scholarly journals Regulation of Type IV Fimbrial Biogenesis in Dichelobacter nodosus

2006 ◽  
Vol 188 (13) ◽  
pp. 4801-4811 ◽  
Author(s):  
Dane Parker ◽  
Ruth M. Kennan ◽  
Garry S. Myers ◽  
Ian T. Paulsen ◽  
J. Glenn Songer ◽  
...  
Keyword(s):  
Transcriptional Profiling ◽  
Extracellular Proteases ◽  
Western Blots ◽  
Dichelobacter Nodosus ◽  
Fimbrial Subunit ◽  
Type Iv ◽  
Genome Wide ◽  
Insertional Inactivation ◽  
Two Component Signal Transduction ◽  
Protease Secretion

ABSTRACT Type IV fimbriae are expressed by several bacterial pathogens and are essential for virulence in Dichelobacter nodosus, which causes ovine footrot. We have identified a two-component signal transduction system (PilR/S) and an alternative sigma factor (σ54) that were shown by insertional inactivation to be required for the regulation of fimbrial biogenesis in D. nodosus. Western blots showed that in both pilR and rpoN mutants, fimbrial subunit production was significantly reduced by a process that was shown to occur at a PilR- and σ54-dependent promoter. The mutants lacked surface fimbriae, which were shown to be required for the adherence of D. nodosus cells to tissue culture monolayers. The reduction in fimbrial subunit production in these mutants also resulted in a concomitant loss of the ability to secrete extracellular proteases. A maltose binding protein-PilR fusion protein was purified and was shown to bind specifically to a region located 234 to 594 bp upstream of the fimA transcriptional start point. To determine additional targets of PilR and σ54, genome-wide transcriptional profiling was performed using a whole-genome oligonucleotide microarray. The results indicated that PilR and σ54 regulated genes other than fimA; these genes appear to encode surface-exposed proteins whose role in virulence is unknown. In conclusion, this study represents a significant advancement in our understanding of how the ability of D. nodosus to cause ovine footrot is regulated, as we have shown that the biogenesis of type IV fimbriae in D. nodosus is regulated by a σ54-dependent PilR/S system that also indirectly controls protease secretion.

scholarly journals The Type IV Fimbrial Subunit Gene (fimA) ofDichelobacter nodosus Is Essential for Virulence, Protease Secretion, and Natural Competence

2001 ◽  
Vol 183 (15) ◽  
pp. 4451-4458 ◽  
Author(s):  
Ruth M. Kennan ◽  
Om P. Dhungyel ◽  
Richard J. Whittington ◽  
John R. Egerton ◽  
Julian I. Rood
Keyword(s):  
Virulence Gene ◽  
Extracellular Proteases ◽  
Subunit Protein ◽  
Dichelobacter Nodosus ◽  
Fimbrial Subunit ◽  
Type Iv ◽  
Virulent Type ◽  
Phenotypic Tests ◽  
Protease Secretion

ABSTRACT Dichelobacter nodosus is the essential causative agent of footrot in sheep. The major D. nodosus-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the fimAgene, which encodes the fimbrial subunit protein, from the virulent type G D. nodosus strain VCS1703A. Detailed analysis of two independently derived fimA mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream fimB gene because insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.

scholarly journals Twitching Motility Is Essential for Virulence in Dichelobacter nodosus

2008 ◽  
Vol 190 (9) ◽  
pp. 3323-3335 ◽  
Author(s):  
Xiaoyan Han ◽  
Ruth M. Kennan ◽  
John K. Davies ◽  
Leslie A. Reddacliff ◽  
Om P. Dhungyel ◽  
...  
Keyword(s):  
Cell Movement ◽  
Foot Rot ◽  
Twitching Motility ◽  
Wild Type ◽  
Cell Adherence ◽  
Secretion Pathway ◽  
Dichelobacter Nodosus ◽  
Fimbrial Subunit ◽  
Type Iv ◽  
Protease Secretion

