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PubMed2005

Requirement of the mymA operon for appropriate cell wall ultrastructure and persistence of Mycobacterium tuberculosis in the spleens of guinea pigs.

We had recently reported that the mymA operon (Rv3083 to Rv3089) of Mycobacterium tuberculosis is regulated by AraC/XylS transcriptional regulator VirS (Rv3082c) and is important for the cell envelope of M. tuberculosis. In this study, we further show that a virS mutant (MtbdeltavirS) and a mymA mutant (Mtbmym::hyg) of M. tuberculosis exhibit reduced contents and altered composition of mycolic acids along with the accumulation of saturated C24 and C26 fatty acids compared to the parental strain. These mutants were markedly more susceptible to major antitubercular drugs at acidic pH and also showed increased sensitivity to detergent (sodium dodecyl sulfate) and to acidic stress than the parental strain. We show that disruption of virS and mymA genes impairs the ability of M. tuberculosis to survive in activated macrophages, but not in resting macrophages, suggesting the importance of the mymA operon in protecting the bacterium against harsher conditions. Infection of guinea pigs with MtbdeltavirS, Mtbmym::hyg, and the parental strain resulted in an approximately 800-fold-reduced bacillary load of the mutant strains compared with the parental strain in spleens, but not in the lungs, of animals at 20 weeks postinfection. Phenotypic traits were fully complemented upon reintroduction of the virS gene into MtbdeltavirS. These observations show the important role of the mymA operon in the pathogenesis of M. tuberculosis at later stages of the disease.

Animals↗

PubMed2005

Swarming differentiation and swimming motility in Bacillus subtilis are controlled by swrA, a newly identified dicistronic operon.

The number and disposition of flagella harbored by eubacteria are regulated by a specific trait successfully maintained over generations. The genes governing the number of flagella in Bacillus subtilis have never been identified, although the ifm locus has long been recognized to influence the motility phenotype of this microorganism. The characterization of a spontaneous ifm mutant of B. subtilis, displaying diverse degrees of cell flagellation in both liquid and solid media, raised the question of how the ifm locus governs the number and assembly of functional flagella. The major finding of this investigation is the characterization of a newly identified dicistronic operon, named swrA, that controls both swimming motility and swarming differentiation in B. subtilis. Functional analysis of the swrA operon allowed swrAA (previously named swrA [D. B. Kearns, F. Chu, R. Rudner, and R. Losick, Mol. Microbiol. 52:357-369, 2004]) to be the first gene identified in B. subtilis that controls the number of flagella in liquid environments and the assembly of flagella in response to cell contact with solid surfaces. Evidence is given that the second gene of the operon, swrAB, is essential for enabling the surface-adhering cells to undergo swarming differentiation. Preliminary data point to a molecular interaction between the two gene products.

Amino Acid Sequence↗

PubMed2005

Regulation of the Bacillus subtilis ytmI operon, involved in sulfur metabolism.

The YtlI regulator of Bacillus subtilis activates the transcription of the ytmI operon encoding an l-cystine ABC transporter, a riboflavin kinase, and proteins of unknown function. The expression of the ytlI gene and the ytmI operon was high with methionine and reduced with sulfate. Using deletions and site-directed mutagenesis, a cis-acting DNA sequence important for YtlI-dependent regulation was identified upstream from the -35 box of ytmI. Gel mobility shift assays confirmed that YtlI specifically interacted with this sequence. The replacement of the sulfur-regulated ytlI promoter by the xylA promoter led to constitutive expression of a ytmI'-lacZ fusion in a ytlI mutant, suggesting that the repression of ytmI expression by sulfate was mainly at the level of YtlI synthesis. We further showed that the YrzC regulator negatively controlled ytlI expression while this repressor also acted on ytmI expression via YtlI. The cascade of regulation observed in B. subtilis is conserved in Listeria spp. Both a YtlI-like regulator and a ytmI-type operon are present in Listeria spp. Indeed, the Lmo2352 protein from Listeria monocytogenes was able to replace YtlI for the activation of ytmI expression and a lmo2352'-lacZ fusion was repressed in the presence of sulfate via YrzC in B. subtilis. A common motif, AT(A/T)ATTCCTAT, was found in the promoter region of the ytlI and lmo2352 genes. Deletion of part of this motif or the introduction of point mutations in this sequence confirmed its involvement in ytlI regulation.

