An Isoflavonoid-Inducible Efflux Pump in Agrobacterium tumefaciens Is Involved in Competitive Colonization of Roots

ABSTRACT Agrobacterium tumefaciens 1D1609, which was originally isolated from alfalfa (Medicago sativa L.), contains genes that increase competitive root colonization on that plant by reducing the accumulation of alfalfa isoflavonoids in the bacterial cells. Mutant strain I-1 was isolated by its isoflavonoid-inducible neomycin resistance following mutagenesis with the transposable promoter probe Tn5-B30. Nucleotide sequence analysis showed the transposon had inserted in the first open reading frame, ifeA, of a three-gene locus (ifeA, ifeB, andifeR), which shows high homology to bacterial efflux pump operons. Assays on alfalfa showed that mutant strain I-1 colonized roots normally in single-strain tests but was impaired significantly (P ≤ 0.01) in competition against wild-type strain 1D1609. Site-directed mutagenesis experiments, which produced strains I-4 (ifeA::gusA) and I-6 (ifeA::Ω-Tc), confirmed the importance ofifeA for competitive root colonization. Exposure to the isoflavonoid coumestrol increased β-glucuronidase activity in strain I-4 21-fold during the period when coumestrol accumulation in wild-type cells declined. In the same test, coumestrol accumulation in mutant strain I-6 did not decline. Expression of the ifeA-gusAreporter was also induced by the alfalfa root isoflavonoids formononetin and medicarpin but not by two triterpenoids present in alfalfa. These results show that an efflux pump can confer measurable ecological benefits on A. tumefaciens in an environment where the inducing molecules are known to be present.

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Root Colonization by Agrobacterium tumefaciens Is Reduced in cel, attB,attD, and attR Mutants

Applied and Environmental Microbiology ◽

10.1128/aem.64.7.2341-2345.1998 ◽

1998 ◽

Vol 64 (7) ◽

pp. 2341-2345 ◽

Cited By ~ 52

Author(s):

Ann G. Matthysse ◽

Susan McMahan

Keyword(s):

Agrobacterium Tumefaciens ◽

Root Length ◽

Root Colonization ◽

Bacterial Pathogenesis ◽

Bacterial Suspension ◽

Cellulose Synthesis ◽

Wild Type ◽

Tomato Roots ◽

Extensive Growth ◽

Number Of Bacteria

ABSTRACT Root colonization by Agrobacterium tumefaciens was measured by using tomato and Arabidopsis thaliana roots dipped in a bacterial suspension and planted in soil. Wild-type bacteria showed extensive growth on tomato roots; the number of bacteria increased from 103 bacteria/cm of root length at the time of inoculation to more than 107 bacteria/cm after 10 days. The numbers of cellulose-minus and nonattachingattB, attD, and attR mutant bacteria were less than 1/10,000th the number of wild-type bacteria recovered from tomato roots. On roots of A. thalianaecotype Landsberg erecta, the numbers of wild-type bacteria increased from about 30 to 8,000 bacteria/cm of root length after 8 days. The numbers of cellulose-minus and nonattaching mutant bacteria were 1/100th to 1/10th the number of wild-type bacteria recovered after 8 days. The attachment of A. tumefaciens to cut A. thaliana roots incubated in 0.4% sucrose and observed with a light microscope was also reduced with cel andatt mutants. These results suggest that cellulose synthesis and attachment genes play a role in the ability of the bacteria to colonize roots, as well as in bacterial pathogenesis.

