scholarly journals Identification and characterization of Photorhabdus temperata mutants altered in hemolysis and virulence

2016 ◽  
Vol 62 (8) ◽  
pp. 657-667
Author(s):  
Christine Chapman ◽  
Louis S. Tisa
Keyword(s):  
Hemolytic Activity ◽  
Entomopathogenic Nematode ◽  
Rna Turnover ◽  
Transposon Insertion ◽  
Physiological Properties ◽  
Delayed Initiation ◽  
Transposon Mutants ◽  
Photorhabdus Temperata

Photorhabdus temperata is a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora and an insect pathogen. This bacterium produces a wide variety of virulence factors and hemolytic activity. The goal of this study was to identify hemolysin-defective mutants and test their virulence. A genetic approach was used to identify mutants with altered hemolytic activity by screening a library of 10 000 P. temperata transposon mutants. Three classes of mutants were identified: (i) defective (no hemolytic activity), (ii) delayed (delayed initiation of hemolytic activity), and (iii) early (early initiation of hemolytic activity). The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis and motility. The hemolysin-defective mutants, P10A-C11, P10A-H12, and P79-B5, had inserts in genes involved in RNA turnover (RNase II and 5′-pentaphospho-5′-adenosine pyrophosphohydrolase) and showed reduced virulence and production of extracellular factors. These data support the role of RNA turnover in insect pathogenesis and other physiological functions.

scholarly journals Characterization of symbiotic and associated bacteria from entomopathogenic nematode Heterorhabditis sp. (nematode: Heterorhabditidae) isolated from India

2020 ◽  
Vol 30 (1) ◽  
Author(s):  
Rashid Pervez ◽  
Showkat Ahmad Lone ◽  
Sasmita Pattnaik
Keyword(s):  
Insect Pests ◽  
Entomopathogenic Nematode ◽  
Alcaligenes Faecalis ◽  
Symbiotic Bacteria ◽  
Rrna Gene ◽  
Main Body ◽  
Associated Bacteria ◽  
Molecular Approaches

Abstract Background Entomopathogenic nematodes (EPNs) harboring symbiotic bacteria are one of the safest alternatives to the chemical insecticides for the control of various insect pests. Infective juveniles of EPNs locate a target insect, enter through the openings, and reach the hemocoel, where they release the symbiotic bacteria and the target gets killed by the virulence factors of the bacteria. Photorhabdus with Heterorhabditis spp. are well documented; little is known about the associated bacteria. Main body In this study, we explored the presence of symbiotic and associated bacteria from Heterorhabditis sp. (IISR-EPN 09) and characterized by phenotypic, biochemical, and molecular approaches. Six bacterial isolates, belonging to four different genera, were recovered and identified as follows: Photorhabdus luminescens, one each strain of Providencia vermicola, Pseudomonas entomophila, Alcaligenes aquatilis, and two strains of Alcaligenes faecalis based on the phenotypic, biochemical criteria and the sequencing of 16S rRNA gene. Conclusion P. luminescens is symbiotically associated with Heterorhabditis sp. (IISR-EPN 09), whereas P. vermicola, P. entomophila, A. aquatilis, and A. faecalis are the associated bacteria. Further studies are needed to determine the exact role of the bacterial associates with the Heterorhabditis sp.

scholarly journals Characterization of RelA in Acinetobacter baumannii

2020 ◽  
Vol 202 (12) ◽  
Author(s):  
María Pérez-Varela ◽  
Aimee R. P. Tierney ◽  
Ju-Sim Kim ◽  
Andrés Vázquez-Torres ◽  
Philip Rather
Keyword(s):  
Acinetobacter Baumannii ◽  
Galleria Mellonella ◽  
Multidrug Resistant ◽  
Cell Physiology ◽  
Content Type ◽  
Content Model ◽  
Transposon Insertion ◽  
Downstream Effectors

ABSTRACT In response to nutrient depletion, the RelA and SpoT proteins generate the signaling molecule (p)ppGpp, which then controls a number of downstream effectors to modulate cell physiology. In Acinetobacter baumannii strain AB5075, a relA ortholog (ABUW_3302) was identified by a transposon insertion that conferred an unusual colony phenotype. An in-frame deletion in relA (ΔrelA) failed to produce detectable levels of ppGpp when amino acid starvation was induced with serine hydroxamate. The ΔrelA mutant was blocked from switching from the virulent opaque colony variant (VIR-O) to the avirulent translucent colony variant (AV-T), but the rate of AV-T to VIR-O switching was unchanged. In addition, the ΔrelA mutation resulted in a pronounced hypermotile phenotype on 0.35% agar plates. This hypermotility was dependent on the activation of a LysR regulator ABUW_1132, which was required for expression of AbaR, a LuxR family quorum-sensing regulator. In the ΔrelA mutant, ABUW_1132 was also required for the increased expression of an operon composed of the ABUW_3766-ABUW_3773 genes required for production of the surfactant-like lipopeptide acinetin 505. Additional phenotypes identified in the ΔrelA mutant included (i) cell elongation at high density, (ii) reduced formation of persister cells tolerant to colistin and rifampin, and (iii) decreased virulence in a Galleria mellonella model. IMPORTANCE Acinetobacter baumannii is a pathogen of worldwide importance. Due to the increasing prevalence of antibiotic resistance, these infections are becoming increasingly difficult to treat. New therapies are required to combat multidrug-resistant isolates. The role of RelA in A. baumannii is largely unknown. This study demonstrates that like in other bacteria, RelA controls a variety of functions, including virulence. Strategies to inhibit the activity of RelA and the resulting production of ppGpp could inhibit virulence and may represent a new therapeutic approach.

