scholarly journals Mannitol 1-Phosphate Metabolism Is Required for Sporulation in Planta of the Wheat Pathogen Stagonospora nodorum

2005 ◽  
Vol 18 (2) ◽  
pp. 110-115 ◽  
Author(s):  
Peter S. Solomon ◽  
Kar-Chun Tan ◽  
Richard P. Oliver
Keyword(s):  
Insertional Mutagenesis ◽  
Redox Regulation ◽  
Expressed Sequence Tag ◽  
Stagonospora Nodorum ◽  
Resonance Spectroscopy ◽  
Plant Interactions ◽  
Wild Type ◽  
In Planta ◽  
Mutant Strains

An expressed sequence tag encoding a putative mannitol 1-phosphate dehydrogenase (Mpd1) has been characterized from the fungal wheat pathogen Stagonospora nodorum. Mpd1 was disrupted by insertional mutagenesis, and the resulting mpd1 strains lacked all detectable NAD-linked mannitol 1-phosphate dehydrogenase activity (EC 1.1.1.17). The growth rates, sporulation, and spore viability of the mutant strains in vitro were not significantly different from the wild type. The viability of the mpd1 spores when subjected to heat stress was comparable to wild type. Characterization of the sugar alcohol content by nuclear magnetic resonance spectroscopy revealed that, when grown on glucose, the mutant strains contained significantly less mannitol, less arabitol, but more trehalose than the wildtype strains. The mannitol content of fructose-grown cultures was normal. No secreted mannitol could be detected in wild type or mutants. Pathogenicity assays revealed the disruption of Mpd1 did not affect lesion development, however the mutants were unable to sporulate. These results throw new light on the role of mannitol in fungal plant interactions, suggesting a role in metabolic and redox regulation during the critical process of sporulation on senescing leaf material.

scholarly journals The Alternative Sigma Factor RpoN Is Required for hrpActivity in Pseudomonas syringae pv. Maculicola and Acts at the Level of hrpL Transcription

2000 ◽  
Vol 182 (12) ◽  
pp. 3508-3516 ◽  
Author(s):  
Erik L. Hendrickson ◽  
Pablo Guevera ◽  
Frederick M. Ausubel
Keyword(s):  
Pseudomonas Syringae ◽  
Transcriptional Activity ◽  
Constitutive Expression ◽  
Wild Type ◽  
In Planta ◽  
Disease Symptoms ◽  
Nicotiana Tobacum ◽  
Mutant Strains

ABSTRACT β-Glucuronidase (uidA) reporter gene fusions were constructed for the hrpZ, hrpL, andhrpS genes from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. These reporters, as well as an avrRpt2-uidA fusion, were used to measure transcriptional activity in ES4326 and a ES4326 rpoNmutant. rpoN was required for the expression ofavrRpt2, hrpZ, and hrpL in vitro in minimal media and in vivo when infiltrated into Arabidopsis thaliana leaves. In contrast, the expression of hrpSwas essentially the same in wild-type and rpoN mutant strains. Constitutive expression of hrpL in anrpoN mutant restored hrpZ transcription to wild-type levels, restored the hypersensitive response when infiltrated into tobacco (Nicotiana tobacum), and partially restored the elicitation of virulence-related symptoms but not growth when infiltrated into Arabidopsis leaves. These data indicate that rpoN-mediated control of hrp gene expression acts at the level of hrpL and that in planta growth of P. syringae is not required for the elicitation of disease symptoms.

scholarly journals Salmonella typhimurium LT2 Catabolizes Propionate via the 2-Methylcitric Acid Cycle

1999 ◽  
Vol 181 (18) ◽  
pp. 5615-5623 ◽  
Author(s):  
Alexander R. Horswill ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Salmonella Typhimurium ◽  
Negative Ion ◽  
Resonance Spectroscopy ◽  
Ionization Mass ◽  
Wild Type ◽  
Acid Cycle ◽  
Mutant Strains ◽  
Methylcitrate Synthase