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Requirement of the Yersinia pseudotuberculosis Effectors YopH and YopE in Colonization and Persistence in Intestinal and Lymph Tissues

Infection and Immunity ◽

10.1128/iai.71.8.4595-4607.2003 ◽

2003 ◽

Vol 71 (8) ◽

pp. 4595-4607 ◽

Cited By ~ 89

Author(s):

Lauren K. Logsdon ◽

Joan Mecsas

Keyword(s):

Mutant Strain ◽

Type Iii Secretion ◽

Yersinia Pseudotuberculosis ◽

Host Responses ◽

Wild Type ◽

Mesenteric Lymph Nodes ◽

Single Strain ◽

Type Iii ◽

Mutant Strains ◽

Redundant Functions

ABSTRACT The gram-negative enteric pathogen Yersinia pseudotuberculosis employs a type III secretion system and effector Yop proteins that are required for virulence. Mutations in the type III secretion-translocation apparatus have been shown to cause defects in colonization of the murine cecum, suggesting roles for one or more effector Yops in the intestinal tract. To investigate this possibility, isogenic yop mutant strains were tested for their ability to colonize and persist in intestinal and associated lymph tissues of the mouse following orogastric inoculation. In single-strain infections, a yopHEMOJ mutant strain was unable to colonize, replicate, or persist in intestinal and lymph tissues. A yopH mutant strain specifically fails to colonize the mesenteric lymph nodes, but yopE and yopO mutant strains showed only minor defects in persistence in intestinal and lymph tissues. While no single Yop was found to be essential for colonization or persistence in intestinal tissues in single-strain infections, the absence of both YopH and YopE together almost eliminated colonization of all tissues, indicating either that these two Yops have some redundant functions or that Y. pseudotuberculosis employs multiple strategies for colonization. In competition infections with wild-type Y. pseudotuberculosis, the presence of wild-type bacteria severely hindered the ability of the yopH, yopE, and yopO mutants to persist in many tissues, suggesting that the wild-type bacteria either fills colonization niches or elicits host responses that the yop mutants are unable to withstand.

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Identification and Functional Characterization of the Novel Edwardsiella tarda Effector EseJ

Infection and Immunity ◽

10.1128/iai.02566-14 ◽

2015 ◽

Vol 83 (4) ◽

pp. 1650-1660 ◽

Cited By ~ 35

Author(s):

Hai-Xia Xie ◽

Jin-Fang Lu ◽

Ying Zhou ◽

Jia Yi ◽

Xiu-Jun Yu ◽

...

Keyword(s):

Mutant Strain ◽

Bacterial Adhesion ◽

Sequence Similarity ◽

Functional Characterization ◽

Lethal Dose ◽

Edwardsiella Tarda ◽

Bacterial Cells ◽

Wild Type ◽

High Sequence Similarity ◽

Content Type

Edwardsiella tardais a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and gastro- and extraintestinal infections in humans. The type III secretion system (T3SS) ofE. tardahas been identified as a key virulence factor that contributes to pathogenesis in fish. However, little is known about the associated effectors translocated by this T3SS. In this study, by comparing the profile of secreted proteins of the wild-type PPD130/91 and its T3SS ATPase ΔesaNmutant, we identified a new effector by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. This effector consists of 1,359 amino acids, sharing high sequence similarity with Orf29/30 ofE. tardastrain EIB202, and is renamed EseJ. The secretion and translocation of EseJ depend on the T3SS. A ΔeseJmutant strain adheres to epithelioma papillosum of carp (EPC) cells 3 to 5 times more extensively than the wild-type strain does. EseJ inhibits bacterial adhesion to EPC cells from within bacterial cells. Importantly, the ΔeseJmutant strain does not replicate efficiently in EPC cells and fails to replicate in J774A.1 macrophages. In infected J774A.1 macrophages, the ΔeseJmutant elicits higher production of reactive oxygen species than wild-typeE. tarda. The replication defect is consistent with the attenuation of the ΔeseJmutant in the blue gourami fish model: the 50% lethal dose (LD50) of the ΔeseJmutant is 2.34 times greater than that of the wild type, and the ΔeseJmutant is less competitive than the wild type in mixed infection. Thus, EseJ represents a novel effector that contributes to virulence by reducing bacterial adhesion to EPC cells and facilitating intracellular bacterial replication.

