The location of the promoter of the fadBA operon was defined, and the transcription direction of this operon, from fadB to fadA, as previously proposed [Yang, S.-Y., et al. (1990) J. Biol. Chem. 265, 10424-10429], was corroborated. The multifunctional protein is composed of 729 amino acid residues and has a calculated Mr of 79,593. A putative NAD-binding beta alpha beta-fold necessary for L-3-hydroxyacyl-CoA dehydrogenase function was found in the central region of the fadB gene product. Sequence analyses suggest that the functional domains of the multifunctional protein are arranged in the order enoyl-CoA hydratase:L-3-hydroxyacyl-CoA dehydrogenase: delta 3-cis-delta 2-trans-enoyl-CoA isomerase and suggest that the genes of the E. coli multifunctional protein and rat peroxisomal trifunctional beta-oxidation enzyme evolved from a common ancestral gene.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed2005

A zinc(II)/lead(II)/cadmium(II)-inducible operon from the Cyanobacterium anabaena is regulated by AztR, an alpha3N ArsR/SmtB metalloregulator.

A novel Zn(II)/Pb(II)/Cd(II)-responsive operon that consists of genes encoding a Zn(II)/Pb(II) CPx-ATPase efflux pump (aztA) and a Zn(II)/Cd(II)/Pb(II)-specific SmtB/ArsR family repressor (aztR) has been identified and characterized from the cyanobacterium Anabaena PCC 7120. In vivo real time quantitative RT-PCR assays reveal that both aztR and aztA expression are induced by divalent metal ions Zn(II), Cd(II), and Pb(II) but not by other divalent [Co(II), Ni(II)] or monovalent metal ions [Cu(I) and Ag(I)]. The introduction of a plasmid containing the azt operon into a Zn(II)/Cd(II)-hypersensitive Escherichia coli strain GG48 functionally restores Zn(II) and Pb(II) resistance with a limited effect on Cd(II) resistance. Gel mobility shift assays and aztR O/P-lacZ induction experiments confirm that AztR is the metal-regulated repressor of this operon. In vitro biochemical and mutagenesis studies indicate that AztR contains a sole metal-binding site, designated the alpha3N site, that binds Zn(II), Cd(II), and Pb(II) with a high affinity. Optical absorption spectra of Co(II)- and Cd(II)-substituted AztR and (113)Cd NMR spectroscopy of (113)Cd(II)-substituted AztR reveal that the sole alpha3N site in AztR is a CadC-like distorted tetrahedral S(3)(N,O) metal site. The first metal-coordination shell in the AztR alpha3N site differs from other alpha3N family members that sense Cd(II)/Pb(II) and those alpha5 repressors that sense Zn(II)/Co(II). Our results reveal that the alpha3N site in AztR mediates derepression of the azt operon in the presence of Zn(II), as well as Cd(II) and Pb(II); this might have provided Anabaena with an evolutionary advantage to adapt to heavy-metal-rich environments, while maintaining homeostasis of an essential metal ion, Zn(II).

Amino Acid Sequence↗

PubMed1998

Isopropylbenzene catabolic pathway in Pseudomonas putida RE204: nucleotide sequence analysis of the ipb operon and neighboring DNA from pRE4.

Pseudomonas putida RE204 employs a set of plasmid-specified enzymes in the catabolism of isopropylbenzene (cumene) and related alkylbenzenes. A 21,768 bp segment of the plasmid pRE4, whose sequence is discussed here, includes the ipb (isopropylbenzene catabolic) operon as well as associated genetic elements. The ipb operon, ipbAaAbAcAdBCEGFHD, encodes enzymes catalyzing the conversion of isopropylbenzene to isobutyrate, pyruvate, and acetyl-coenzyme A as well as an outer membrane protein (IpbH) of uncertain function. These gene products are 75 to 91% identical to those encoded by other isopropylbenzene catabolic operons and are somewhat less similar to analogous proteins of related pathways for the catabolism of mono-substituted benzenes. Upstream of ipbAa, ipbR encodes a positive regulatory protein which has about 56% identity to XylS regulatory proteins of TOL (xylene/toluate) catabolic plasmids. This similarity and that of the DNA sequence in the proposed ipb operator-promoter region (ipbOP) to the same region of the xyl meta operon (xylOmPm) suggest that, although the IpbR and XylS regulatory proteins recognize very different inducers, their interactions with DNA to activate gene expression are similar. Upstream of ipbR is an 1196 bp insertion sequence, IS1543, related to IS52 and IS1406. Separating ipbR from ipbAa are 3 additional tightly clustered IS elements. These are IS1544, related to IS1543, IS52, and other members of the IS5 family; IS1545, related to IS1240; and IS1546, related to IS1491. Encompassing the ipb catabolic genes and the other genetic elements and separated from each other by 18,492 bp, are two identical, directly repeated 1007 bp DNA segments. Homologous recombination between these segments appears to be responsible for the occasional deletion of the intervening DNA from pRE4.

Amino Acid Sequence↗

PubMed2001

Functional analysis of a chromosomal arsenic resistance operon in Pseudomonas fluorescens strain MSP3.

We reported earlier about the detection of a chromosomally located arsenic operon (arsRBC) in a gram-negative bacterium Pseudomonas fluorescens strain MSP3, which showed resistance to elevated levels of sodium arsenate and sodium arsenite. The genes for arsenic resistance were cloned into the HindIII site of pBluescript vector producing three clones MSA1, MSA2 and MSI3 conferring resistance to sodium arsenate and arsenite salts. They were further sub-cloned to delineate the insert size and the sub-clones were designated as MSA11, MSA12 and MSI13. The sub-clone pMSA12 (2.6 kb) fragment was further packaged into EcoRI-PstI site of M13mp19 and sequenced. Nucleotide sequencing revealed the presence of three open reading frames homologous to the arsR, arsB and arsC genes of arsenic resistance. Three cistrons of the ars operon encoded polypeptides ArsR, ArsB and ArsC with molecular weights ranging approximately 12, 37and 24 kDa, respectively. These polypeptides were visualized on SDS-PAGE stained with Coomassie blue and measured in a densitometer. The arsenic resistance operon (arsRBC) of strain MSP3 plasmid pMSA12 consists of 3 genes namely, arsR--encoding a repressor regulatory protein, arsB--the determinant of the membrane efflux protein that confers resistance by pumping arsenic from the cells and arsC--a small cytoplasmic polypeptide required for arsenate resistance only, not for arsenite resistance. ArsB protein is believed to use the cell membrane potential to drive the efflux of intracellular arsenite ions. ArsC encodes for the enzyme arsenate reductase which reduces intracellular As(V) (arsenate) to more toxic As(III) (arsenite) and is subsequently extruded from the cell. The arsenate reductase activity was present in the soluble cytoplasmic fraction in E. coli clones. In the context of specified function of the arsenic operon encoded proteins, uptake and efflux mechanisms were studied in the wild strain and the arsenate/arsenite clones. The cell free filtrates of the arsenate clones (MSA11 and MSA12) obtained from P. fluorescens containing the arsC gene showed that arsenate reduction requires glutathione reductase, glutathione (GSH), glutaredoxin and ArsC protein. The protein was purified in an active form and a spectrophotometric assay was developed in which the oxidation of NADPH was coupled to reduction of arsenate. The molecular weights and the location of the polypeptides were obtained from Coomassie stained SDS-PAGE of extracellular and intracellular fractions of the cells.

Arsenates↗