High Genetic Variability of the agr Locus in Staphylococcus Species

ABSTRACT The agr quorum-sensing and signal transduction system was initially described in Staphylococcus aureus, where four distinct allelic variants have been sequenced. Western blotting suggests the presence of homologous loci in many other staphylococci, and this has been confirmed for S. epidermidis and S. lugdunensis. In this study we isolated agr-like loci from a range of staphylococci by using PCR amplification from primers common to the six published agr sequences and bracketing the most variable region, associated with quorum-sensing specificity. Positive amplifications were obtained from 14 of 34 staphylococcal species or subspecies tested. Sequences of the amplicons identified 24 distinct variants which exhibited extensive sequence divergence with only 10% of the nucleotides absolutely conserved on multiple alignment. This variability involved all three open reading frames involved in quorum sensing and signal transduction. However, these variants retained several protein signatures, including the conserved cysteine residue of the autoinducing peptide, with the exception of S. intermedius of pigeon origin, which contained a serine in place of cysteine at this position. We discuss hypotheses on the mode of action and the molecular evolution of the agr locus based on comparisons between the newly determined sequences.

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The staphylococcal saeRS system coordinates environmental signals with agr quorum sensing

Microbiology ◽

10.1099/mic.0.26575-0 ◽

2003 ◽

Vol 149 (10) ◽

pp. 2709-2717 ◽

Cited By ~ 126

Author(s):

Richard P. Novick ◽

Dunrong Jiang

Keyword(s):

Quorum Sensing ◽

Exponential Phase ◽

Transcriptional Level ◽

Signal Transduction System ◽

Environmental Signals ◽

Sensing System ◽

Regulatory Strategy ◽

Quorum Sensing System ◽

Two Component Signal Transduction ◽

Reading Frames

sae is a two-component signal transduction system in Staphylococcus aureus that regulates the expression of many virulence factors at the transcriptional level and appears to act synergistically with agr in some cases. In this study, the interactions between sae and agr have been characterized in some detail. It was found that the sae locus is larger and more complex than originally envisioned, in that it is expressed from several promoters, giving rise to four or five transcripts, at least three of which are initiated upstream of saeRS and contain two additional reading frames, here designated saeP and saeQ, which are likely to have important roles in sae function. The upstream transcripts are induced during exponential phase concomitantly with the onset of RNAIII synthesis and their induction requires the agr effector, RNAIII, but is blocked by several environmental signals that override the effects of RNAIII. saeR is also required for the induction of these transcripts, so that the sae locus contains an autoinduction circuit. It is suggested that sae is downstream of agr in the exoprotein activation pathway (and also epistatic with agr), that it coordinates the effects of environmental signals with the agr quorum-sensing system, and therefore that it is a key intermediary in the overall regulatory strategy by which S. aureus senses and responds to its environment.

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The Previously Unidentified, Divergent Badnavirus Species Cacao red vein-banding virus is Associated with Cacao Swollen Shoot Disease in Nigeria

Plant Disease ◽

10.1094/pdis-09-18-1561-re ◽

2019 ◽

Vol 103 (6) ◽

pp. 1302-1308 ◽

Cited By ~ 2

Author(s):

Nomatter Chingandu ◽

Lelia Dongo ◽

Osman A. Gutierrez ◽

Judith K. Brown

Keyword(s):

Phylogenetic Analyses ◽

Pcr Amplification ◽

West African ◽

Open Reading Frames ◽

Nucleotide Identity ◽

Genome Sequences ◽

Foliar Symptoms ◽

Field Samples ◽

Polymerase Chain ◽

Reading Frames

Cacao swollen shoot disease (CSSD) of Theobroma cacao was reported in Nigeria in 1944; however, no badnaviral genome sequences have been found associated with the symptomatic trees. In 2017, leaf samples (n = 18) were collected from cacao trees from Osun and Oyo, Nigeria showing foliar symptoms that included red vein-banding and shoot swelling, and variable secondary mosaic, mottling, and fern-like pattern symptoms. Abutting primers designed around previously determined 500-bp intergenic region sequences were used for polymerase chain reaction (PCR) amplification. Of the 18 samples, 9 yielded an approximately 7,000-bp, apparently genome-size product. The nine genomes were sequenced and found to encode four open reading frames, and to share 86 to 99% nucleotide identity. Pairwise analysis of the Nigerian genomes with 21 previously reported CSSD badnaviruses, at the complete genome and reverse-transcription ribonuclease H (1,230 bp) sequence levels, indicated 71 to 75 and 72 to 76% nucleotide identity, respectively. Phylogenetic analysis of the nine complete genomes indicated that the closest relatives of the divergent Nigerian isolates were previously described West African CSSD badnaviruses. Based on pairwise comparisons and phylogenetic analyses, the Nigerian CSSD isolates constitute a previously unrecognized Badnavirus sp., herein named Cacao red vein-banding virus (CRVBV). Primers designed based on the CRVBV genome sequences amplified a 1,068-bp fragment from 16 of 18 field samples tested by PCR, suggesting the possible existence of additional CRVBV variants.