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Involvement of the Reserve Material Poly-β-Hydroxybutyrate in Azospirillum brasilense Stress Endurance and Root Colonization

Applied and Environmental Microbiology ◽

10.1128/aem.69.6.3244-3250.2003 ◽

2003 ◽

Vol 69 (6) ◽

pp. 3244-3250 ◽

Cited By ~ 119

Author(s):

Daniel Kadouri ◽

Edouard Jurkevitch ◽

Yaacov Okon

Keyword(s):

Plant Growth ◽

Mutant Strain ◽

Azospirillum Brasilense ◽

Type Strain ◽

Wild Type Strain ◽

Root Colonization ◽

Cell Aggregation ◽

Wild Type ◽

Phb Production ◽

Promote Plant Growth

ABSTRACT When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-β-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.

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A Set of Genes Encoding a Second Toluene Efflux System in Pseudomonas putida DOT-T1E Is Linked to the tod Genes for Toluene Metabolism

Journal of Bacteriology ◽

10.1128/jb.182.4.937-943.2000 ◽

2000 ◽

Vol 182 (4) ◽

pp. 937-943 ◽

Cited By ~ 92

Author(s):

Gilberto Mosqueda ◽

Juan-Luis Ramos

Keyword(s):

Pseudomonas Putida ◽

Culture Medium ◽

Efflux Pump ◽

Site Directed Mutagenesis ◽

Solvent Tolerance ◽

Directed Mutagenesis ◽

Wild Type ◽

Efflux System ◽

Host Chromosome ◽

Knock Out

ABSTRACT Sequence analysis in Pseudomonas putida DOT-T1E revealed a second toluene efflux system for toluene metabolism encoded by the ttgDEF genes, which are adjacent to thetod genes. The ttgDEF genes were expressed in response to the presence of aromatic hydrocarbons such as toluene and styrene in the culture medium. To characterize the contribution of the TtgDEF system to toluene tolerance in P. putida, site-directed mutagenesis was used to knock out the gene in the wild-type DOT-T1E strain and in a mutant derivative, DOT-T1E-18. This mutant carried a Tn5 insertion in the ttgABCgene cluster, which encodes a toluene efflux pump that is synthesized constitutively. For site-directed mutagenesis, a cassette to knock out the ttgD gene and encoding resistance to tellurite was constructed in vitro and transferred to the corresponding host chromosome via the suicide plasmid pKNG101. Successful replacement of the wild-type sequences with the mutant cassette was confirmed by Southern hybridization. A single ttgD mutant, DOT-T1E-1, and a double mutant with knock outs in the ttgD andttgA genes, DOT-T1E-82, were obtained and characterized for toluene tolerance. This was assayed by the sudden addition of toluene (0.3% [vol/vol]) to the liquid culture medium of cells growing on Luria-Bertani (LB) medium (noninduced) or on LB medium with toluene supplied via the gas phase (induced). Induced cells of the singlettgD mutant were more sensitive to sudden toluene shock than were the wild-type cells; however, noninduced wild-type andttgD mutant cells were equally tolerant to toluene shock. Noninduced cells of the double DOT-T1E-82 mutant did not survive upon sudden toluene shock; however, they still remained viable upon sudden toluene shock if they had been previously induced. These results are discussed in the context of the use of multiple efflux pumps involved in solvent tolerance in P. putida DOT-T1E.