ATP-Binding Cassette Transporters↗

PubMed2005

Regulation of tryptophan operon expression in the archaeon Methanothermobacter thermautotrophicus.

Conserved trp genes encode enzymes that catalyze tryptophan biosynthesis in all three biological domains, and studies of their expression in Bacteria and eukaryotes have revealed a variety of different regulatory mechanisms. The results reported here provide the first detailed description of an archaeal trp gene regulatory system. We have established that the trpEGCFBAD operon in Methanothermobacter thermautotrophicus is transcribed divergently from a gene (designated trpY) that encodes a tryptophan-sensitive transcription regulator. TrpY binds to TRP box sequences (consensus, TGTACA) located in the overlapping promoter regions between trpY and trpE, inhibiting trpY transcription in the absence of tryptophan and both trpY and trpEGCFBAD transcription in the presence of tryptophan. TrpY apparently inhibits trpY transcription by blocking RNA polymerase access to the site of trpY transcription initiation and represses trpEGCFBAD transcription by preventing TATA box binding protein (TBP) binding to the TATA box sequence. Given that residue 2 (W2) is the only tryptophan in TrpY and in TrpY homologues in other Euryarchaea and that there is only one tryptophan codon in the entire trpEGCFBAD operon (trpB encodes W175), expression of the trp operon may also be regulated in vivo by the supply of charged tRNA(Trp) available to translate the second codon of the trpY mRNA.

Euryarchaeota↗

PubMed2005

Mutations that activate the silent bgl operon of Escherichia coli confer a growth advantage in stationary phase.

Wild-type strains of Escherichia coli are unable to utilize aromatic beta-glucosides such as arbutin and salicin because the major genetic system that encodes the functions for their catabolism, the bgl operon, is silent and uninducible. We show that strains that carry an activated bgl operon exhibit a growth advantage over the wild type in stationary phase in the presence of the rpoS819 allele that causes attenuated rpoS regulon expression. Our results indicate a possible evolutionary advantage in retaining the silent bgl operon by wild-type bacteria.

Alleles↗

PubMed2005

Transcriptional polarity in rRNA operons of Escherichia coli nusA and nusB mutant strains.

Synthesis of ribosomes in Escherichia coli requires an antitermination system that modifies RNA polymerase to achieve efficient transcription of the genes specifying 16S, 23S, and 5S rRNA. This modification requires nucleotide signals in the RNA and specific transcription factors, such as NusA and NusB. Transcription of rrn operons in strains lacking the ability to produce either NusA or NusB was examined by electron microscopy. The distribution and numbers of RNA polymerase molecules on rrn operons were determined for each mutant. Compared to the wild type, the 16S gene in the nusB mutant strain had an equivalent number of RNA polymerase molecules, but the number of RNA polymerase molecules was reduced 1.4-fold for the nusA mutant. For both mutant strains, there were twofold-fewer RNA polymerase molecules on the 23S RNA gene than for the wild type. Overall, the mutant strains each had 1.6-fold-fewer RNA polymerase molecules on their rrn operons than did the wild type. To determine if decreased transcription of the 23S gene observed by electron microscopy also affected the 30S/50S ribosomal subunit ratio, ribosome profiles were examined by sucrose gradient analysis. The 30S/50S ratio increased 2.5- to 3-fold for the nus mutant strains over that for wild-type cells. Thus, strains carrying either a nusA mutation or a nusB mutation have defects in transcription of 23S rRNA.

