scholarly journals A ClpB Chaperone Knockout Mutant of Mesorhizobium ciceri Shows a Delay in the Root Nodulation of Chickpea Plants

2012 ◽  
Vol 25 (12) ◽  
pp. 1594-1604 ◽  
Author(s):  
Clarisse Brígido ◽  
Marta Robledo ◽  
Esther Menéndez ◽  
Pedro F. Mateos ◽  
Solange Oliveira
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Fluorescent Protein ◽  
Root Nodules ◽  
Strain Analysis ◽  
Wild Type ◽  
Knockout Mutant ◽  
Mesorhizobium Ciceri ◽  
Acid Shock ◽  
Green Fluorescent

Several molecular chaperones are known to be involved in bacteria stress response. To investigate the role of chaperone ClpB in rhizobia stress tolerance as well as in the rhizobia-plant symbiosis process, the clpB gene from a chickpea microsymbiont, strain Mesorhizobium ciceri LMS-1, was identified and a knockout mutant was obtained. The ClpB knockout mutant was tested to several abiotic stresses, showing that it was unable to grow after a heat shock and it was more sensitive to acid shock than the wild-type strain. A plant-growth assay performed to evaluate the symbiotic performance of the clpB mutant showed a higher proportion of ineffective root nodules obtained with the mutant than with the wild-type strain. Nodulation kinetics analysis showed a 6- to 8-day delay in nodule appearance in plants inoculated with the ΔclpB mutant. Analysis of nodC gene expression showed lower levels of transcript in the ΔclpB mutant strain. Analysis of histological sections of nodules formed by the clpB mutant showed that most of the nodules presented a low number of bacteroids. No differences in the root infection abilities of green fluorescent protein–tagged clpB mutant and wild-type strains were detected. To our knowledge, this is the first study that presents evidence of the involvement of the chaperone ClpB from rhizobia in the symbiotic nodulation process.

scholarly journals Sphingolipid C-9 Methyltransferases Are Important for Growth and Virulence but Not for Sensitivity to Antifungal Plant Defensins in Fusarium graminearum

2008 ◽  
Vol 8 (2) ◽  
pp. 217-229 ◽  
Author(s):  
Vellaisamy Ramamoorthy ◽  
Edgar B. Cahoon ◽  
Mercy Thokala ◽  
Jagdeep Kaur ◽  
Jia Li ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Fungal Growth ◽  
Wild Type ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Disease Symptoms ◽  
Plant Defensins ◽  
Antifungal Action

ABSTRACT The C-9-methylated glucosylceramides (GlcCers) are sphingolipids unique to fungi. They play important roles in fungal growth and pathogenesis, and they act as receptors for some antifungal plant defensins. We have identified two genes, FgMT1 and FgMT2, that each encode a putative sphingolipid C-9 methyltransferase (C-9-MT) in the fungal pathogen Fusarium graminearum and complement a Pichia pastoris C-9-MT-null mutant. The ΔFgmt1 mutant produced C-9-methylated GlcCer like the wild-type strain, PH-1, whereas the ΔFgmt2 mutant produced 65 to 75% nonmethylated and 25 to 35% methylated GlcCer. No ΔFgmt1ΔFgmt2 double-knockout mutant producing only nonmethylated GlcCer could be recovered, suggesting that perhaps C-9-MTs are essential in this pathogen. This is in contrast to the nonessential nature of this enzyme in the unicellular fungus P. pastoris. The ΔFgmt2 mutant exhibited severe growth defects and produced abnormal conidia, while the ΔFgmt1 mutant grew like the wild-type strain, PH-1, under the conditions tested. The ΔFgmt2 mutant also exhibited drastically reduced disease symptoms in wheat and much-delayed disease symptoms in Arabidopsis thaliana. Surprisingly, the ΔFgmt2 mutant was less virulent on different host plants tested than the previously characterized ΔFggcs1 mutant, which lacks GlcCer synthase activity and produces no GlcCer at all. Moreover, the ΔFgmt1 and ΔFgmt2 mutants, as well as the P. pastoris strain in which the C-9-MT gene was deleted, retained sensitivity to the antifungal plant defensins MsDef1 and RsAFP2, indicating that the C-9 methyl group is not a critical structural feature of the GlcCer receptor required for the antifungal action of plant defensins.