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Pathogenic potential of a collagenase gene fromAeromonas veronii

Canadian Journal of Microbiology ◽

10.1139/w07-109 ◽

2008 ◽

Vol 54 (1) ◽

pp. 1-10 ◽

Cited By ~ 22

Author(s):

Hyun-Ja Han ◽

Tatsuo Taki ◽

Hidehiro Kondo ◽

Ikuo Hirono ◽

Takashi Aoki

Keyword(s):

Mutant Strain ◽

Type Strain ◽

Pathogenic Bacteria ◽

Wild Type Strain ◽

Collagenolytic Activity ◽

Bacterial Cells ◽

Wild Type ◽

Pathogenic Potential ◽

Collagenase Gene

The role of collagenase as a mechanism of bacterial pathogenicity in some pathogenic bacteria has been reported. The information on the role of collagenase in Aeromonas spp. pathogenesis is scant. In the present study, a mutant Aeromonas veronii RY001 that is deficient in the putative collagenase gene acg was constructed and compared with the wild-type strain for virulence factors. Bacterial cells and cell-free extracellular products of the mutant had significantly less collagenolytic activity, but there were not significant differences in caseinolytic, gelatinolytic, and elastolytic activities. Adhesion and invasion abilities of the mutant strain on epithelioma papillosum of carp cells was only 56% of that of the wild-type strain, and the cytotoxicity of the mutant strain to epithelioma papillosum of carp cells was only 42% of that of the wild-type strain. The LD50values of the wild-type strain were determined as 1.6 × 106and 3.5 × 105cfu in goldfish and mice, respectively, whereas the mutant RY001 strain showed slightly higher values (i.e., 2.8 × 106and 1.4 × 106cfu in goldfish and mice, respectively). These results indicated the involvement of the collagenase gene in the pathogenesis of A. veronii.

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Helicobacter pylori and Complex Gangliosides

Infection and Immunity ◽

10.1128/iai.72.3.1519-1529.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1519-1529 ◽

Cited By ~ 37

Author(s):

Niamh Roche ◽

Jonas Ångström ◽

Marina Hurtig ◽

Thomas Larsson ◽

Thomas Borén ◽

...

Keyword(s):

Helicobacter Pylori ◽

Sialic Acid ◽

Mutant Strain ◽

Carbohydrate Chain ◽

Proton Nuclear Magnetic Resonance ◽

Bacterial Cells ◽

Wild Type ◽

Knockout Mutant ◽

Factors Affecting ◽

H Pylori

ABSTRACT Recognition of sialic acid-containing glycoconjugates by the human gastric pathogen Helicobacter pylori has been repeatedly demonstrated. To investigate the structural requirements for H. pylori binding to complex gangliosides, a large number of gangliosides were isolated and characterized by mass spectrometry and proton nuclear magnetic resonance. Ganglioside binding of sialic acid-recognizing H. pylori strains (strains J99 and CCUG 17874) and knockout mutant strains with the sialic acid binding adhesin SabA or the NeuAcα3Galβ4GlcNAcβ3Galβ4GlcNAcβ-binding neutrophil-activating protein HPNAP deleted was investigated using the thin-layer chromatogram binding assay. The wild-type bacteria bound to N-acetyllactosamine-based gangliosides with terminal α3-linked NeuAc, while gangliosides with terminal NeuGcα3, NeuAcα6, or NeuAcα8NeuAcα3 were not recognized. The factors affecting binding affinity were identified as (i) the length of the N-acetyllactosamine carbohydrate chain, (ii) the branches of the carbohydrate chain, and (iii) fucose substitution of the N-acetyllactosamine core chain. While the J99/NAP− mutant strain displayed a ganglioside binding pattern identical to that of the parent J99 wild-type strain, no ganglioside binding was obtained with the J99/SabA− mutant strain, demonstrating that the SabA adhesin is the sole factor responsible for the binding of H. pylori bacterial cells to gangliosides.