Centrifugation, Density Gradient↗

PubMed2005

Effects of tryptophan starvation on levels of the trp RNA-binding attenuation protein (TRAP) and anti-TRAP regulatory protein and their influence on trp operon expression in Bacillus subtilis.

The anti-TRAP protein (AT), encoded by the rtpA gene of Bacillus subtilis, can bind to and inhibit the tryptophan-activated trp RNA-binding attenuation protein (TRAP). AT binding can prevent TRAP from promoting transcription termination in the leader region of the trp operon, thereby increasing trp operon expression. We show here that AT levels continue to increase as tryptophan starvation becomes more severe, whereas the TRAP level remains relatively constant and independent of tryptophan starvation. Assuming that the functional form of AT is a trimer, we estimate that the ratios of AT trimers per TRAP molecule are 0.39 when the cells are grown under mild tryptophan starvation conditions, 0.83 under more severe starvation conditions, and approximately 2.0 when AT is expressed maximally. As the AT level is increased, a corresponding increase is observed in the anthranilate synthase level. When AT is expressed maximally, the anthranilate synthase level is about 70% of the level observed in a strain lacking TRAP. In a nutritional shift experiment where excess phenylalanine and tyrosine could potentially starve cells of tryptophan, both the AT level and anthranilate synthase activity were observed to increase. Expression of the trp operon is clearly influenced by the level of AT.

Anthranilate Synthase↗

PubMed1969

Temperature-sensitive repression of the tryptophan operon in Escherichia coli.

Mutants of Escherichia coli exhibiting temperature-sensitive repression of the tryptophan operon have been isolated among the revertants of a tryptophan auxotroph, trpS5, that produces an altered tryptophanyl transfer ribonucleic acid (tRNA) synthetase. Unlike the parental strain, these mutants grew in the absence of tryptophan at high but not at low temperature. When grown at 43.5 C with excess tryptophan (repression conditions), they produced 10 times more anthranilate synthetase than when grown at 36 C or lower temperatures. Similar, though less striking, temperature-sensitivity was observed with respect to the formation of tryptophan synthetase. Transduction mapping by phage P1 revealed that these mutants carry a mutation cotransducible with thr at 60 to 80%, in addition to trpS5, and that the former mutation is primarily responsible for the temperature-sensitive repression. These results suggest that the present mutants represent a novel type of mutation of the classical regulatory gene trpR, which probably determines the structure of a protein involved in repression of the tryptophan operon. In agreement with this conclusion, tRNA of several trpR mutants was found to be normal with respect to its tryptophan acceptability. It was also shown that the trpS5 allele, whether present in trpR or trpR(+) strains, produced appreciably higher amounts of anthranilate synthetase than the corresponding trpS(+) strains under repression conditions. This was particularly true at higher temperatures. These results provide further evidence for our previous conclusion that tryptophanyl-tRNA synthetase is somehow involved in repression of this operon.

Chromosome Mapping↗

PubMed1974

Isolation of a lambda dv plasmid carrying the bacterial gal operon.

A lambdadvgal plasmid carrying genes for controlled plasmid replication from phage lambda and the bacterial gal operon was isolated as a deletion mutant of phage lambdagalq4, which carries the gal operon between lambda genes P and Q. The plasmid DNA was found in cell extracts as covalently closed circular molecules. The plasmid was characterized by using genetic crosses, digestion with the specific endonuclease EcoRI, sucrose gradient centrifugation, and electron microscopy. In one clone analyzed, the plasmid was a complete dimer (O(lambda)P(lambda)galO(lambda)P(lambda)gal); in a subclone derived from it, the plasmid was a partial dimer with only one copy of gal (O(lambda)P(lambda)O(lambda)P(lambda)gal). The partial dimer may be a recombination product of the complete dimer, since test crosses show that the gal and lambda sequences in the plasmid can be separated by recombination. Analyses of the EcoRI digests of plasmid DNAs indicated one cleavage site per lambda gene sequence and none in the gal operon. A lambdadvgal monomer was approximately 6.7 x 10(6) daltons and the lambda gene and gal components were 3.9 x 10(6) and 2.8 x 10(6) daltons, respectively. The lambdadvgal plasmid can be introduced into a new bacterial host by transfection at an efficiency of 10(-6) per DNA molecule.