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Genome-Scale Cloning and Expression of Individual Open Reading Frames Using Topoisomerase I-Mediated Ligation

Genome Research ◽

10.1101/gr.9.4.383 ◽

1999 ◽

Vol 9 (4) ◽

pp. 383-392

Author(s):

John A. Heyman ◽

Jeremiah Cornthwaite ◽

Luis Foncerrada ◽

Jeremiah R. Gilmore ◽

Erin Gontang ◽

...

Keyword(s):

Topoisomerase I ◽

Room Temperature ◽

Pcr Amplification ◽

Open Reading Frames ◽

Cloning And Expression ◽

Dna Topoisomerase ◽

Specific Restriction ◽

Genome Scale ◽

Reading Frames

The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors. This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme’s high substrate specificity ensures a low rate of vector-alone transformants. We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs. Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.

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Forced Evolution Reveals the Importance of Short Open Reading Frame A and Secondary Structure in the Cauliflower Mosaic Virus 35S RNA Leader

Journal of Virology ◽

10.1128/jvi.72.5.4157-4169.1998 ◽

1998 ◽

Vol 72 (5) ◽

pp. 4157-4169 ◽

Cited By ~ 29

Author(s):

Mikhail M. Pooggin ◽

Thomas Hohn ◽

Johannes Fütterer

Keyword(s):

Secondary Structure ◽

Mosaic Virus ◽

Pcr Amplification ◽

Cauliflower Mosaic Virus ◽

Open Reading Frames ◽

Reading Frame ◽

Short Open Reading Frame ◽

The Stability ◽

Virus Replication Cycle ◽

Reading Frames

ABSTRACT Cauliflower mosaic virus pregenomic 35S RNA begins with a long leader sequence containing an extensive secondary structure and up to nine short open reading frames (sORFs), 2 to 35 codons in length. To test whether any of these sORFs are required for virus viability, their start codons were mutated either individually or in various combinations. The resulting viral mutants were tested for infectivity on mechanically inoculated turnip plants. Viable mutants were passaged several times, and the stability of the introduced mutations was analyzed by PCR amplification and sequencing. Mutations at the 5′-proximal sORF A and in the center of the leader resulted in delayed symptom development and in the appearance of revertants. In the central leader region, the predicted secondary structure, rather than the sORF organization, was restored, while true reversions or second-site substitutions in response to mutations of sORF A restored this sORF. Involvement of sORF A and secondary structure of the leader in the virus replication cycle, and especially in translation of the 35S RNA via ribosome shunting, is discussed.

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Identification of a Two-Component Signal Transduction System fromCorynebacterium diphtheriae That Activates Gene Expression in Response to the Presence of Heme and Hemoglobin

Journal of Bacteriology ◽

10.1128/jb.181.17.5330-5340.1999 ◽

1999 ◽

Vol 181 (17) ◽

pp. 5330-5340 ◽

Cited By ~ 55

Author(s):

Michael P. Schmitt

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Response Regulator ◽

Open Reading Frames ◽

Sensor Kinase ◽

Dependent Manner ◽

Signal Transduction System ◽

Kinase Gene ◽

Two Component ◽

Two Component Signal Transduction

ABSTRACT Corynebacterium diphtheriae, the causative agent of diphtheria, utilizes various host compounds to acquire iron. TheC. diphtheriae hmuO gene encodes a heme oxygenase that is involved in the utilization of heme and hemoglobin as iron sources. Transcription of the hmuO gene in C. diphtheriae is controlled under a dual regulatory mechanism in which the diphtheria toxin repressor protein (DtxR) and iron repress expression while either heme or hemoglobin is needed to activate transcription. In this study, two clones isolated from a C. diphtheriae chromosomal library were shown to activate transcription from the hmuO promoter in Escherichia coli. Sequence analysis revealed that these activator clones each carried distinct genes whose products had significant homology to response regulators of two-component signal transduction systems. Located upstream from each of these response regulator homologs are partial open reading frames that are predicted to encode the C-terminal portions of sensor kinases. The full-length sensor kinase gene for each of these systems was cloned from the C. diphtheriaechromosome, and constructs each carrying one complete sensor kinase gene and its cognate response regulator were constructed. One of these constructs, pTSB20, which carried the response regulator (chrA) and its cognate sensor kinase (chrS), was shown to strongly activate transcription from the hmuOpromoter in a heme-dependent manner in E. coli. A mutation in chrA (chrAD50N), which changed a conserved aspartic acid residue at position 50, the presumed site of phosphorylation by ChrS, to an asparagine, abolished heme-dependent activation. These findings suggest that the sensor kinase ChrS is involved in the detection of heme and the transduction of this signal, via a phosphotransfer mechanism, to the response regulator ChrA, which then activates transcription of the hmuO promoter. This is the first report of a bacterial two-component signal transduction system that controls gene expression through a heme-responsive mechanism.