scholarly journals Environmental Regulation of Exopolysaccharide Production in Sinorhizobium meliloti

2000 ◽  
Vol 182 (3) ◽  
pp. 599-606 ◽  
Author(s):  
Kiprian E. Mendrygal ◽  
Juan E. González
Keyword(s):  
Molecular Weight ◽  
Type Strain ◽  
Sinorhizobium Meliloti ◽  
Wild Type Strain ◽  
Root Nodules ◽  
Weight Fraction ◽  
Wild Type ◽  
Exopolysaccharide Production ◽  
Successful Establishment ◽  
High Phosphate

ABSTRACT Exopolysaccharide production by Sinorhizobium melilotiis required for invasion of root nodules on alfalfa and successful establishment of a nitrogen-fixing symbiosis between the two partners.S. meliloti wild-type strain Rm1021 requires production of either succinoglycan, a polymer of repeating octasaccharide subunits, or EPS II, an exopolysaccharide of repeating dimer subunits. The reason for the production of two functional exopolysaccharides is not clear. Earlier reports suggested that low-phosphate conditions stimulate the production of EPS II in Rm1021. We found that phosphate concentrations determine which exopolysaccharide is produced by S. meliloti. The low-phosphate conditions normally found in the soil (1 to 10 μM) stimulate EPS II production, while the high-phosphate conditions inside the nodule (20 to 100 mM) block EPS II synthesis and induce the production of succinoglycan. Interestingly, the EPS II produced by S. meliloti in low-phosphate conditions does not allow the invasion of alfalfa nodules. We propose that this invasion phenotype is due to the lack of the active molecular weight fraction of EPS II required for nodule invasion. An analysis of the function of PhoB in this differential exopolysaccharide production is presented.

scholarly journals Genome Shuffling Improves Degradation of the Anthropogenic Pesticide Pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723

2004 ◽  
Vol 70 (4) ◽  
pp. 2391-2397 ◽  
Author(s):  
MingHua Dai ◽  
Shelley D. Copley
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Constitutive Expression ◽  
Strain Analysis ◽  
Genome Shuffling ◽  
Original Strain ◽  
Negative Bacterium ◽  
Wild Type ◽  
Enhanced Resistance ◽  
Sphingobium Chlorophenolicum

ABSTRACT Pentachlorophenol (PCP), a highly toxic anthropogenic pesticide, can be mineralized by Sphingobium chlorophenolicum, a gram-negative bacterium isolated from PCP-contaminated soil. However, degradation of PCP is slow and S. chlorophenolicum cannot tolerate high levels of PCP. We have used genome shuffling to improve the degradation of PCP by S. chlorophenolicum. We have obtained several strains that degrade PCP faster and tolerate higher levels of PCP than the wild-type strain. Several strains obtained after the third round of shuffling can grow on one-quarter-strength tryptic soy broth plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of PCP at concentrations higher than 0.6 mM. Some of the mutants are able to completely degrade 3 mM PCP in one-quarter-strength tryptic soy broth, whereas no degradation can be achieved by the wild-type strain. Analysis of several improved strains suggests that the improved phenotypes are due to various combinations of mutations leading to an enhanced growth rate, constitutive expression of the PCP degradation genes, and enhanced resistance to the toxicity of PCP and its metabolites.

scholarly journals VdSNF1, the Sucrose Nonfermenting Protein Kinase Gene of Verticillium dahliae, Is Required for Virulence and Expression of Genes Involved in Cell-Wall Degradation

2011 ◽  
Vol 24 (1) ◽  
pp. 129-142 ◽  
Author(s):  
Aliki K. Tzima ◽  
Epaminondas J. Paplomatas ◽  
Payungsak Rauyaree ◽  
Manuel D. Ospina-Giraldo ◽  
Seogchan Kang
Keyword(s):  
Cell Wall ◽  
Verticillium Dahliae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Catabolic Repression ◽  
Kinase Gene ◽  
Fluid Medium