Centrifugation, Density Gradient↗

PubMed2004

Characterization of Paenibacillus popilliae rRNA operons.

The terminal 39 nucleotides on the 3' end of the 16S rRNA gene, along with the complete DNA sequences of the 5S rRNA, 23S rRNA, tRNA(Ile), and tRNA(Ala) genes were determined for Paenibacillus popilliae using strains NRRL B-2309 and Dutky 1. Southern hybridization analysis with a 16S rDNA hybridization probe and restriction-digested genomic DNA demonstrated 8 copies of the 16S rRNA gene in P. popilliae strains KLN 3 and Dutky 1. Additionally, the 23S rRNA gene in P. popilliae strains NRRL B-2309, KLN 3, and Dutky 1 was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to occur as 8 copies. It was concluded that these 3 P. popilliae strains contained 8 rrn operons. The 8 operon copies were preferentially located on approximately one-half of the chromosome and were organized into 3 different patterns of genes, as follows: 16S-23S-5S, 16S-ala-23S-5S, and 16S-5S-ile-ala-23S-5S. This is the first report to identify a 5S rRNA gene between the 16S and 23S rRNA genes of a bacterial rrn operon. Comparative analysis of the nucleotides on the 3' end of the 16S rRNA gene suggests that translation of P. popilliae mRNA may occur in Bacillus subtilis and Escherichia coli.

Bacillus subtilis↗

PubMed1997

Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators.

The xyl genes of Pseudomonas putida TOL plasmid that specify catabolism of toluene and xylenes are organized in four transcriptional units: the upper-operon xylUWCAMBN for conversion of toluene/xylenes into benzoate/alkylbenzoates; the meta-operon xylXYZLTEGFJQKIH, which encodes the enzymes for further conversion of these compounds into Krebs cycle intermediates; and xylS and xylR, which are involved in transcriptional control. The XylS and XylR proteins are members of the XylS/AraC and NtrC families, respectively, of transcriptional regulators. The xylS gene is constitutively expressed at a low level from the Ps2 promoter. The XylS protein is activated by interaction with alkylbenzoates, and this active form stimulates transcription from Pm by sigma70- or sigmaS-containing RNA polymerase (the meta loop). The xylR gene is also expressed constitutively. The XylR protein, which in the absence of effectors binds in a nonactive form to target DNA sequences, is activated by aromatic hydrocarbons and ATP; it subsequently undergoes multimerization and structural changes that result in stimulation of transcription from Pu of the upper operon. This latter process is assisted by the IHF protein and mediated by sigma54-containing RNA polymerase. Once activated, the XylR protein also stimulates transcription from the Ps1 promoter of xylS without interfering with expression from Ps2. This process is assisted by the HU protein and is mediated by sigma54-containing RNA polymerase. As a consequence of hyperexpression of the xylS gene, the XylS protein is hyperproduced and stimulates transcription from Pm even in the absence of effectors (the cascade loop). The two sigma54-dependent promoters are additionally subject to global (catabolite repression) control.

Bacterial Proteins↗

PubMed2006

OpWise: operons aid the identification of differentially expressed genes in bacterial microarray experiments.

BACKGROUND: Differentially expressed genes are typically identified by analyzing the variation between replicate measurements. These procedures implicitly assume that there are no systematic errors in the data even though several sources of systematic error are known. RESULTS: OpWise estimates the amount of systematic error in bacterial microarray data by assuming that genes in the same operon have matching expression patterns. OpWise then performs a Bayesian analysis of a linear model to estimate significance. In simulations, OpWise corrects for systematic error and is robust to deviations from its assumptions. In several bacterial data sets, significant amounts of systematic error are present, and replicate-based approaches overstate the confidence of the changers dramatically, while OpWise does not. Finally, OpWise can identify additional changers by assigning genes higher confidence if they are consistent with other genes in the same operon. CONCLUSION: Although microarray data can contain large amounts of systematic error, operons provide an external standard and allow for reasonable estimates of significance. OpWise is available at http://microbesonline.org/OpWise.

