scholarly journals Identification of a Third Sulfate Activation System in Sinorhizobium sp. Strain BR816: the CysDN Sulfate Activation Complex

2003 ◽  
Vol 69 (4) ◽  
pp. 2006-2014 ◽  
Author(s):  
Carla Snoeck ◽  
Christel Verreth ◽  
Ismael Hernández-Lucas ◽  
Esperanza Martínez-Romero ◽  
Jos Vanderleyden
Keyword(s):  
Growth Characteristics ◽  
Open Reading Frames ◽  
Lag Phase ◽  
Sulfur Source ◽  
Nod Factors ◽  
Wild Type ◽  
Triple Mutant ◽  
Nod Factor ◽  
Growth Defects ◽  
Sulfate Activation

ABSTRACT Sinorhizobium sp. strain BR816 possesses two nodPQ copies, providing activated sulfate (3′-phosphoadenosine-5′-phosphosulfate [PAPS]) needed for the biosynthesis of sulfated Nod factors. It was previously shown that the Nod factors synthesized by a nodPQ double mutant are not structurally different from those of the wild-type strain. In this study, we describe the characterization of a third sulfate activation locus. Two open reading frames were fully characterized and displayed the highest similarity with the Sinorhizobium meliloti housekeeping ATP sulfurylase subunits, encoded by the cysDN genes. The growth characteristics as well as the levels of Nod factor sulfation of a cysD mutant (FAJ1600) and a nodP1 nodQ2 cysD triple mutant (FAJ1604) were determined. FAJ1600 shows a prolonged lag phase only with inorganic sulfate as the sole sulfur source, compared to the wild-type parent. On the other hand, FAJ1604 requires cysteine for growth and produces sulfate-free Nod factors. Apigenin-induced nod gene expression for Nod factor synthesis does not influence the growth characteristics of any of the strains studied in the presence of different sulfur sources. In this way, it could be demonstrated that the “household” CysDN sulfate activation complex of Sinorhizobium sp. strain BR816 can additionally ensure Nod factor sulfation, whereas the symbiotic PAPS pool, generated by the nodPQ sulfate activation loci, can be engaged for sulfation of amino acids. Finally, our results show that rhizobial growth defects are likely the reason for a decreased nitrogen fixation capacity of bean plants inoculated with cysD mutant strains, which can be restored by adding methionine to the plant nutrient solution.

scholarly journals Involvement of Salicylic Acid in the Establishment of the Rhizobium meliloti-Alfalfa Symbiosis

1998 ◽  
Vol 11 (2) ◽  
pp. 153-155 ◽  
Author(s):  
F. Martínez-Abarca ◽  
J. A. Herrera-Cervera ◽  
P. Bueno ◽  
J. Sanjuan ◽  
T. Bisseling ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Rhizobium Meliloti ◽  
Significant Inhibition ◽  
Nod Factors ◽  
Wild Type ◽  
Nod Factor ◽  
Rhizobium Strains ◽  
Meliloti Strain

Inoculation of alfalfa plants with either incompatible Rhizobium or a Rhizobium mutant blocked in Nod factor synthesis led to an accumulation of salicylic acid in roots, in contrast to plants inoculated with a wild-type (compatible) R. meliloti strain. When salicylic acid was exogenously applied prior to inoculation of alfalfa plants with either purified Nod factor or compatible Rhizobium strains, a significant inhibition of nodule primordia formation and a reduction of the number of emerging nodules, respectively, as well as a delay in nodule visualization, were observed. These results suggest an involvement of Rhizobium-synthesized Nod factors in the inhibition of salicylic acid-mediated defense in legumes.

scholarly journals The Rhizobium etli metZ Gene Is Essential for Methionine Biosynthesis and Nodulation of Phaseolus vulgaris

1999 ◽  
Vol 12 (1) ◽  
pp. 24-34 ◽  
Author(s):  
Rosarita Taté ◽  
Anna Riccio ◽  
Emilia Caputo ◽  
Maurizio laccarino ◽  
Eduardo J. Patriarca
Keyword(s):  
Phaseolus Vulgaris ◽  
Mutant Strain ◽  
Wild Type Strain ◽  
Homologous Gene ◽  
Sulfur Source ◽  
Rhizobium Etli ◽  
Nod Factors ◽  
Wild Type ◽  
Root Cells ◽  
Rnase Protection

