scholarly journals Fate of a Pseudomonas savastanoi pv. savastanoi Type III Secretion System Mutant in Olive Plants (Olea europaea L.)

2010 ◽  
Vol 76 (11) ◽  
pp. 3611-3619 ◽  
Author(s):  
Isabel P�rez-Mart�nez ◽  
Luis Rodr�guez-Moreno ◽  
Lotte Lambertsen ◽  
Isabel M. Matas ◽  
Jes�s Murillo ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Fluorescent Protein ◽  
Bacterial Pathogen ◽  
Membrane Vesicles ◽  
Tumor Formation ◽  
Outer Membrane Vesicles ◽  
Phylogenetic Position ◽  
Wild Type

ABSTRACT Pseudomonas savastanoi pv. savastanoi strain NCPPB 3335 is a model bacterial pathogen for studying the molecular basis of disease production in woody hosts. We report the sequencing of the hrpS-to-hrpZ region of NCPPB 3335, which has allowed us to determine the phylogenetic position of this pathogen with respect to previously sequenced Pseudomonas syringae hrp clusters. In addition, we constructed a mutant of NCPPB 3335, termed T3, which carries a deletion from the 3′ end of the hrpS gene to the 5′ end of the hrpZ operon. Despite its inability to multiply in olive tissues and to induce tumor formation in woody olive plants, P. savastanoi pv. savastanoi T3 can induce knot formation on young micropropagated olive plants. However, the necrosis and formation of internal open cavities previously reported in knots induced by the wild-type strain were not observed in those induced by P. savastanoi pv. savastanoi T3. Tagging of P. savastanoi pv. savastanoi T3 with green fluorescent protein (GFP) allowed real-time monitoring of its behavior on olive plants. In olive plant tissues, the wild-type strain formed aggregates that colonized the intercellular spaces and internal cavities of the hypertrophic knots, while the mutant T3 strain showed a disorganized distribution within the parenchyma of the knot. Ultrastructural analysis of knot sections revealed the release of extensive outer membrane vesicles from the bacterial cell surface of the P. savastanoi pv. savastanoi T3 mutant, while the wild-type strain exhibited very few vesicles. This phenomenon has not been described before for any other bacterial phytopathogen during host infection.

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nayeong Kim ◽  
Hyo Jeong Kim ◽  
Man Hwan Oh ◽  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Antimicrobial Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

scholarly journals The sensor kinase BfmS controls production of outer membrane vesicles in Acinetobacter baumannii

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
Seung Il Kim ◽  
Joo Hee Son ◽  
Shukho Kim ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Type Strain ◽  
Wild Type Strain ◽  
A549 Cells ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Sensor Kinase ◽  
Wild Type

Abstract Background Acinetobacter baumannii is an important opportunistic pathogen responsible for various nosocomial infections. The BfmRS two-component system plays a role in pathogenesis and antimicrobial resistance of A. baumannii via regulation of bacterial envelope structures. This study investigated the role of the sensor kinase, BfmS, in localization of outer membrane protein A (OmpA) in the outer membrane and production of outer membrane vesicles (OMVs) using wild-type A. baumannii ATCC 17978, ΔbfmS mutant, and bfmS-complemented strains. Results The ΔbfmS mutant showed hypermucoid phenotype in the culture plates, growth retardation under static culture conditions, and reduced susceptibility to aztreonam and colistin compared to the wild-type strain. The ΔbfmS mutant produced less OmpA in the outer membrane but released more OmpA via OMVs than the wild-type strain, even though expression of ompA and its protein production were not different between the two strains. The ΔbfmS mutant produced 2.35 times more OMV particles and 4.46 times more OMV proteins than the wild-type stain. The ΔbfmS mutant OMVs were more cytotoxic towards A549 cells than wild-type strain OMVs. Conclusions The present study demonstrates that BfmS controls production of OMVs in A. baumannii. Moreover, BfmS negatively regulates antimicrobial resistance of A. baumannii and OMV-mediated host cell cytotoxicity. Our results indicate that BfmS negatively controls the pathogenic traits of A. baumannii via cell envelope structures and OMV production.

scholarly journals Contribution of Nitrate Assimilation to the Fitness of Pseudomonas syringae pv. syringae B728a on Plants

2012 ◽  
Vol 79 (2) ◽  
pp. 678-687 ◽  
Author(s):  
Audrey Parangan-Smith ◽  
Steven Lindow
Keyword(s):  
Nitrate Reductase ◽  
Nitrogen Source ◽  
Pseudomonas Syringae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Fluorescent Protein ◽  
Nitrate Assimilation ◽  
Wild Type ◽  
Content Type ◽  
Nitrogenous Compounds

ABSTRACTThe ability ofPseudomonas syringaepv. syringae to use nitrate as a nitrogen source in culture and on leaves was assessed. Substantial amounts of leaf surface nitrate were detected directly and by use of a bioreporter of nitrate on bean plants grown with a variety of nitrogen sources. While a nitrate reductase mutant,P. syringaeΔnasB, exhibited greatly reduced growth in culture with nitrate as the sole nitrogen source, it exhibited population sizes similar to those of the wild-type strain on leaves. However, the growth of the ΔnasBmutant was much less than that of the wild-type strain when cultured in bean leaf washings supplemented with glucose, suggesting thatP. syringaeexperiences primarily carbon-limited and only secondarily nitrogen-limited growth on bean leaves. Only a small proportion of the cells of a green fluorescent protein (GFP)-basedP. syringaenitrate reductase bioreporter, LK2(pOTNas4), exhibited fluorescence on leaves. This suggests that only a subset of cells experience high nitrate levels or that nitrate assimilation is repressed by the presence of ammonium or other nitrogenous compounds in many leaf locations. While only a subpopulation ofP. syringaeconsumes nitrate at a given time on the leaves, the ability of those cells to consume this resource would be strongly beneficial to those cells, especially in environments in which nitrate is the most abundant form of nitrogen.