ABSTRACT We previously identified the prpBCDE operon, which encodes catabolic functions required for propionate catabolism inSalmonella typhimurium. Results from13C-labeling experiments have identified the route of propionate breakdown and determined the biochemical role of each Prp enzyme in this pathway. The identification of catabolites accumulating in wild-type and mutant strains was consistent with propionate breakdown through the 2-methylcitric acid cycle. Our experiments demonstrate that the α-carbon of propionate is oxidized to yield pyruvate. The reactions are catalyzed by propionyl coenzyme A (propionyl-CoA) synthetase (PrpE), 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (probably PrpD), 2-methylisocitrate hydratase (probably PrpD), and 2-methylisocitrate lyase (PrpB). In support of this conclusion, the PrpC enzyme was purified to homogeneity and shown to have 2-methylcitrate synthase activity in vitro.1H nuclear magnetic resonance spectroscopy and negative-ion electrospray ionization mass spectrometry identified 2-methylcitrate as the product of the PrpC reaction. Although PrpC could use acetyl-CoA as a substrate to synthesize citrate, kinetic analysis demonstrated that propionyl-CoA is the preferred substrate.

scholarly journals Mannitol is required for asexual sporulation in the wheat pathogen Stagonospora nodorum (glume blotch)

2006 ◽  
Vol 399 (2) ◽  
pp. 231-239 ◽  
Author(s):  
Peter S. Solomon ◽  
Ormonde D. C. Waters ◽  
Cordula I. Jörgens ◽  
Rohan G. T. Lowe ◽  
Judith Rechberger ◽  
...  
Keyword(s):  
Double Mutant ◽  
Physiological Role ◽  
Stagonospora Nodorum ◽  
In Planta ◽  
Mannitol Dehydrogenase ◽  
Glume Blotch ◽  
Dehydrogenase Gene ◽  
Mannitol Metabolism ◽  
Mutant Strains

The physiological role of the mannitol cycle in the wheat pathogen Stagonospora nodorum (glume blotch) has been investigated by reverse genetics and metabolite profiling. A putative mannitol 2-dehydrogenase gene (Mdh1) was cloned by degenerate PCR and disrupted. The resulting mutated mdh1 strains lacked all detectable NADPH-dependent mannitol dehydrogenase activity. The mdh1 strains were unaffected for mannitol production but, surprisingly, were still able to utilize mannitol as a sole carbon source, suggesting a hitherto unknown mechanism for mannitol catabolism. The mutant strains were not compromised in their ability to cause disease or sporulate. To further our understanding of mannitol metabolism, a previously developed mannitol-1-phosphate dehydrogenase (gene mpd1) disruption construct [Solomon, Tan and Oliver (2005) Mol. Plant–Microbe Interact. 18, 110–115] was introduced into the mutated mdh1 background, resulting in a strain lacking both enzyme activities. The mpd1mdh1 strains were unable to grow on mannitol and produced only trace levels of mannitol. The double-mutant strains were unable to sporulate in vitro when grown on minimal medium for extended periods. Deficiency in sporulation was correlated with the depletion of intracellular mannitol pools. Significantly sporulation could be restored with the addition of mannitol. Pathogenicity of the double mutant was not compromised, although, like the previously characterized mpd1 mutants, the strains were unable to sporulate in planta. These findings not only question the currently hypothesized pathways of mannitol metabolism, but also identify for the first time that mannitol is required for sporulation of a filamentous fungus.

scholarly journals Putative Surface Proteins Encoded within a Novel Transferable Locus Confer a High-Biofilm Phenotype to Enterococcus faecalis

2006 ◽  
Vol 188 (6) ◽  
pp. 2063-2072 ◽  
Author(s):  
Preeti M. Tendolkar ◽  
Arto S. Baghdayan ◽  
Nathan Shankar
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecalis ◽  
Insertional Mutagenesis ◽  
Surface Proteins ◽  
Conjugative Plasmid ◽  
Sequence Information ◽  
Wild Type ◽  
Abiotic Surfaces ◽  
Mutant Bank

ABSTRACT Enterococci are opportunistic pathogens and among the leading causes of nosocomial infections. Enterococcus faecalis, the dominant species among infection-derived isolates, has recently been recognized as capable of forming biofilms on abiotic surfaces in vitro as well as on indwelling medical devices. A few bacterial factors known to contribute to biofilm formation in E. faecalis have been characterized. To identify additional factors which may be important to this process, we utilized a Tn917-based insertional mutagenesis strategy to generate a mutant bank in a high-biofilm-forming E. faecalis strain, E99. The resulting mutant bank was screened for mutants exhibiting a significantly reduced ability to form biofilms. One mutant, P101D12, which showed greater than 70% reduction in its ability to form biofilms compared to the wild-type parent, was further characterized. The single Tn917 insertion in P101D12 was mapped to a gene, bee-2, encoding a probable cell wall-anchored protein. Sequence information for the region flanking bee-2 revealed that this gene was a member of a locus (termed the bee locus for biofilm enhancer in enterococcus) comprised of five genes encoding three putative cell wall-anchored proteins and two probable sortases. Contour-clamped homogeneous electric field gel and Southern hybridization analyses suggested that the bee locus is likely harbored on a large conjugative plasmid. Filter mating assays using wild-type E99 or mutant P101D12 as a donor confirmed that the bee locus could transfer conjugally at high frequency to recipient E. faecalis strains. This represents the first instance of the identification of a mobile genetic element conferring biofilm-forming property in E. faecalis.

Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster 

2003 ◽  
Vol 99 (4) ◽  
pp. 867-875 ◽  
Author(s):  
Sumiko Gamo ◽  
Junya Tomida ◽  
Katsuyuki Dodo ◽  
Dai Keyakidani ◽  
Hitoshi Matakatsu ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Double Mutant ◽  
Developmental Stages ◽  
Northern Blotting ◽  
General Anesthetics ◽  
Wild Type ◽  
Mutant Strains ◽  
Low Expression

Background Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity. Methods Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311). Results Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains. Conclusion ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.

scholarly journals Triple Gene Block Protein Interactions Involved in Movement of Barley Stripe Mosaic Virus

2008 ◽  
Vol 82 (10) ◽  
pp. 4991-5006 ◽  
Author(s):  
Hyoun-Sub Lim ◽  
Jennifer N. Bragg ◽  
Uma Ganesan ◽  
Diane M. Lawrence ◽  
Jialin Yu ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Triple Gene Block ◽  
Cell Movement ◽  
Wild Type Virus ◽  
Barley Stripe Mosaic Virus ◽  
Wild Type ◽  
In Planta ◽  
Gene Block ◽  
Physical Interactions

ABSTRACT Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.

scholarly journals A Polyamine Oxidase from Selaginella lepidophylla (SelPAO5) can Replace AtPAO5 in Arabidopsis through Converting Thermospermine to Norspermidine instead to Spermidine

Plants ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 99 ◽  
Author(s):  
G. H. M. Sagor ◽  
Tomonobu Kusano ◽  
Thomas Berberich
Keyword(s):  
Normal Values ◽  
Normal Growth ◽  
Polyamine Oxidase ◽  
Loss Of Function ◽  
Wild Type ◽  
In Planta ◽  
Selaginella Lepidophylla ◽  
Growth Phenotype ◽  
Polyamine Oxidases

Of the five polyamine oxidases in Arabidopsis thaliana, AtPAO5 has a substrate preference for the tetraamine thermospermine (T-Spm) which is converted to triamine spermidine (Spd) in a back-conversion reaction in vitro. A homologue of AtPAO5 from the lycophyte Selaginella lepidophylla (SelPAO5) back-converts T-Spm to the uncommon polyamine norspermidine (NorSpd) instead of Spd. An Atpao5 loss-of-function mutant shows a strong reduced growth phenotype when growing on a T-Spm containing medium. When SelPAO5 was expressed in the Atpao5 mutant, T-Spm level decreased to almost normal values of wild type plants, and NorSpd was produced. Furthermore the reduced growth phenotype was cured by the expression of SelPAO5. Thus, a NorSpd synthesis pathway by PAO reaction and T-Spm as substrate was demonstrated in planta and the assumption that a balanced T-Spm homeostasis is needed for normal growth was strengthened.

scholarly journals Recovery of Fusarium oxysporum Fo47 Mutants Affected in Their Biocontrol Activity After Transposition of the Fot1 Element

2002 ◽  
Vol 92 (9) ◽  
pp. 936-945 ◽  
Author(s):  
Sophie Trouvelot ◽  
Chantal Olivain ◽  
Ghislaine Recorbet ◽  
Quirico Migheli ◽  
Claude Alabouvette
Keyword(s):  
Fusarium Oxysporum ◽  
Insertional Mutagenesis ◽  
Wild Type Strain ◽  
Growth Habit ◽  
Antagonistic Activity ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Biocontrol Activity