A mutant strain (CTNUX23) of Rhizobium etli carrying Tn5 unable to grow with sulfate as the sole sulfur source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a metZ (O-succinylhomoserine sulfhydrylase)-homologous gene. The CTNUX23 mutant strain had a growth dependency for methionine, although cystathionine or homocysteine, but not homoserine or O-succinylhomoserine, allowed growth of the mutant. RNase protection assays showed that the metZ-like gene had a basal level of expression in methionine- or cysteine-grown cells, which was induced when sulfate or thiosulfate was used. The metZ gene was cloned from the parent wild-type strain, CE3, and the resulting plasmid pAR204 relieved, after transformation, the methionine auxotrophy of both strains CTNUX23 of R. etli and PAO503(metZ) of Pseudomonas aeruginosa. Unlike strain CE3 or CTNUX23 (pAR204), strain CTNUX23 showed undetectable levels of O-succinylhomoserine sulfhydrylase activity. Strain CTNUX23 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) or to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris, unless methionine was added to the growth medium. These data and our previous results support the notion that cysteine or glutathione, but not methionine, is supplied by the root cells to bacteria growing inside the plant.

scholarly journals PmrA(Con) Confers pmrHFIJKL-Dependent EGTA and Polymyxin Resistance on msbB Salmonella by Decorating Lipid A with Phosphoethanolamine

2007 ◽  
Vol 189 (14) ◽  
pp. 5161-5169 ◽  
Author(s):  
Sean R. Murray ◽  
Robert K. Ernst ◽  
David Bermudes ◽  
Samuel I. Miller ◽  
K. Brooks Low
Keyword(s):  
Structural Analysis ◽  
Essential Role ◽  
Lipid A ◽  
Biosynthetic Enzymes ◽  
Strain Atcc ◽  
Loss Of Function ◽  
Wild Type ◽  
Triple Mutant ◽  
Growth Defects ◽  
Rich Media

ABSTRACT Mutations in pmrA were recombined into Salmonella strain ATCC 14028 msbB to determine if pmrA-regulated modifications of lipopolysaccharide could suppress msbB growth defects. A mutation that functions to constitutively activate pmrA [pmrA(Con)] suppresses msbB growth defects on EGTA-containing media. Lipid A structural analysis showed that Salmonella msbB pmrA(Con) strains, compared to Salmonella msbB strains, have increased amounts of palmitate and phosphoethanolamine but no aminoarabinose addition, suggesting that aminoarabinose is not incorporated into msbB lipid A. Surprisingly, loss-of-function mutations in the aminoarabinose biosynthetic genes restored EGTA and polymyxin sensitivity to Salmonella msbB pmrA(Con) strains. These blocks in aminoarabinose biosynthesis also prevented lipid A phosphoethanolamine incorporation and reduced the levels of palmitate addition, indicating previously unknown roles for the aminoarabinose biosynthetic enzymes. Lipid A structural analysis of the EGTA- and polymyxin-resistant triple mutant msbB pmrA(Con) pagP::Tn10, which contains phosphoethanolamine but no palmitoylated lipid A, suggests that phosphoethanolamine addition is sufficient to confer EGTA and polymyxin resistance on Salmonella msbB strains. Additionally, palmitoylated lipid A was observed only in wild-type Salmonella grown in the presence of salt in rich media. Thus, we correlate EGTA resistance and polymyxin resistance with phosphoethanolamine-decorated lipid A and demonstrate that the aminoarabinose biosynthetic proteins play an essential role in lipid A phosphoethanolamine addition and affect lipid A palmitate addition in Salmonella msbB strains.

scholarly journals Responsiveness to exogenous cAMP of a Saccharomyces cerevisiae strain conferred by naturally occurring alleles of PDE1 and PDE2.