Verticillium dahliae is a soilborne fungus causing vascular wilt in a diverse array of plant species. Its virulence has been attributed, among other factors, to the activity of hydrolytic cell wall–degrading enzymes (CWDE). The sucrose nonfermenting 1 gene (VdSNF1), which regulates catabolic repression, was disrupted in V. dahliae tomato race 1. Expression of CWDE in the resulting mutants was not induced in inductive medium and in simulated xylem fluid medium. Growth of the mutants was significantly reduced when grown with pectin or galactose as a carbon source whereas, with glucose, sucrose, and xylose, they grew similarly to wild-type and ectopic transformants. The mutants were severely impaired in virulence on tomato and eggplant (final disease severity reduced by an average of 87%). Microscopic observation of the infection behavior of a green fluorescent protein (gfp)-labeled VdSNF1 mutant (70ΔSF-gfp1) showed that it was defective in initial colonization of roots. Cross sections of tomato stem at the cotyledonary level showed that 70ΔSF-gfp1 colonized xylem vessels considerably less than the wild-type strain. The wild-type strain heavily colonized xylem vessels and adjacent parenchyma cells. Quantification of fungal biomass in plant tissues further confirmed reduced colonization of roots, stems, and cotyledons by 70ΔSF-gfp1 relative to that by the wild-type strain.

scholarly journals Inactivation of the Pst System Reduces the Virulence of an Avian Pathogenic Escherichia coli O78 Strain

2005 ◽  
Vol 73 (7) ◽  
pp. 4138-4145 ◽  
Author(s):  
Martin G. Lamarche ◽  
Charles M. Dozois ◽  
France Daigle ◽  
Mélissa Caza ◽  
Roy Curtiss ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Surface Composition ◽  
Constitutive Expression ◽  
Rabbit Serum ◽  
Wild Type ◽  
E Coli ◽  
Acid Shock ◽  
Pst System

ABSTRACT Escherichia coli O78 strains are frequently associated with extraintestinal diseases, such as airsacculitis and septicemia, in poultry, livestock, and humans. To understand the influence of the pst operon in the virulence of E. coli, we introduced mutations into the pst genes of the avian pathogenic E. coli (APEC) O78:K80 strain χ7122 by allelic exchange. The mutation of pst genes led to the constitutive expression of the Pho regulon. Furthermore, the virulence of APEC strain χ7122 in a chicken infection model was attenuated by inactivation of the Pst system. The pst mutant caused significantly fewer extraintestinal lesions in infected chickens, and bacterial numbers isolated from different tissues after infection were significantly lower for the mutant than for the wild-type strain. Moreover, resistance to the bactericidal effects of rabbit serum and acid shock was impaired in the pst mutant, in contrast to the wild-type strain. In addition, the MIC of polymyxin was twofold lower for the mutant than for the wild-type strain. Although the pst mutant demonstrated an increased susceptibility to rabbit serum, this strain was not killed by chicken serum, suggesting the presence of differences in host innate immune defenses and complement-mediated killing. In APEC O78 strain χ7122, a functional Pst system is required for full virulence and resistance to acid shock and polymyxin. Our results suggest that the mutation of pst genes induces a deregulation of phosphate sensing and changes in the cell surface composition that lead to decreased virulence, indicating the importance of the Pst system for the virulence of pathogenic E. coli strains from different hosts.

scholarly journals 4-Chlorobenzoate Uptake in Comamonas sp. Strain DJ-12 Is Mediated by a Tripartite ATP-Independent Periplasmic Transporter

2006 ◽  
Vol 188 (24) ◽  
pp. 8407-8412 ◽  
Author(s):  
Jong-Chan Chae ◽  
Gerben J. Zylstra
Keyword(s):  
Growth Rate ◽  
Gene Cluster ◽  
Type Strain ◽  
Wild Type Strain ◽  
Proton Motive Force ◽  
Significant Inhibition ◽  
Motive Force ◽  
Wild Type ◽  
Knockout Mutant