Algorithms↗

PubMed2004

Inter-genomic displacement via lateral gene transfer of bacterial trp operons in an overall context of vertical genealogy.

BACKGROUND: The growing conviction that lateral gene transfer plays a significant role in prokaryote genealogy opens up a need for comprehensive evaluations of gene-enzyme systems on a case-by-case basis. Genes of tryptophan biosynthesis are frequently organized as whole-pathway operons, an attribute that is expected to facilitate multi-gene transfer in a single step. We have asked whether events of lateral gene transfer are sufficient to have obscured our ability to track the vertical genealogy that underpins tryptophan biosynthesis. RESULTS: In 47 complete-genome Bacteria, the genes encoding the seven catalytic domains that participate in primary tryptophan biosynthesis were distinguished from any paralogs or xenologs engaged in other specialized functions. A reliable list of orthologs with carefully ascertained functional roles has thus been assembled and should be valuable as an annotation resource. The protein domains associated with primary tryptophan biosynthesis were then concatenated, yielding single amino-acid sequence strings that represent the entire tryptophan pathway. Lateral gene transfer of several whole-pathway trp operons was demonstrated by use of phylogenetic analysis. Lateral gene transfer of partial-pathway trp operons was also shown, with newly recruited genes functioning either in primary biosynthesis (rarely) or specialized metabolism (more frequently). CONCLUSIONS: (i) Concatenated tryptophan protein trees are congruent with 16S rRNA subtrees provided that the genomes represented are of sufficiently close phylogenetic spacing. There are currently seven tryptophan congruency groups in the Bacteria. Recognition of a succession of others can be expected in the near future, but ultimately these should coalesce to a single grouping that parallels the 16S rRNA tree (except for cases of lateral gene transfer). (ii) The vertical trace of evolution for tryptophan biosynthesis can be deduced. The daunting complexities engendered by paralogy, xenology, and idiosyncrasies of nomenclature at this point in time have necessitated an expert-assisted manual effort to achieve a correct analysis. Once recognized and sorted out, paralogy and xenology can be viewed as features that enrich evolutionary histories.

Bacteria↗

PubMed2003

Lateral gene transfer and ancient paralogy of operons containing redundant copies of tryptophan-pathway genes in Xylella species and in heterocystous cyanobacteria.

BACKGROUND: Tryptophan-pathway genes that exist within an apparent operon-like organization were evaluated as examples of multi-genic genomic regions that contain phylogenetically incongruous genes and coexist with genes outside the operon that are congruous. A seven-gene cluster in Xylella fastidiosa includes genes encoding the two subunits of anthranilate synthase, an aryl-CoA synthetase, and trpR. A second gene block, present in the Anabaena/Nostoc lineage, but not in other cyanobacteria, contains a near-complete tryptophan operon nested within an apparent supraoperon containing other aromatic-pathway genes. RESULTS: The gene block in X. fastidiosa exhibits a sharply delineated low-GC content. This, as well as bias of codon usage and 3:1 dinucleotide analysis, strongly implicates lateral gene transfer (LGT). In contrast, parametric studies and protein tree phylogenies did not support the origination of the Anabaena/Nostoc gene block by LGT. CONCLUSIONS: Judging from the apparent minimal amelioration, the low-GC gene block in X. fastidiosa probably originated by LGT at a relatively recent time. The surprising inability to pinpoint a donor lineage still leaves room for alternative, albeit less likely, explanations other than LGT. On the other hand, the large Anabaena/Nostoc gene block does not seem to have arisen by LGT. We suggest that the contemporary Anabaena/Nostoc array of divergent paralogs represents an ancient ancestral state of paralog divergence, with extensive streamlining by gene loss occurring in the lineage of descent representing other (unicellular) cyanobacteria.