Genetics ◽  
1993 ◽  
Vol 135 (2) ◽  
pp. 321-326 ◽  
Author(s):  
H Mitsuzawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Slow Growth ◽  
Loss Of Function ◽  
Wild Type ◽  
Triple Mutant ◽  
Apparent Absence ◽  
Camp Pathway ◽  
Naturally Occurring ◽  
Elevated Activity ◽  
Growth Phenotype

Abstract The Saccharomyces cerevisiae strain P-28-24C, from which cAMP requiring mutants derived, responded to exogenously added cAMP. Upon the addition of cAMP, this strain showed phenotypes shared by mutants with elevated activity of the cAMP pathway. Genetic analysis involving serial crosses of this strain to a strain with another genetic background revealed that the responsiveness to cAMP results from naturally occurring loss-of-function alleles of PDE1 and PDE2, which encode low and high affinity cAMP phosphodiesterases, respectively. In addition, P-28-24C was found to carry a mutation conferring slow growth that lies in CYR1, which encodes adenylate cyclase, and the slow growth phenotype caused by the cyr1 mutation was suppressed by the pde2 mutation. Therefore P-28-24C is fortuitously a pde1 pde2 cyr1 triple mutant. Responsiveness to cAMP conferred by pde mutations suggests that S. cerevisiae cells are permeable to cAMP to some extent and that the apparent absence of effect of exogenously added cAMP on wild-type cells is due to immediate degradation by cAMP phosphodiesterases.

scholarly journals Rsp5p, a New Link between the Actin Cytoskeleton and Endocytosis in the Yeast Saccharomyces cerevisiae

2002 ◽  
Vol 22 (20) ◽  
pp. 6946-6948 ◽  
Author(s):  
Joanna Kamińska ◽  
Beata Gajewska ◽  
Anita K. Hopper ◽  
Teresa ˙Zołądek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Growth Defects ◽  
Genes Encoding ◽  
Cytoskeleton Dynamics

ABSTRACT Rsp5p is an ubiquitin-protein ligase of Saccharomyces cerevisiae that has been implicated in numerous processes including transcription, mitochondrial inheritance, and endocytosis. Rsp5p functions at multiple steps of endocytosis, including ubiquitination of substrates and other undefined steps. We propose that one of the roles of Rsp5p in endocytosis involves maintenance and remodeling of the actin cytoskeleton. We report the following. (i) There are genetic interactions between rsp5 and several mutant genes encoding actin cytoskeletal proteins. rsp5 arp2, rsp5 end3, and rsp5 sla2 double mutants all show synthetic growth defects. Overexpressed wild-type RSP5 or mutant rsp5 genes with lesions of some WW domains suppress growth defects of arp2 and end3 cells. The defects in endocytosis, actin cytoskeleton, and morphology of arp2 are also suppressed. (ii) Rsp5p and Sla2p colocalize in abnormal F-actin-containing clumps in arp2 and pan1 mutants. Immunoprecipitation experiments confirmed that Rsp5p and Act1p colocalize in pan1 mutants. (iii) Rsp5p and Sla2p coimmunoprecipitate and partially colocalize to punctate structures in wild-type cells. These studies provide the first evidence for an interaction of an actin cytoskeleton protein with Rsp5p. (iv) rsp5-w1 mutants are resistant to latrunculin A, a drug that sequesters actin monomers and depolymerizes actin filaments, consistent with the fact that Rsp5p is involved in actin cytoskeleton dynamics.

scholarly journals Actin-dependent regulation of the cardiac Na+/Ca2+ exchanger

2006 ◽  
Vol 290 (3) ◽  
pp. C691-C701 ◽  
Author(s):  
Madalina Condrescu ◽  
John P. Reeves
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cytochalasin D ◽  
Lag Phase ◽  
Cell Contact ◽  
Wild Type ◽  
Filamentous Actin ◽  
Surface Distribution ◽  
Reverse Mode ◽  
Hydrophilic Domain

In the present study, the bovine cardiac Na+/Ca2+ exchanger (NCX1.1) was expressed in Chinese hamster ovary cells. The surface distribution of the exchanger protein, externally tagged with the hemagglutinin (HA) epitope, was associated with underlying actin filaments in regions of cell-to-cell contact and also along stress fibers. After we treated cells with cytochalasin D, NCX1.1 protein colocalized with patches of fragmented filamentous actin (F-actin). In contrast, an HA-tagged deletion mutant of NCX1.1 that was missing much of the exchanger's central hydrophilic domain Δ(241–680) did not associate with F-actin. In cells expressing the wild-type exchanger, cytochalasin D inhibited allosteric Ca2+ activation of NCX activity as shown by prolongation of the lag phase of low Ca2+ uptake after initiation of the reverse (i.e., Ca2+ influx) mode of NCX activity. Other agents that perturbed F-actin structure (methyl-β-cyclodextrin, latrunculin B, and jasplakinolide) also increased the duration of the lag phase. In contrast, when reverse-mode activity was initiated after allosteric Ca2+ activation, both cytochalasin D and methyl-β-cyclodextrin (Me-β-CD) stimulated NCX activity by ∼70%. The activity of the Δ(241–680) mutant, which does not require allosteric Ca2+ activation, was also stimulated by cytochalasin D and Me-β-CD. The increased activity after these treatments appeared to reflect an increased amount of exchanger protein at the cell surface. We conclude that wild-type NCX1.1 associates with the F-actin cytoskeleton, probably through interactions involving the exchanger's central hydrophilic domain, and that this association interferes with allosteric Ca2+ activation.