ABSTRACTType IV fimbriae are essential virulence factors ofDichelobacter nodosus, the principal causative agent of ovine foot rot. ThefimAfimbrial subunit gene is required for virulence, butfimAmutants exhibit several phenotypic changes and it is not certain if the effects on virulence result from the loss of type IV fimbria-mediated twitching motility, cell adherence, or reduced protease secretion. We showed that mutation of either thepilTorpilUgene eliminated the ability to carry out twitching motility. However, thepilTmutants displayed decreased adhesion to epithelial cells and reduced protease secretion, whereas thepilUmutants had wild-type levels of extracellular protease secretion and adherence. These data provided evidence that PilT is required for the type IV fimbria-dependent protease secretion pathway inD. nodosus. It was postulated that sufficient fimbrial retraction must occur in thepilUmutants to allow protease secretion, but not twitching motility, to take place. Although no cell movement was detected in apilUmutant ofD. nodosus, aberrant motion was detected in an equivalent mutant ofPseudomonas aeruginosa. These observations explain how inD. nodosusprotease secretion can occur in apilUmutant but not in apilTmutant. In addition, virulence studies with sheep showed that both thepilTandpilUmutants were avirulent, providing evidence that mutation of the type IV fimbrial system affects virulence by eliminating twitching motility, not by altering cell adherence or protease secretion.

scholarly journals Type IV Fimbrial Biogenesis Is Required for Protease Secretion and Natural Transformation in Dichelobacter nodosus

2007 ◽  
Vol 189 (14) ◽  
pp. 5022-5033 ◽  
Author(s):  
Xiaoyan Han ◽  
Ruth M. Kennan ◽  
Dane Parker ◽  
John K. Davies ◽  
Julian I. Rood
Keyword(s):  
Natural Transformation ◽  
Secretion System ◽  
Extracellular Protease ◽  
Classical Type ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Dichelobacter Nodosus ◽  
Type Iv ◽  
Type Ii Secretion System ◽  
Protease Secretion

ABSTRACT The objective of this study was to develop an understanding of the molecular mechanisms by which type IV fimbrial biogenesis, natural transformation, and protease secretion are linked in the ovine foot rot pathogen, Dichelobacter nodosus. We have shown that like the D. nodosus fimbrial subunit FimA, the pilin-like protein PilE and the FimN, FimO, and FimP proteins, which are homologs of PilB, PilC, and PilD from Pseudomonas aeruginosa, are essential for fimbrial biogenesis and natural transformation, indicating that transformation requires an intact type IV fimbrial apparatus. The results also showed that extracellular protease secretion in the fimN, fimO, fimP, and pilE mutants was significantly reduced, which represents the first time that PilB, PilC, and PilE homologs have been shown to be required for the secretion of unrelated extracellular proteins in a type IV fimbriate bacterium. Quantitative real-time PCR analysis of the three extracellular protease genes aprV2, aprV5, and bprV showed that the effects on protease secretion were not mediated at the transcriptional level. Bioinformatic analysis did not identify a classical type II secretion system, and the putative fimbrial biogenesis gene pilQ was the only outer membrane secretin gene identified. Based on these results, it is postulated that in D. nodosus, protease secretion occurs by a type II secretion-related process that directly involves components of the type IV fimbrial biogenesis machinery, which represents the only type II secretion system encoded by the small genome of this highly evolved pathogen.

scholarly journals Pseudomonas stutzeri Has Two Closely RelatedpilA Genes (Type IV Pilus Structural Protein) with Opposite Influences on Natural Genetic Transformation

2001 ◽  
Vol 183 (7) ◽  
pp. 2359-2366 ◽  
Author(s):  
Stefan Graupner ◽  
Wilfried Wackernagel
Keyword(s):  
Genetic Transformation ◽  
Pseudomonas Stutzeri ◽  
Leader Peptide ◽  
Duplex Dna ◽  
Dna Uptake ◽  
Structural Protein ◽  
Dichelobacter Nodosus ◽  
Type Iv ◽  
Insertional Inactivation ◽  
Natural Genetic Transformation

ABSTRACT Pseudomonas stutzeri has type IV pili for which the pilA gene (here termed pilAI) provides the structural protein and which are required for DNA uptake and natural genetic transformation. Downstream of pilAIwe identified a gene, termed pilAII, coding for a deduced protein with a size similar to that of PilAI with 55% amino acid sequence identity and with a typical leader peptide including a leader peptidase cleavage site. Fusions to lacZ revealed that pilAII is expressed only about 10% compared topilAI, although the genes are cotranscribed as shown by reverse transcription-PCR. Surprisingly, insertional inactivation ofpilAII produced a hypertransformation phenotype giving about 16-fold-increased transformation frequencies. Hypertransformation also occurred in pilAI pilAII double mutants expressing heterologous pilA genes of nontransformable bacteria, like Pseudomonas aeruginosa or Dichelobacter nodosus. The overexpression of pilAII decreased transformation up to 5,000-fold compared to that of thepilAII mutant. However, neither inactivation ofpilAII nor its overexpression affected the amounts of [3H]thymidine-labeled DNA that were competence-specifically bound and taken up by the cells. In thepilAII mutant, the transformation by purified single-stranded DNA (which depends on comA andexbB, as does transformation by duplex DNA) was also increased 17-fold. It is concluded that PilAII suppresses a step in transformation after the uptake of duplex DNA into the cell and perhaps before its translocation into the cytoplasm. The idea that the degree of the transformability of cells could be permanently adjusted by the expression level of an antagonistic protein is discussed.