Amino Acid Sequence↗

PubMed2005

Transcriptional analysis of the ylaABCD operon of Bacillus subtilis encoding a sigma factor of extracytoplasmic function family.

The ylaABCD operon of Bacillus subtilis contains four predicted ORFs in the order ylaA, ylaB, ylaC and ylaD, where ylaC is assumed to code for a sigma factor of the extracytoplasmic function (ECF) family. Predicted YlaD may function as the anti-YlaC factor as it has an oxidative stress sensing domain similar to that of the RsrA, which is the anti-sigma factor of SigR, an ECF sigma of Streptomyces coelicolor. Northern blot analysis of the ylaABCD operon revealed two transcriptional products resulting from a distal promoter upstream of ylaA and from an internal promoter located at the first codon of ylaC. Both transcription start sites were determine by primer extension and 5'-RACE PCR. The transcription from the distal promoter was initiated by over-expression of YlaC on a multi-copy plasmid and depended on YlaC. DNA sequences of the -35 and -10 regions were similar to those recognized by other ECF sigmas of B. subtilis. On the other hand the transcription from the internal promoter was induced by oxidative stress and depended on Spx, which is an oxidative stress responding factor interacting with the alpha subunit of RNA polymerase core enzyme. The latter transcription depended possibly on SigA. We could not detect translation of YlaC from this transcript. Experiments with ylaD-disruption or co-overexpression of ylaD with ylaC suggested that YlaD functions as the anti-YlaC factor. Although YlaD has an oxidative stress sensing domain, oxidative stress did not induce the whole ylaABCD operon.

Bacillus subtilis↗

PubMed1997

Characteristics of Escherichia coli HB101 and Pseudomonas putida PpY101 harboring a recombinant plasmid with tandem insertion of the mercury resistance operon.

We constructed the plasmid pSUPmer2 by inserting tandem copies of the mercury resistance (mer) operon into a broad host range-vector, and introduced it into Escherichia coli HB101 and Pseudomonas putida PpY101 to increase their mercury resistance. Strains harboring plasmid pSUPmer2 had higher mercury resistance and mercuric reductase activity than those strains harboring the plasmid pSUPmer which had one copy of the mer operon. Mercury resistance of P. putida PpY101 was significantly increased by tandem insertion of the mer operon.

Cell Division↗

PubMed2004

Genetic analysis of an incomplete bio operon in a biotin auxotrophic strain of Bacillus subtilis natto OK2.

We describe the genetic analysis of the bio operon of the biotin auxotrophic Bacillus subtilis natto OK2 strain. The OK2 strain would only cross-feed with the Escherichia coli bioB mutant and also grew well in medium containing dethiobiotin. Sequencing analysis revealed two significant genetic alterations in the bioW and bioF genes within the bio operon of the OK2 strain. Complementation analysis with B. subtilis 168 bio mutants demonstrated that only the bioB gene could complement, but other bio operon genes could not. A bio(+) transformant, isolated from an OK2 strain, has biotin autotrophy.

Bacillus subtilis↗

PubMed2004

Eukaryotic operon genes can define highly conserved syntenies.

The synteny conservation of the members of eukaryotic operons was investigated by mapping their orthologues in Drosophila, human, and other eukaryotes. While the homologues of the operon members are generally not linked, some examples of highly conserved syntenies were found. The most significant synteny involves two members of one C. elegans operon, encoding fibrillarin and ribosomal protein S16. Their homologues are linked in human, mouse, Drosophila, Anopheles gambiae, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Plasmodium falciparum, and Guillardia theta, but not in five other genomes. The distances between the genes are larger than in the nematode, suggesting the prevalence of intrachromosomal rearrangements.

Animals↗