scholarly journals Generation of an Isogenic Collection of Yeast Actin Mutants and Identification of Three Interrelated Phenotypes

Genetics ◽  
2001 ◽  
Vol 157 (2) ◽  
pp. 533-543
Author(s):  
Johanna L Whitacre ◽  
Dana A Davis ◽  
Kurt A Toenjes ◽  
Sharon M Brower ◽  
Alison E M Adams
Keyword(s):  
Crystal Structure ◽  
Growth Rates ◽  
Small Region ◽  
Wild Type ◽  
Large Collection ◽  
Growth Defects ◽  
Cytoskeletal Function ◽  
Highly Correlated ◽  
The Relationship ◽  
Mutant Cells

Abstract A large collection of yeast actin mutations has been previously isolated and used in numerous studies of actin cytoskeletal function. However, the various mutations have been in congenic, rather than isogenic, backgrounds, making it difficult to compare the subtle phenotypes that are characteristic of these mutants. We have therefore placed 27 mutations in an isogenic background. We used a subset of these mutants to compare the degree to which different actin alleles are defective in sporulation, endocytosis, and growth on NaCl-containing media. We found that the three phenotypes are highly correlated. The correlations are specific and not merely a reflection of general growth defects, because the phenotypes are not correlated with growth rates under normal conditions. Significantly, those actin mutants exhibiting the most severe phenotypes in all three processes have altered residues that cluster to a small region of the actin crystal structure previously defined as the fimbrin (Sac6p)-binding site. We examined the relationship between endocytosis and growth on salt and found that shifting wild-type or actin mutant cells to high salt reduces the rate of α-factor internalization. These results suggest that actin mutants may be unable to grow on salt because of additive endocytic defects (due to mutation and salt).

scholarly journals Yeasts as Complementary Model Systems for the Study of the Pathological Repercussions of Enhanced Synphilin-1 Glycation and Oxidation

2021 ◽  
Vol 22 (4) ◽  
pp. 1677
Author(s):  
David Seynnaeve ◽  
Daniel P. Mulvihill ◽  
Joris Winderickx ◽  
Vanessa Franssens
Keyword(s):  
Lewy Bodies ◽  
Inhibitory Effect ◽  
Model Systems ◽  
Wild Type ◽  
Glyoxalase System ◽  
Inclusion Formation ◽  
Growth Defects ◽  
Yeast Strains ◽  
Autophagic Degradation ◽  
Detrimental Impact

Synphilin-1 has previously been identified as an interaction partner of α-Synuclein (αSyn), a primary constituent of neurodegenerative disease-linked Lewy bodies. In this study, the repercussions of a disrupted glyoxalase system and aldose reductase function on Synphilin-1 inclusion formation characteristics and cell growth were investigated. To this end, either fluorescent dsRed-tagged or non-tagged human SNCAIP, which encodes the Synphilin-1 protein, was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast strains devoid of enzymes Glo1, Glo2, and Gre3. Presented data shows that lack of Glo2 and Gre3 activity in S. cerevisiae increases the formation of large Synphilin-1 inclusions. This correlates with enhanced oxidative stress levels and an inhibitory effect on exponential growth, which is most likely caused by deregulation of autophagic degradation capacity, due to excessive Synphilin-1 aggresome build-up. These findings illustrate the detrimental impact of increased oxidation and glycation on Synphilin-1 inclusion formation. Similarly, polar-localised inclusions were observed in wild-type S. pombe cells and strains deleted for either glo1+ or glo2+. Contrary to S. cerevisiae, however, no growth defects were observed upon expression of SNCAIP. Altogether, our findings show the relevance of yeasts, especially S. cerevisiae, as complementary models to unravel mechanisms contributing to Synphilin-1 pathology in the context of neurodegenerative diseases.