ABSTRACT The fcb gene cluster involved in the hydrolytic dehalogenation of 4-chlorobenzoate is organized in the order fcbB-fcbA-fcbT1-fcbT2-fcbT3-fcbC in Comamonas sp. strain DJ-12. The genes are operonic and inducible with 4-chloro-, 4-iodo-, and 4-bromobenzoate. The fcbT1, fcbT2, and fcbT3 genes encode a transporter in the secondary TRAP (tripartite ATP-independent periplasmic) family. An fcbT1T2T3 knockout mutant shows a much slower growth rate on 4-chlorobenzoate compared to the wild type. 4-Chlorobenzoate is transported into the wild-type strain five times faster than into the fcbT1T2T3 knockout mutant. Transport of 4-chlorobenzoate shows significant inhibition by 4-bromo-, 4-iodo-, and 4-fluorobenzoate and mild inhibition by 3-chlorobenzoate, 2-chlorobenzoate, 4-hydroxybenzoate, 3-hydroxybenzoate, and benzoate. Uptake of 4-chlorobenzoate is significantly inhibited by ionophores which collapse the proton motive force.

The groEL2 gene, but not groEL1, is required for biosynthesis of the secondary metabolite myxovirescin in Myxococcus xanthus DK1622

Microbiology ◽  
2014 ◽  
Vol 160 (3) ◽  
pp. 488-495 ◽  
Author(s):  
Yan Wang ◽  
Xi Li ◽  
Wenyan Zhang ◽  
Xiuwen Zhou ◽  
Yue-zhong Li
Keyword(s):  
Secondary Metabolite ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Myxococcus Xanthus ◽  
Domain Swapping ◽  
Wild Type ◽  
Zone Of Inhibition ◽  
Knockout Mutant ◽  
E Coli

Myxococcus xanthus DK1622 possesses two copies of the groEL gene: groEL1, which participates in development, and groEL2, which is involved in the predatory ability of cells. In this study, we determined that the groEL2 gene is required for the biosynthesis of the secondary metabolite myxovirescin (TA), which plays essential roles in predation. The groEL2-knockout mutant strain was defective in producing a zone of inhibition and displayed decreased killing ability against Escherichia coli, while the groEL1-knockout mutant strain exhibited little difference from the wild-type strain DK1622. HPLC revealed that deletion of the groEL2 gene blocked the production of TA, which was present in the groEL1-knockout mutant. The addition of exogenous TA rescued the inhibition and killing abilities of the groEL2-knockout mutant against E. coli. Analysis of GroEL domain-swapping mutants indicated that the C-terminal equatorial domain of GroEL2 was essential for TA production, while the N-terminal equatorial or apical domains of GroEL2 were not sufficient to rescue TA production of the groEL2 knockout.

scholarly journals mhpTEncodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

2013 ◽  
Vol 79 (20) ◽  
pp. 6362-6368 ◽  
Author(s):  
Ying Xu ◽  
Bing Chen ◽  
Hongjun Chao ◽  
Ning-Yi Zhou
Keyword(s):  
Escherichia Coli ◽  
Wild Type Strain ◽  
Fluorescent Protein ◽  
Gene Knockout ◽  
Transport Activity ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent ◽  
K 12

ABSTRACTEscherichia coliK-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome,mhpTwas proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated thatmhpTis essential for 3HPP catabolism inE. coliK-12 W3110 at pH 8.2. Uptake assays with14C-labeled substrates demonstrated that strain W3110 and strain W3110ΔmhpTcontaining recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110ΔmhpTcontaining the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital forE. coliK-12 W3110 growth on this substrate under basic conditions.

scholarly journals Virulence Traits of a Serogroup C Meningococcus and IsogeniccssAMutant, Defective in Surface-Exposed Sialic Acid, in a Murine Model of Meningitis

2019 ◽  
Vol 87 (4) ◽  
Author(s):  
Roberta Colicchio ◽  
Chiara Pagliuca ◽  
Susanna Ricci ◽  
Elena Scaglione ◽  
Denis Grandgirard ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Corpus Callosum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Lethal Dose ◽  
Sialic Acids ◽  
Wild Type ◽  
Knockout Mutant ◽  
Content Type

