Expression of a Serratia marcescens Chitinase Gene in Sinorhizobium fredii USDA191 and Sinorhizobium meliloti RCR2011 Impedes Soybean and Alfalfa Nodulation

A gene encoding chitinase from Serratia marcescens BJL200 was cloned into a broad-host-range vector (pRK415) and mobilized into Sinorhizobium fredii USDA191. Chitinolytic activity was detected in S. fredii USDA191 transconjugants that carried the S. marcescens chiB gene. Chitinase-producing S. fredii USDA191 formed nodules on soybean cultivar McCall. However, there was a delay in nodule formation and a marked decrease in the total number of nodules formed by the chitinase-producing S. fredii in comparison with the wild-type strain. Expression of chitinase in S. meliloti RCR2011 also impeded alfalfa nodulation. Thin-layer chromatography of 14C-labeled Nod factors from chitinase-producing S. fredii USDA191 revealed hydrolysis of lipochitooligosaccharides.

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Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product

Microbiology ◽

10.1099/mic.0.26476-0 ◽

2003 ◽

Vol 149 (10) ◽

pp. 2931-2939 ◽

Cited By ~ 63

Author(s):

Alex K. Jaques ◽

Tamo Fukamizo ◽

Diana Hall ◽

Richard C. Barton ◽

Gemma M. Escott ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Wild Type Strain ◽

Chitinase Activity ◽

Gene Replacement ◽

Wild Type ◽

Gene Encoding ◽

Batch Cultures ◽

Putative Active Site ◽

Hydrolysis Of

The gene encoding a major, inducible 45 kDa chitinase of Aspergillus fumigatus was cloned and analysis of the deduced amino acid sequence identified a chitinase of the fungal/bacterial class which was designated ChiB1. Recombinant ChiB1, expressed in Pichia pastoris, was shown to function by a retaining mechanism of action. That is, the β-conformation of the chitin substrate linkage was preserved in the product in a manner typical of family 18 chitinases. Cleavage patterns with the N-acetylglucosamine (GlcNAc) oligosaccharide substrates GlcNAc4, GlcNAc5 and GlcNAc6 indicated that the predominant reaction involved hydrolysis of GlcNAc2 from the non-reducing end of each substrate. Products of transglycosylation were also identified in each incubation. Following disruption of chiB1 by gene replacement, growth and morphology of disruptants and of the wild-type strain were essentially identical. However, during the autolytic phase of batch cultures the level of chitinase activity in culture filtrate from a disruptant was much lower than the activity from the wild-type. The search for chitinases with morphogenetic roles in filamentous fungi should perhaps focus on chitinases of the fungal/plant class although such an investigation will be complicated by the identification of at least 11 putative active site domains for family 18 chitinases in the A. fumigatus TIGR database (http://www.tigr.org/).

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Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

Scientific Reports ◽

10.1038/s41598-020-80125-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

José Francisco Cruz-Pérez ◽

Roxana Lara-Oueilhe ◽

Cynthia Marcos-Jiménez ◽

Ricardo Cuatlayotl-Olarte ◽

María Luisa Xiqui-Vázquez ◽

...

Keyword(s):

Azospirillum Brasilense ◽

Type Strain ◽

Wild Type Strain ◽

Soil Conditions ◽

Wild Type ◽

Gene Encoding ◽

Wheat Roots ◽

Genes Encoding ◽

In The Wild ◽

Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

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Genetic Analysis of Mutations Affecting pckA Regulation in Rhizobium (Sinorhizobium) meliloti

Genetics ◽

10.1093/genetics/147.4.1521 ◽

1997 ◽

Vol 147 (4) ◽

pp. 1521-1531 ◽

Cited By ~ 2

Author(s):

Magne Østerås ◽

Shelley A P O'Brien ◽

Turlough M Finan

Keyword(s):

Sinorhizobium Meliloti ◽

Phosphoenolpyruvate Carboxykinase ◽

Root Nodules ◽

Genetic Regulation ◽

Carbon Sources ◽

Dicarboxylic Acids ◽

Phenotypic Analysis ◽

Gene Encoding ◽

Type Locus ◽

Rapid Induction

Abstract The enzyme phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step in the gluconeogenic pathway in most organisms. We are examining the genetic regulation of the gene encoding Pck, pckA, in Rhizobium (now Sinorhizobium) meliloti. This bacterium forms N2-fixing root nodules on alfalfa, and the major energy sources supplied to the bacteria within these nodules are C4-dicarboxylic acids such as malate and succinate. R. meliloti cells growing in glucose minimal medium show very low pckA expression whereas addition of succinate to this medium results in a rapid induction of pckA transcription. We identified spontaneous mutations (rpk) that alter the regulation of pckA expression such that pckA is expressed in media containing the non-inducing carbon sources lactose and glucose. Genetic and phenotypic analysis allowed us to differentiate at least four rpk mutant classes that map to different locations on the R. meliloti chromosome. The wild-type locus corresponding to one of these rpk loci was cloned by complementation, and two Tn5 insertions within the insert DNA that no longer complemented the rpk mutation were identified. The nucleotide sequence of this region revealed that both Tn5 insertions lay within a gene encoding a protein homologous to the Ga1R/LacI family of transcriptional regulators that are involved in metabolism.