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Massively parallel DNA target capture using Long Adapter Single Stranded Oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology

10.22541/au.162075978.89906964/v1 ◽

2021 ◽

Author(s):

LORENZO TOSI ◽

Lamia Chkaiban ◽

H. Benjamin Larman ◽

Jeffrey Rosenfeld ◽

Biju Parekkadan

Keyword(s):

Pcr Amplification ◽

Plasmid Vector ◽

Open Reading Frames ◽

Dna Oligonucleotides ◽

Target Capture ◽

Specificity And Sensitivity ◽

Long Read ◽

Dna Template ◽

Dna Recombinase ◽

Reading Frames

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the backbone for the mature LASSO probe through Cre-Loxp intramolecular recombination. This strategy generates high quality LASSO probes libraries (~46% of probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 E.coli ORFs spanning from 400- to 4,000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260-fold. Here, we show that LASSO probes obtained in this manner, are able to capture full-length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning of human sequences.

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Sequence analysis of the equid herpesvirus 2 chemokine receptor homologues E1, ORF74 and E6 demonstrates high sequence divergence between field isolates

Journal of General Virology ◽

10.1099/vir.0.82942-0 ◽

2007 ◽

Vol 88 (9) ◽

pp. 2450-2462 ◽

Cited By ~ 19

Author(s):

Emma L. Sharp ◽

Helen E. Farrell ◽

Kerstin Borchers ◽

Edward C. Holmes ◽

Nicholas J. Davis-Poynter

Keyword(s):

Chemokine Receptors ◽

Sequence Data ◽

Sequence Divergence ◽

Gene Families ◽

Open Reading Frames ◽

Amino Acid Difference ◽

Transmembrane Receptors ◽

Equid Herpesvirus ◽

High Sequence Divergence ◽

Reading Frames

Equid herpesvirus 2 (EHV-2), in common with other members of the subfamily Gammaherpesvirinae, encodes homologues of cellular seven-transmembrane receptors (7TMR), namely open reading frames (ORFs) E1, 74 and E6, which each show some similarity to cellular chemokine receptors. Whereas ORF74 and E6 are members of gammaherpesvirus-conserved 7TMR gene families, E1 is currently unique to EHV-2. To investigate their genetic variability, EHV-2 7TMRs from a panel of equine gammaherpesvirus isolates were sequenced. A region of gB was sequenced to provide comparative sequence data. Phylogenetic analysis revealed six ‘genogroups’ for E1 and four for ORF74, which exhibited approximately 10–38 and 11–27 % amino acid difference between groups, respectively. In contrast, E6 was highly conserved, with two genogroups identified. The greatest variation was observed within the N-terminal domains and other extracellular regions. Nevertheless, analysis of the number of non-synonymous (d N) and synonymous (d S) substitutions per site generally supported the hypothesis that the 7TMRs are under negative selective pressure to retain functionally important residues, although some site-specific positive selection (d N>d S) was also observed. Collectively, these data are consistent with transmembrane and cytoplasmic domains being less tolerant of mutations with adverse effects upon function. Finally, there was no evidence for genetic linkage between the different gB, E1, ORF74 and E6 genotypes, suggesting frequent intergenic recombination between different EHV-2 strains.

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The effect of mosquito passage on the La Crosse virus genotype

Journal of General Virology ◽

10.1099/0022-1317-82-12-2919 ◽

2001 ◽

Vol 82 (12) ◽

pp. 2919-2926 ◽

Cited By ~ 6

Author(s):

Monica K. Borucki ◽

Brian J. Kempf ◽

Carol D. Blair ◽

Barry J. Beaty

Keyword(s):