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The Effect of Cellulose Overproduction on Binding and Biofilm Formation on Roots by Agrobacterium tumefaciens

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-1002 ◽

2005 ◽

Vol 18 (9) ◽

pp. 1002-1010 ◽

Cited By ~ 71

Author(s):

Ann G. Matthysse ◽

Mazz Marry ◽

Leonard Krall ◽

Mitchell Kaye ◽

Bronwyn E. Ramey ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Biofilm Formation ◽

Root Colonization ◽

Viable Cell ◽

Bacterial Attachment ◽

Chemical Fractionation ◽

Wild Type ◽

Ciona Savignyi ◽

Cellulose Synthase Gene ◽

Plant Surfaces

Agrobacterium tumefaciens growing in liquid attaches to the surface of tomato and Arabidopsis thaliana roots, forming a biofilm. The bacteria also colonize roots grown in sterile quartz sand. Attachment, root colonization, and biofilm formation all were markedly reduced in celA and chvB mutants, deficient in production of cellulose and cyclic β-(1,2)-D-glucans, respectively. We have identified two genes (celG and celI) in which mutations result in the overproduction of cellulose as judged by chemical fractionation and methylation analysis. Wild-type and chvB mutant strains carrying a cDNA clone of a cellulose synthase gene from the marine urochordate Ciona savignyi also overproduced cellulose. The overproduction in a wild-type strain resulted in increased biofilm formation on roots, as evaluated by light microscopy, and levels of root colonization intermediate between those of cellulose-minus mutants and the wild type. Overproduction of cellulose by a nonattaching chvB mutant restored biofilm formation and bacterial attachment in microscopic and viable cell count assays and partially restored root colonization. Although attachment to plant surfaces was restored, overproduction of cellulose did not restore virulence in the chvB mutant strain, suggesting that simple bacterial binding to plant surfaces is not sufficient for pathogenesis.

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The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

Journal of Bacteriology ◽

10.1128/jb.00762-07 ◽

2007 ◽

Vol 189 (18) ◽

pp. 6704-6713 ◽

Cited By ~ 36

Author(s):

Brian M. Thompson ◽

Lashanda N. Waller ◽

Karen F. Fox ◽

Alvin Fox ◽

George C. Stewart

Keyword(s):

Bacillus Anthracis ◽

Mutant Strain ◽

Basal Layer ◽

Structural Defects ◽

Bacterial Cells ◽

Wild Type ◽

Fatal Disease ◽

Specific Location ◽

Immunofluorescence Studies ◽

Kinetics Of

ABSTRACT Anthrax is a highly fatal disease caused by the gram-positive, endospore-forming, rod-shaped bacterium Bacillus anthracis. Spores, rather than vegetative bacterial cells, are the source of anthrax infections. Spores of B. anthracis are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. Filaments of the hair-like nap are made up largely of a single collagen-like glycoprotein called BclA. A second glycoprotein, BclB, has been identified in the exosporium layer. The specific location of this glycoprotein within the exosporium layer and its role in the biology of the spore are unknown. We created a mutant strain of B. anthracis ΔSterne that carries a deletion of the bclB gene. The mutant was found to possess structural defects in the exosporium layer of the spore (visualized by electron microscopy, immunofluorescence, and flow cytometry) resulting in an exosporium that is more fragile than that of a wild-type spore and is easily lost. Immunofluorescence studies also indicated that the mutant strain produced spores with increased levels of the BclA glycoprotein accessible to the antibodies on the surface. The resistance properties of the mutant spores were unchanged from those of the wild-type spores. A bclB mutation did not affect spore germination or kinetics of spore survival within macrophages. BclB plays a key role in the formation and maintenance of the exosporium structure in B. anthracis.

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Improvement in the Thermostability of d-Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.05566-11 ◽

2011 ◽

Vol 77 (20) ◽

pp. 7316-7320 ◽

Cited By ~ 31

Author(s):

Jin-Geun Choi ◽

Yo-Han Ju ◽

Soo-Jin Yeom ◽

Deok-Kun Oh

Keyword(s):

Enzyme Activity ◽

Agrobacterium Tumefaciens ◽

Operation Time ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Conversion Yield ◽

Content Type ◽

Aromatic Stacking

ABSTRACTThe S213C, I33L, and I33L S213C variants ofd-psicose 3-epimerase fromAgrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer ofd-psicose.

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