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Mutation in the ntrR Gene, a Member of the vap Gene Family, Increases the Symbiotic Efficiency of Sinorhizobium meliloti

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.7.887 ◽

2001 ◽

Vol 14 (7) ◽

pp. 887-894 ◽

Cited By ~ 25

Author(s):

Boglárka Oláh ◽

Erno Kiss ◽

Zoltán Györgypál ◽

Judit Borzi ◽

Gyöngyi Cinege ◽

...

Keyword(s):

Nitrogen Fixation ◽

Pathogenic Bacteria ◽

Sinorhizobium Meliloti ◽

Root Nodules ◽

Nifh Gene ◽

Dry Weight ◽

Nodule Occupancy ◽

Gene Encoding ◽

Symbiotic Efficiency ◽

Host Interactions

In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the “ntrPR operon,” which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.

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Antifeedant Effects and Repellent Activity of Loline Alkaloids from Endophyte-Infected Tall Fescue against Horn Flies, Haematobia irritans (Diptera: Muscidae)

Molecules ◽

10.3390/molecules26040817 ◽

2021 ◽

Vol 26 (4) ◽

pp. 817

Author(s):

Javier Espinoza ◽

Manuel Chacón-Fuentes ◽

Andrés Quiroz ◽

Leonardo Bardehle ◽

Paul Escobar-Bahamondes ◽

...

Keyword(s):

Tall Fescue ◽

Preparative Thin Layer Chromatography ◽

Repellent Activity ◽

Choice Test ◽

Haematobia Irritans ◽

Major Pest ◽

Horn Flies ◽

Layer Chromatography ◽

Hydrolysis Of ◽

Spatial Repellency

Haematobia irritans is an obligate bloodsucking ectoparasite of cattle and is the global major pest of livestock production. Currently, H. irritans management is largely dependent upon broad-spectrum pesticides, which lately has led to the development of insecticide resistance. Thus, alternative control methods are necessary. Endophyte-infected grasses have been studied as an alternative due to their capability to biosynthesize alkaloids associated with anti-insect activities. Thus, the main aim of this study was to evaluate the antifeedant and repellent activity of lolines obtained from endophyte-infected tall fescue against H. irritans adults in laboratory conditions. The alkaloid extract (ALKE) was obtained by acid–base extraction. N-formyl loline (NFL) and N-acetyl loline (NAL) were isolated by preparative thin layer chromatography (pTLC) and column chromatography (CC), and the loline was prepared by acid hydrolysis of a NFL/NAL mixture. Loline identification was performed by gas chromatography coupled to mass spectrometry (GC/MS). Feeding behavior was evaluated by a non-choice test, and olfactory response was evaluated using a Y-tube olfactometer. Accordingly, all samples showed antifeedant activities. NFL was the most antifeedant compound at 0.5 µg/µL and 1.0 µg/µL, and it was statistically equal to NAL but different to loline; however, NAL was not statistically different to loline. NFL and NAL at 0.25 µg/µL were more active than loline. All samples except loline exhibited spatial repellency in the olfactometer. Thus, the little or non-adverse effects for cattle and beneficial activities of those lolines make them suitable candidates for horn fly management.

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Evidence for a Type III Secretion System in Aeromonas salmonicida subsp. salmonicida

Journal of Bacteriology ◽

10.1128/jb.184.21.5966-5970.2002 ◽

2002 ◽

Vol 184 (21) ◽

pp. 5966-5970 ◽

Cited By ~ 74

Author(s):

Sarah E. Burr ◽

Katja Stuber ◽

Thomas Wahli ◽

Joachim Frey

Keyword(s):

Type Iii Secretion ◽

Type Strain ◽

Wild Type Strain ◽

Type Iii Secretion System ◽

Secretion System ◽

Aeromonas Salmonicida ◽

Broad Host Range ◽

Wild Type ◽

Secretion Systems ◽

Type Iii

ABSTRACT Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis, is an important fish pathogen. We have screened this bacterium with a broad-host-range probe directed against yscV, the gene that encodes the archetype of a highly conserved family of inner membrane proteins found in every known type III secretion system. This has led to the identification of seven open reading frames that encode homologues to proteins functioning within the type III secretion systems of Yersinia species. Six of these proteins are encoded by genes comprising a virA operon. The A. salmonicida subsp. salmonicida yscV homologue, ascV, was inactivated by marker replacement mutagenesis and used to generate an isogenic ascV mutant. Comparison of the extracellular protein profiles from the ascV mutant and the wild-type strain indicates that A. salmonicida subsp. salmonicida secretes proteins via a type III secretion system. The recently identified ADP-ribosylating toxin AexT was identified as one such protein. Finally, we have compared the toxicities of the wild-type A. salmonicida subsp. salmonicida strain and the ascV mutant against RTG-2 rainbow trout gonad cells. While infection with the wild-type strain results in significant morphological changes, including cell rounding, infection with the ascV mutant has no toxic effect, indicating that the type III secretion system we have identified plays an important role in the virulence of this pathogen.