scholarly journals Quorum Sensing Regulates Exopolysaccharide Production, Motility, and Virulence in Pseudomonas syringae

2005 ◽  
Vol 18 (7) ◽  
pp. 682-693 ◽  
Author(s):  
Beatriz Quiñones ◽  
Glenn Dulla ◽  
Steven E. Lindow
Keyword(s):  
Quorum Sensing ◽  
Pseudomonas Syringae ◽  
Type Strain ◽  
Double Mutant ◽  
Wild Type Strain ◽  
Homoserine Lactone ◽  
Brown Spot ◽  
Wild Type ◽  
In Planta ◽  
Alginate Production

The N-acyl homoserine lactone (AHL)-mediated quorumsensing system in the phytopathogen Pseudomonas syringae pv. syringae requires the AHL synthase AhlI and the regulator AhlR, and is additionally subject to regulation by AefR. The contribution of quorum sensing to the expression of a variety of traits expected to be involved in epiphytic fitness and virulence of P. syringae were examined. Both an aefR- mutant and an ahlR- double mutant, deficient in AHL production, were significantly impaired in alginate production and had an increased susceptibility to hydrogen peroxide compared with the wild-type strain. These mutants were hypermotile in culture, invaded leaves more rapidly, and caused an increased incidence of brown spot lesions on bean leaves after a 48-h moist incubation. Interestingly, an aefR- mutant was both the most motile and virulent. Like the wild-type strain, the AHL-deficient mutant strains incited water-soaked lesions on bean pods. However, lesions caused by an ahlI- ahlR- double mutant were larger, whereas those incited by an aefR- mutant were smaller. In contrast, tissue maceration of pods, which occurs at a later stage of infection, was completely abolished in the AHL-deficient mutants. Both the incidence of disease and in planta growth of P. syringae pv. tabaci were greatly reduced in transgenic tobacco plants that produced AHL compared with wild-type plants. These results demonstrate that quorum sensing in P. syringae regulates traits that contribute to epiphytic fitness as well as to distinct stages of disease development during plant infection.

scholarly journals Novel Toluene Elimination System in a Toluene-Tolerant Microorganism

2000 ◽  
Vol 182 (22) ◽  
pp. 6451-6455 ◽  
Author(s):  
Hideki Kobayashi ◽  
Katsuyuki Uematsu ◽  
Hisako Hirayama ◽  
Koki Horikoshi
Keyword(s):  
Membrane Proteins ◽  
Cell Membrane ◽  
Outer Membrane ◽  
Parent Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Wild Type ◽  
Sensitive Mutant

ABSTRACT In studies of Pseudomonas putida IH-2000, a toluene-tolerant microorganism, membrane vesicles (MVs) were found to be released from the outer membrane when toluene was added to the culture. These MVs were found to be composed of phospholipids, lipopolysaccharides (LPS), and very low amounts of outer membrane proteins. The MVs also contained a higher concentration of toluene molecules (0.172 ± 0.012 mol/mol of lipid) than that found in the cell membrane. In contrast to the wild-type strain, the toluene-sensitive mutant strain 32, which differs from the parent strain in LPS and outer membrane proteins, did not release MVs from the outer membrane. The toluene molecules adhering to the outer membrane are eliminated by the shedding of MVs, and this system appears to serve as an important part of the toluene tolerance system of IH-2000.

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 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 The chvH Locus ofAgrobacterium Encodes a Homologue of an Elongation Factor Involved in Protein Synthesis

2001 ◽  
Vol 183 (1) ◽  
pp. 36-45 ◽  
Author(s):  
Wen-Tao Peng ◽  
Lois M. Banta ◽  
Trevor C. Charles ◽  
Eugene W. Nester
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Single Copy ◽  
Tumor Formation ◽  
Wild Type ◽  
Chromosomal Locus ◽  
Gene Encoding ◽  
Elongation Factor P

ABSTRACT The virulence of Agrobacterium tumefaciens depends on both chromosome- and Ti plasmid-encoded gene products. In this study, we characterize a chromosomal locus, chvH, previously identified by TnphoA mutagenesis and shown to be required for tumor formation. Through DNA sequencing and comparison of the sequence with identified sequences in the database, we show that this locus encodes a protein similar in sequence to elongation factor P, a protein thought to be involved in peptide bond synthesis inEscherichia coli. The analysis of vir-lacZ andvir-phoA translational fusions as well as Western immunoblotting revealed that the expression of Vir proteins such as VirE2 was significantly reduced in the chvH mutant compared with the wild-type strain. The E. coli efp gene complemented detergent sensitivity, virulence, and expression of VirE2 in the chvH mutant, suggesting that chvH andefp are functionally homologous. As expected, ChvH exerts its activity at the posttranscriptional level. Southern analysis suggests that the gene encoding this elongation factor is present as a single copy in A. tumefaciens. We constructed achvH deletion mutant in which a 445-bp fragment within its coding sequence was deleted and replaced with an omega fragment. On complex medium, this mutant grew more slowly than the wild-type strain, indicating that elongation factor P is important but not essential for the growth of Agrobacterium.

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