scholarly journals Complementation Analysis of the Dichelobacter nodosus fimN, fimO, and fimP Genes inPseudomonas aeruginosa and Transcriptional Analysis of thefimNOP Gene Region

1998 ◽  
Vol 66 (1) ◽  
pp. 297-304 ◽  
Author(s):  
Joanne L. Johnston ◽  
Stephen J. Billington ◽  
Volker Haring ◽  
Julian I. Rood
Keyword(s):  
Transcriptional Analysis ◽  
Gene Region ◽  
Reading Frame ◽  
Protective Antigens ◽  
Dichelobacter Nodosus ◽  
Fimbrial Subunit ◽  
Type Iv ◽  
Solid Media ◽  
Prepilin Peptidase ◽  
Polar Type

ABSTRACT The causative agent of ovine footrot, the gram-negative anaerobeDichelobacter nodosus, produces polar type IV fimbriae, which are the major protective antigens. The D. nodosusgenes fimN, fimO, and fimP are homologs of the Pseudomonas aeruginosa fimbrial assembly genes, pilB, pilC, and pilD, respectively. Both the pilD and fimP genes encode prepilin peptidases that are responsible for cleavage of the leader sequence from the immature fimbrial subunit. To investigate the functional similarity of the fimbrial biogenesis systems from these organisms, the D. nodosus genes were introduced intoP. aeruginosa strains carrying mutations in the homologous genes. Analysis of the resultant derivatives showed that thefimP gene complemented a pilD mutant ofP. aeruginosa for both fimbrial assembly and protein secretion. However, the fimN and fimO genes did not complement pilB or pilC mutants, respectively. These results suggest that although the PilD prepilin peptidase can be functionally replaced by the heterologous FimP protein, the function of the PilB and PilC proteins may require binding or catalytic domains specific for the P. aeruginosafimbrial assembly system. The transcriptional organization and regulation of the fimNOP gene region were also examined. The results of reverse transcriptase PCR and primer extension analysis suggested that these genes form an operon transcribed from two ς70-type promoters located upstream of ORFM, an open reading frame proximal to fimN. Transcription of theD. nodosus fimbrial subunit was found to increase in cells grown on solid media, and it was postulated that this regulatory effect may be of significance in the infected footrot lesion.

scholarly journals Genome-wide transcriptional profiling of Botrytis cinerea genes targeting plant cell walls during infections of different hosts

2014 ◽  
Vol 5 ◽  
Author(s):  
Barbara Blanco-Ulate ◽  
Abraham Morales-Cruz ◽  
Katherine C. H. Amrine ◽  
John M. Labavitch ◽  
Ann L. T. Powell ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Plant Cell ◽  
Cell Walls ◽  
Transcriptional Profiling ◽  
Plant Cell Walls ◽  
Genome Wide

scholarly journals A description of large-scale metabolomics studies: increasing value by combining metabolomics with genome-wide SNP genotyping and transcriptional profiling

2012 ◽  
Vol 215 (1) ◽  
pp. 17-28 ◽  
Author(s):  
Georg Homuth ◽  
Alexander Teumer ◽  
Uwe Völker ◽  
Matthias Nauck
Keyword(s):  
Blood Cells ◽  
Large Scale ◽  
Genetic Factors ◽  
Association Studies ◽  
Transcriptional Profiling ◽  
Genome Wide Association Studies ◽  
Protein Levels ◽  
Future Developments ◽  
Genome Wide ◽  
Metabolome Data

The metabolome, defined as the reflection of metabolic dynamics derived from parameters measured primarily in easily accessible body fluids such as serum, plasma, and urine, can be considered as the omics data pool that is closest to the phenotype because it integrates genetic influences as well as nongenetic factors. Metabolic traits can be related to genetic polymorphisms in genome-wide association studies, enabling the identification of underlying genetic factors, as well as to specific phenotypes, resulting in the identification of metabolome signatures primarily caused by nongenetic factors. Similarly, correlation of metabolome data with transcriptional or/and proteome profiles of blood cells also produces valuable data, by revealing associations between metabolic changes and mRNA and protein levels. In the last years, the progress in correlating genetic variation and metabolome profiles was most impressive. This review will therefore try to summarize the most important of these studies and give an outlook on future developments.