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Effects of a Nod-factor-overproducing strain of Sinorhizobium meliloti on the expression of the ENOD40 gene in Melilotus alba

Canadian Journal of Botany ◽

10.1139/b02-076 ◽

2002 ◽

Vol 80 (9) ◽

pp. 907-915 ◽

Cited By ~ 6

Author(s):

Walter F Giordano ◽

Michelle R Lum ◽

Ann M Hirsch

Keyword(s):

Lateral Root ◽

Sinorhizobium Meliloti ◽

Cortical Cell ◽

Host Cells ◽

Nodule Development ◽

Nodule Formation ◽

Additional Increase ◽

Nod Factor ◽

Melilotus Alba ◽

Different Strains

We have initiated studies on the molecular biology and genetics of white sweetclover (Melilotus alba Desr.) and its responses to inoculation with the nitrogen-fixing symbiont Sinorhizobium meliloti. Early nodulin genes such as ENOD40 serve as markers for the transition from root to nodule development even before visible stages of nodule formation are evident. Using Northern blot analysis, we found that the ENOD40 gene was expressed within 6 h after inoculation with two different strains of S. meliloti, one of which overproduces symbiotic Nod factors. Inoculation with this strain resulted in an additional increase in ENOD40 gene expression over a typical wild-type S. meliloti strain. Moreover, the increase in mRNA brought about by the Nod-factor-overproducing strain 24 h after inoculation was correlated with lateral root formation by using whole-mount in situ hybridization to localize ENOD40 transcripts in lateral root meristems and by counting lateral root initiation sites. Cortical cell divisions were not detected. We also found that nodulation occurred more rapidly on white sweetclover in response to the Nod-factor-overproducing strain, but ultimately there was no difference in nodulation efficiency in terms of nodule number or the number of roots nodulated by the two strains. Also, the two strains could effectively co-colonize the host when inoculated together, although a few host cells were occupied by both strains.Key words: ENOD40, Nod factor, Melilotus, Sinorhizobium, symbiosis.

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Characterization of a Unique Chromosomal Copper Resistance Gene Cluster from Xanthomonas campestris pv. vesicatoria

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.8284-8291.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 8284-8291 ◽

Cited By ~ 44

Author(s):

Huseyin Basim ◽

Gerald V. Minsavage ◽

Robert E. Stall ◽

Jaw-Fen Wang ◽

Savita Shanker ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Resistance Genes ◽

Xanthomonas Campestris ◽

Sinorhizobium Meliloti ◽

The United States ◽

Copper Resistance ◽

Pcr Analysis ◽

Pepper Plant ◽

Gene Encoding ◽

Copper Resistance Genes

ABSTRACT We characterized the copper resistance genes in strain XvP26 of Xanthomonas campestris pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in Xanthomonas and Pseudomonas strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in Pseudomonas syringae pv. tomato and Escherichia coli, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in E. coli and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in Sinorhizobium meliloti, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant X. campestris pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in X. campestris pv. vesicatoria.

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The use of bioluminescence as a reporter to study the adherence of the plant growth promoting rhizopseudomonads 7NSK2 and ANP15 to canola roots

Canadian Journal of Microbiology ◽

10.1139/m93-046 ◽

1993 ◽

Vol 39 (3) ◽

pp. 329-334 ◽

Cited By ~ 15

Author(s):

J. Boelens ◽

D. Zoutman ◽

J. Campbell ◽

W. Verstraete ◽

W. Paranchych

Keyword(s):

Plant Growth ◽

Freundlich Isotherm ◽

Root Surface ◽

Broad Host Range ◽

Bacterial Bioluminescence ◽

Range Vector ◽

Plant Growth Promoting ◽

Specific Receptors ◽

Growth Promoting ◽

Kinetics Of

The adherence of the plant growth promoting rhizopseudomonads Pseudomonas aeruginosa 7NSK2 and Pseudomonas fluorescens ANP15 to canola roots (Brassica campestris L. c.v. Tobin) was examined by means of a bacterial bioluminescence system. The bioluminescence broad host range vector pDLUX-I was constructed from pLAFR-I and the lux A–E genes of Vibrio fischerii. This vector was conjugally transferred into the plant growth promoting rhizopseudomonads 7NSK2 and ANP15. The transformed strains were constitutively bioluminescent at an optimal temperature of 21 °C. The measured bioluminescence was directly proportional to the density of the bacteria in suspension and was the same for both planktonic and sessile bacteria adhering to the root surface. The adherence of the plant growth promoting rhizopseudomonads was proportional to the density of the bacterial inoculum, approached saturation at 60 min, and was reversible. The kinetics of the microbial adhesion was described by a Freundlich isotherm suggesting that the adherence of the bacteria to the canola root surface does not involve specific receptors. We conclude that the pDLUX-I vector is an easy and accurate way to study the kinetics of microbial adherence to the rhizoplane.Key words: rhizopseudomonads, bioluminescence, adhesion, plant growth promotion.not available

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