scholarly journals Enhancer Requirement for Murine Cytomegalovirus Growth and Genetic Complementation by the Human Cytomegalovirus Enhancer

1998 ◽  
Vol 72 (11) ◽  
pp. 8502-8509 ◽  
Author(s):  
Ana Angulo ◽  
Martin Messerle ◽  
Ulrich H. Koszinowski ◽  
Peter Ghazal
Keyword(s):  
Regulatory Region ◽  
Open Reading Frames ◽  
Productive Infection ◽  
Murine Cytomegalovirus ◽  
Growth Defect ◽  
Wild Type ◽  
Sequence Composition ◽  
Culture Cells ◽  
Sequence Elements ◽  
Human Cmv

ABSTRACT The cytomegalovirus (CMV) enhancer is a highly complex regulatory region containing multiple elements that interact with a variety of host-encoded transcription factors. Many of these sequence elements are conserved among the different species strains of CMV, although the arrangement of the various elements and overall sequence composition of the CMV enhancers differ remarkably. To delineate the importance of this region to a productive infection and to explore the possibility of generating a murine CMV (MCMV) under the control of human CMV (HCMV) genetic elements, the MCMV enhancer was resected and replaced either with nonregulatory sequences or with paralogous sequences from HCMV. The effects of these various deletions and substitutions on viral growth in transfected or infected tissue-culture cells were evaluated. We found that mutations in MCMV that eliminate or substitute for the enhancer with nonregulatory sequences showed a severe deficiency in virus synthesis. This growth defect is effectively complemented by the homologous MCMV enhancer as well as the HCMV enhancer. In the latter case, the chimeric viruses (hybrid MCMV strains) containing the molecularly shuffled human enhancer exhibit infectious kinetics similar to that of parental wild-type and wild-type revertant MCMV. These results also show that open reading frames m124, m124.1, and m125 located within the enhancer region are nonessential for growth of MCMV in cells. Most importantly, we conclude that the enhancer of MCMV is required for optimal infection and that its diverged human counterpart can advantageously replace its role in promoting viral infectivity.

scholarly journals Polymorphisms in Pilin Glycosylation Locus of Neisseria meningitidis Expressing Class II Pili

2001 ◽  
Vol 69 (6) ◽  
pp. 3597-3604 ◽  
Author(s):  
Charlene M. Kahler ◽  
Larry E. Martin ◽  
Yih-Ling Tzeng ◽  
Yoon K. Miller ◽  
Kerith Sharkey ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Human Serum ◽  
Class Ii ◽  
Open Reading Frames ◽  
Normal Human Serum ◽  
Wild Type ◽  
Experimental Conditions ◽  
Type Iv ◽  
Insertional Inactivation ◽  
Normal Human

ABSTRACT We have located a locus, pgl, in Neisseria meningitidis strain NMB required for the glycosylation of class II pili. Between five and eight open reading frames (ORFs) (pglF, pglB, pglC, pglB2, orf2, orf3, orf8, and avtA) were present in the pgl clusters of different meningococcal isolates. The Class I pilus-expressing strains Neisseria gonorrhoeae MS11 and N. meningitidis MC58 each contain a pgl cluster in which orf2 andorf3 have been deleted. Strain NMB and other meningococcal isolates which express class II type IV pili contained pglclusters in which pglB had been replaced bypglB2 and an additional novel ORF, orf8, had been inserted between pglB2 and pglC. Insertional inactivation of the eight ORFs of the pglcluster of strain NMB showed that pglF, pglB2, pglC, andpglD, but not orf2, orf3, orf8, andavtA, were necessary for pilin glycosylation. Pilin glycosylation was not essential for resistance to normal human serum, as pglF and pglD mutants retained wild-type levels of serum resistance. Although pglB2 andpglC mutants were significantly sensitive to normal human serum under the experimental conditions used, subsequent examination of the encapsulation phenotypes revealed that pglB2 andpglC mutants expressed almost 50% less capsule than wild-type NMB. A mutation in orf3, which did not affect pilin glycosylation, also resulted in a 10% reduction in capsule expression and a moderately serum sensitive phenotype. On the basis of these results we suggest that pilin glycosylation may proceed via a lipid-linked oligosaccharide intermediate and that blockages in this pathway may interfere with capsular transport or assembly.

Sign in / Sign up

Export Citation Format

Share Document