scholarly journals Closed-reference metatranscriptomics enables in planta profiling of putative virulence activities in the grapevine trunk-disease complex

2017 ◽  
Author(s):  
Abraham Morales-Cruz ◽  
Gabrielle Allenbeck ◽  
Rosa Figueroa-Balderas ◽  
Vanessa E. Ashworth ◽  
Daniel P. Lawrence ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Transcriptional Profiling ◽  
Underlying Disease ◽  
In Planta ◽  
Dna And Rna ◽  
Trunk Disease ◽  
Specificity And Sensitivity ◽  
Genome Wide ◽  
Field Samples ◽  
Trunk Diseases

SummaryGrapevines, like other perennial crops, are affected by so-called ‘trunk diseases’, which damage the trunk and other woody tissues. Mature grapevines typically contract more than one trunk disease and often multiple grapevine trunk pathogens (GTPs) are recovered from infected tissues. The co-existence of different GTP species in complex and dynamic microbial communities complicates the study of the molecular mechanisms underlying disease development especially under vineyard conditions. The objective of this study was to develop and optimize a community-level transcriptomics (i.e., metatranscriptomics) approach that can monitor simultaneously the virulence activities of multiple GTPs in planta. The availability of annotated genomes for the most relevant co-infecting GTPs in diseased grapevine wood provided the unprecedented opportunity to generate a multi-species reference for mapping and quantifying DNA and RNA sequencing reads. We first evaluated popular sequence read mappers using permutations of multiple simulated datasets. Alignment parameters of the selected mapper were optimized to increase the specificity and sensitivity for its application to metagenomics and metatranscriptomics analyses. Initial testing on grapevine wood experimentally inoculated with individual GTPs confirmed the validity of the method. Using naturally-infected field samples expressing a variety of trunk disease symptoms, we show that our approach provides quantitative assessments of species composition as well as genome-wide transcriptional profiling of potential virulence factors, namely cell wall degradation, secondary metabolism and nutrient uptake for all co-infecting GTPs.

scholarly journals Genome-wide identification and transcriptional analyses of MATE transporter genes in root tips of wild Cicer spp. under aluminium stress

2020 ◽  
Author(s):  
Xia Zhang ◽  
Brayden Weir ◽  
Hongru Wei ◽  
Zhiwei Deng ◽  
Xiaoqi Zhang ◽  
...  
Keyword(s):  
Root Growth ◽  
Root Elongation ◽  
Transcriptional Profiling ◽  
Functional Characterization ◽  
Relative Reduction ◽  
Root Tips ◽  
Al Tolerance ◽  
Genome Wide ◽  
Encoding Genes ◽  
Al Treatment

AbstractChickpea is an economically important legume crop with high nutritional value in human diets. Aluminium-toxicity poses a significant challenge for the yield improvement of this increasingly popular crop in acidic soils. The wild progenitors of chickpea may provide a more diverse gene pool for Al-tolerance in chickpea breeding. However, the genetic basis of Al-tolerance in chickpea and its wild relatives remains largely unknown. Here, we assessed the Al-tolerance of six selected wild Cicer accessions by measuring the root elongation in solution culture under control (0 µM Al3+) and Al-treatment (30 µM Al3+) conditions. Al-treatment significantly reduced the root elongation in all target lines compared to the control condition after 2-day’s growth. However, the relative reduction of root elongation in different lines varied greatly: 3 lines still retained significant root growth under Al-treatment, whilst another 2 lines displayed no root growth at all. We performed genome-wide identification of multidrug and toxic compound extrusion (MATE) encoding genes in the Cicer genome. A total of 56 annotated MATE genes were identified, which divided into 4 major phylogeny groups (G1-4). Four homologues to lupin LaMATE (> 50% aa identity; named CaMATE1-4) were clustered with previously characterised MATEs related to Al-tolerance in various other plants. qRT-PCR showed that CaMATE2 transcription in root tips was significantly up-regulated upon Al-treatment in all target lines, whilst CaMATE1 was up-regulated in all lines except Bari2_074 and Deste_064, which coincided with the lines displaying no root growth under Al-treatment. Transcriptional profiling in five Cicer tissues revealed that CaMATE1 is specifically transcribed in the root tissue, further supporting its role in Al-detoxification in roots. This first identification of MATE-encoding genes associated with Al-tolerance in Cicer paves the ways for future functional characterization of MATE genes in Cicer spp., and to facilitate future design of gene-specific markers for Al-tolerant line selection in chickpea breeding programs.

Sign in / Sign up

Export Citation Format

Share Document