ABSTRACTIn serogroup CNeisseria meningitidis, thecssA(siaA) gene codes for an UDP-N-acetylglucosamine 2-epimerase that catalyzes the conversion of UDP-N-acetyl-α-d-glucosamine intoN-acetyl-d-mannosamine and UDP in the first step in sialic acid biosynthesis. This enzyme is required for the biosynthesis of the (α2→9)-linked polysialic acid capsule and for lipooligosaccharide (LOS) sialylation. In this study, we have used a reference serogroup C meningococcal strain and an isogeniccssAknockout mutant to investigate the pathogenetic role of surface-exposed sialic acids in a model of meningitis based on intracisternal inoculation of BALB/c mice. Results confirmed the key role of surface-exposed sialic acids in meningococcal pathogenesis. The 50% lethal dose (LD50) of the wild-type strain 93/4286 was about four orders of magnitude lower than that of thecssAmutant. Compared to the wild-type strain, the ability of this mutant to replicate in brain and spread systemically was severely impaired. Evaluation of brain damage evidenced a significant reduction in cerebral hemorrhages in mice infected with the mutant in comparison with the levels in those challenged with the wild-type strain. Histological analysis showed the typical features of bacterial meningitis, including inflammatory cells in the subarachnoid, perivascular, and ventricular spaces especially in animals infected with the wild type. Noticeably, 80% of mice infected with the wild-type strain presented with massive bacterial localization and accompanying inflammatory infiltrate in thecorpus callosum, indicating high tropism of meningococci exposing sialic acids toward this brain structure and a specific involvement of thecorpus callosumin the mouse model of meningococcal meningitis.

scholarly journals F240 of β2-Tubulin Explains why Fusarium graminearum is Less Sensitive to Carbendazim than Botrytis cinerea

2018 ◽  
Vol 108 (3) ◽  
pp. 352-361 ◽  
Author(s):  
Yuanye Zhu ◽  
Xiaoyu Liang ◽  
Yanjun Li ◽  
Yabing Duan ◽  
Zhitian Zheng ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Binding Affinity ◽  
Fusarium Graminearum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Fluorescent Protein ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
Benzimidazole Fungicides ◽  
Β Tubulin

β-Tubulin is the target of benzimidazole fungicides, the most widely used of which is carbendazim (methyl benzimidazol-2-ylcarbamate [MBC]). MBC sensitivity is determined by the differential affinity of MBC for β-tubulins. However, the mechanism of less sensitivity of Fusarium graminearum to MBC compared with other fungi, including Botrytis cinerea, Colletotrichum gloeosporioides, and Sclerotinia sclerotiorum, remains exclusive. Alignment of β-tubulin amino acid sequences showed that position 240 of β-tubulins is leucine (L) in most pathogenic fungi but is phenylalanine (F) in the Fgβ2-tubulin of the F. graminearum wild type. The effective concentration resulting in 50% inhibition (EC50) value of MBC against the Fgβ2F240L mutant of F. graminearum is 0.047 μg/ml, which was 10-fold lower than that of wild-type strain 2021. Moreover, The EC50 value of MBC against the BcβL“240”F (actually position 232) mutant of Botrytis cinerea was 0.44 μg/ml, which was ninefold higher than that of B. cinerea wild-type strain Bt4-1. In response to MBC treatment (0.15 μg/ml), microtubules were clearly visible in Fgβ2-enhanced green fluorescent protein (EGFP) but not in Fgβ2F240L-EGFP. Moreover, a molecular docking assay indicated that F240L mutation created a pi-pi interaction between Fgβ2-tubulin and MBC and increased the binding affinity of Fgβ2-tubulin to MBC. Our results suggest that F240 is responsible for the naturally less MBC sensitivity in F. graminearum compared with B. cinerea, C. gloeosporioides, and S. sclerotiorum by decreasing the binding affinity between Fgβ2-tubulin and MBC.

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