To investigate the biocontrol mechanisms by which the antagonistic Fusarium oxysporum strain Fo47 is active against Fusarium wilt, a Fot1 transposon-mediated insertional mutagenesis approach was adopted to generate mutants affected in their antagonistic activity. Ninety strains in which an active Fot1 copy had transposed were identified with a phenotypic assay for excision and tested for their biocontrol activity against F. oxysporum f. sp. lini on flax in greenhouse experiments. Sixteen strains were affected in their capacity to protect flax plants, either positively (more antagonistic than Fo47) or negatively (less antagonistic). The molecular characterization of these mutants confirms the excision of Fot1 and its reinsertion in most of the cases. Moreover, we demonstrate that other transposable elements such as Fot2, impala, and Hop have no transposition activity in the mutant genomes. The phenotypic characterization of these mutants shows that they are affected neither in their in vitro growth habit nor in their competitiveness in soil compared with wild-type strain Fo47. These results show that mutants are not impaired in their saprophytic phase and suggest that the altered biocontrol phenotype should likely be expressed during the interaction with the host plant.

scholarly journals Glucomannan-Mediated Attachment of Rhizobium leguminosarum to Pea Root Hairs Is Required for Competitive Nodule Infection

2008 ◽  
Vol 190 (13) ◽  
pp. 4706-4715 ◽  
Author(s):  
Alan Williams ◽  
Adam Wilkinson ◽  
Martin Krehenbrink ◽  
Daniela M. Russo ◽  
Angeles Zorreguieta ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Rhizobium Leguminosarum ◽  
Root Hairs ◽  
The Other ◽  
Plant Interactions ◽  
Wild Type ◽  
Polysaccharide Synthesis ◽  
Surface Polysaccharides

ABSTRACT The Rhizobium leguminosarum biovar viciae genome contains several genes predicted to determine surface polysaccharides. Mutants predicted to affect the initial steps of polysaccharide synthesis were identified and characterized. In addition to the known cellulose (cel) and acidic exopolysaccharide (EPS) (pss) genes, we mutated three other loci; one of these loci (gmsA) determines glucomannan synthesis and one (gelA) determines a gel-forming polysaccharide, but the role of the other locus (an exoY-like gene) was not identified. Mutants were tested for attachment and biofilm formation in vitro and on root hairs; the mutant lacking the EPS was defective for both of these characteristics, but mutation of gelA or the exoY-like gene had no effect on either type of attachment. The cellulose (celA) mutant attached and formed normal biofilms in vitro, but it did not form a biofilm on root hairs, although attachment did occur. The cellulose-dependent biofilm on root hairs appears not to be critical for nodulation, because the celA mutant competed with the wild-type for nodule infection. The glucomannan (gmsA) mutant attached and formed normal biofilms in vitro, but it was defective for attachment and biofilm formation on root hairs. Although this mutant formed nodules on peas, it was very strongly outcompeted by the wild type in mixed inoculations, showing that glucomannan is critical for competitive nodulation. The polysaccharide synthesis genes around gmsA are highly conserved among other rhizobia and agrobacteria but are absent from closely related bacteria (such as Brucella spp.) that are not normally plant associated, suggesting that these genes may play a wide role in bacterium-plant interactions.

scholarly journals Salmonella enterica Serovar Enteritidis Pathogenicity Island 1 Is Not Essential for but Facilitates Rapid Systemic Spread in Chickens

2009 ◽  
Vol 77 (7) ◽  
pp. 2866-2875 ◽  
Author(s):  
Taseen S. Desin ◽  
Po-King S. Lam ◽  
Birgit Koch ◽  
Claudia Mickael ◽  
Emil Berberov ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Systemic Infection ◽  
Poultry Meat ◽  
Wild Type ◽  
Secretion Systems ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Systemic Spread ◽  
Mutant Strains ◽  
Vitro Analysis

ABSTRACT Salmonella enterica subsp. enterica serovar Enteritidis is a leading cause of human food-borne illness that is mainly associated with the consumption of contaminated poultry meat and eggs. To cause infection, S. Enteritidis is known to use two type III secretion systems, which are encoded on two salmonella pathogenicity islands, SPI-1 and SPI-2, the first of which is thought to play a major role in invasion and bacterial uptake. In order to study the role of SPI-1 in the colonization of chicken, we constructed deletion mutants affecting the complete SPI-1 region (40 kb) and the invG gene. Both ΔSPI-1 and ΔinvG mutant strains were impaired in the secretion of SipD, a SPI-1 effector protein. In vitro analysis using polarized human intestinal epithelial cells (Caco-2) revealed that both mutant strains were less invasive than the wild-type strain. A similar observation was made when chicken cecal and small intestinal explants were coinfected with the wild-type and ΔSPI-1 mutant strains. Oral challenge of 1-week-old chicken with the wild-type or ΔSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection of the liver and spleen was delayed in birds that were challenged with the ΔSPI-1 strain. These data demonstrate that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of the organism in chickens.

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