Cell Culture ◽

Variable Region ◽

Open Reading Frames ◽

La Crosse Virus ◽

Virus Genotype ◽

Genetic Changes ◽

Viral Genomes ◽

Virus Rna ◽

Different Strains ◽

Reading Frames

The genetic consequences of passing three different strains of La Crosse (LAC) virus orally and transovarially in Aedes triseriatus mosquitoes were examined. Two of the LAC strains (WT LAC and LAC ORI) had been passaged numerous times in cell culture; the third strain (SM1-78) had been passaged only once in suckling mice. Genetic changes were monitored in three regions of the LAC genome after oral infection and dissemination in the mosquito, and transovarial transmission (TOT) of the virus to progeny. Sequence analyses were used to characterize the genetic changes occurring in regions of G1, G2 and N open reading frames (ORFs) during passage. Only one mutation was detected in the G1 ORF of SM1-78 virus after mosquito passage; however, numerous nucleotide and amino acid substitutions were detected in the G1 ORF of WT LAC and LAC ORI (cell culture-adapted viruses). In contrast to G1, the N and G2 ORF sequences examined were stable. Mutations introduced into viral genomes during replication in parental mosquitoes were expressed in progeny mosquitoes following TOT. Genetic diversity of virus populations from a single mosquito was examined by single-strand conformation polymorphisms analysis of the variable region of glycoprotein G1. LAC virus RNA genotype diversity was greatest in virus that infected and replicated in the midgut, and declined as virus disseminated from the midgut and infected ovaries and salivary glands.

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The Tomato Kinome and the Tomato Kinase Library ORFeome: Novel Resources for the Study of Kinases and Signal Transduction in Tomato and Solanaceae Species

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-13-0218-ta ◽

2014 ◽

Vol 27 (1) ◽

pp. 7-17 ◽

Cited By ~ 14

Author(s):

Dharmendra K. Singh ◽

Mauricio Calviño ◽

Elizabeth K. Brauer ◽

Noe Fernandez-Pozo ◽

Susan Strickler ◽

...

Keyword(s):

Signal Transduction ◽

Pseudomonas Syringae ◽

Large Scale ◽

Protein Microarray ◽

Open Reading Frames ◽

In Planta ◽

Solanaceae Species ◽

Novel Associations ◽

Reading Frames ◽

Scale Format

Protein kinase–driven phosphorylation constitutes the core of cellular signaling. Kinase components of signal transduction pathways are often targeted for inactivation by pathogens. The study of kinases and immune signal transduction in the model crop tomato (Solanum lycopersicum) would benefit from the availability of community-wide resources for large scale and systems-level experimentation. Here, we defined the tomato kinome and performed a comprehensive comparative analysis of the tomato kinome and 15 other plant species. We constructed a tomato kinase library (TOKN 1.0) of over 300 full-length open reading frames (ORF) cloned into a recombination-based vector. We developed a high-throughput pipeline to isolate and transform tomato protoplasts. A subset of the TOKN 1.0 library kinases were expressed in planta, were purified, and were used to generate a functional tomato protein microarray. All resources created were utilized to test known and novel associations between tomato kinases and Pseudomonas syringae DC3000 effectors in a large-scale format. Bsk7 was identified as a component of the plant immune response and a candidate effector target. These resources will enable comprehensive investigations of signaling pathways and host-pathogen interactions in tomato and other Solanaceae spp.

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Genetic Divergence with Emergence of Novel Phenotypic Variants of Equine Arteritis Virus during Persistent Infection of Stallions

Journal of Virology ◽

10.1128/jvi.73.5.3672-3681.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 3672-3681 ◽

Cited By ~ 60

Author(s):

Jodi F. Hedges ◽

Udeni B. R. Balasuriya ◽

Peter J. Timoney ◽

William H. McCollum ◽

N. James MacLachlan

Keyword(s):

Persistent Infection ◽

Reproductive Tract ◽

Variable Region ◽

Open Reading Frames ◽

Equine Arteritis Virus ◽

Phenotypic Properties ◽

Equine Viral Arteritis ◽

Venereal Infection ◽

Reading Frames

ABSTRACT The persistently infected carrier stallion is the critical natural reservoir of equine arteritis virus (EAV), as venereal infection of mares frequently occurs after breeding to such stallions. Two Thoroughbred stallions that were infected during the 1984 outbreak of equine viral arteritis in central Kentucky subsequently became long-term EAV carriers. EAV genomes amplified from the semen of these two stallions were compared by sequence analysis of the six 3′ open reading frames (ORFs 2 through 7), which encode the four known structural proteins and two uncharacterized glycoproteins. The major variants of the EAV population that sequentially arose within the reproductive tract of each carrier stallion varied by approximately 1% per year, and the heterogeneity of the viral quasispecies increased during the course of long-term persistent infection. The various ORFs of the dominant EAV variants evolved independently, and there was apparently strong selective pressure on the uncharacterized GP3 protein during persistent infection. Amino acid changes also occurred in the V1 variable region of the GL protein. This region has been previously identified as a crucial neutralization domain, and selective pressures exerted on the V1 region during persistent EAV infection led to the emergence of virus variants with distinct neutralization properties. Thus, evolution of the EAV quasispecies that occurs during persistent infection of the stallion clearly can influence viral phenotypic properties such as neutralization and perhaps virulence.

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