scholarly journals Identification of Xylella fastidiosa Antivirulence Genes: Hemagglutinin Adhesins Contribute to X. fastidiosa Biofilm Maturation and Colonization and Attenuate Virulence

2005 ◽  
Vol 18 (8) ◽  
pp. 856-868 ◽  
Author(s):  
Magalie R. Guilhabert ◽  
Bruce C. Kirkpatrick
Keyword(s):  
Xylella Fastidiosa ◽  
Plant Diseases ◽  
Plant Interactions ◽  
Wild Type ◽  
In Planta ◽  
Transposon Tn5 ◽  
Transposon Insertion ◽  
Transposon Mutants ◽  
Major Impediment

Xylella fastidosa, a gram-negative, xylem-limited bacterium, is the causal agent of several economically important plant diseases, including Pierce's disease (PD) and citrus variegated chlorosis (CVC). Until recently, the inability to transform or produce transposon mutants of X. fastidosa had been a major impediment to identifying X. fastidosa genes that mediate pathogen and plant interactions. A random transposon (Tn5) library of X. fastidosa was constructed and screened for mutants showing more severe symptoms and earlier grapevine death (hypervirulence) than did vines infected with the wild type. Seven hypervirulent mutants identified in this screen moved faster and reached higher populations than the wild type in grapevines. These results suggest that X. fastidosa attenuates its virulence in planta and that movement is important in X. fastidosa virulence. The mutated genes were sequenced and none had been described previously as antivirulence genes, although six of them showed similarity with genes of known functions in other organisms. One transposon insertion inactivated a hemagglutinin adhesin gene (PD2118), which we named HxfA. Another mutant in a second putative X. fastidosa hemagglutinin gene, PD1792 (HxfB), was constructed, and further characterization of these hxf mutants suggests that X. fastidosa hemagglutinins mediate contact between X. fastidosa cells, which results in colony formation and biofilm maturation within the xylem vessels.

scholarly journals Isolation and Characterization of Xenorhabdus nematophila Transposon Insertion Mutants Defective in Lipase Activity against Tween

2009 ◽  
Vol 191 (16) ◽  
pp. 5325-5331 ◽  
Author(s):  
Gregory R. Richards ◽  
Eugenio I. Vivas ◽  
Aaron W. Andersen ◽  
Delmarie Rivera-Santos ◽  
Sara Gilmore ◽  
...  
Keyword(s):  
Lipase Activity ◽  
Metabolic Pathways ◽  
Lipase Production ◽  
Xenorhabdus Nematophila ◽  
Transposon Insertion ◽  
Isolation And Characterization ◽  
Transposon Mutants ◽  
Conserved Gene ◽  
Insertion Mutants

ABSTRACT We identified Xenorhabdus nematophila transposon mutants with defects in lipase activity. One of the mutations, in yigL, a conserved gene of unknown function, resulted in attenuated virulence against Manduca sexta insects. We discuss possible connections between lipase production, YigL, and specific metabolic pathways.

scholarly journals Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis

2015 ◽  
Vol 197 (13) ◽  
pp. 2201-2216 ◽  
Author(s):  
Sheldon Hurst ◽  
Holli Rowedder ◽  
Brandye Michaels ◽  
Hannah Bullock ◽  
Ryan Jackobeck ◽  
...  
Keyword(s):  
Biological Control Agent ◽  
Entomopathogenic Nematode ◽  
Draft Genome ◽  
Control Agent ◽  
Content Type ◽  
Transposon Insertion ◽  
Defective Mutant ◽  
Rnase Ii ◽  
Mutualistic Association ◽  
Photorhabdus Temperata

ABSTRACTThe entomopathogenic nematodeHeterorhabditis bacteriophoraforms a specific mutualistic association with its bacterial partnerPhotorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome ofP. temperataand identify genes that plays a role in the pathogenesis of thePhotorhabdus-Heterorhabditissymbiosis. A draft genome sequence ofP. temperatastrain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000P. temperatatransposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions.IMPORTANCEThe relationship betweenPhotorhabdusand entomopathogenic nematodeHeterorhabditisrepresents a well-known mutualistic system that has potential as a biological control agent. The elucidation of the genome of the bacterial partner and role that RNase II plays in its life cycle has provided a greater understanding ofPhotorhabdusas both an insect pathogen and a nematode symbiont.

scholarly journals Listeria monocytogenes relA and hpt Mutants Are Impaired in Surface-Attached Growth and Virulence

2002 ◽  
Vol 184 (3) ◽  
pp. 621-628 ◽  
Author(s):  
Clare M. Taylor ◽  
Mark Beresford ◽  
Harry A. S. Epton ◽  
David C. Sigee ◽  
Gilbert Shama ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Stringent Response ◽  
Physiological Adaptation ◽  
Salvage Pathway ◽  
Purine Salvage ◽  
Biofilm Growth ◽  
Transposon Insertion ◽  
New Information

ABSTRACT We describe here the identification and characterization of two Listeria monocytogenes (Tn917-LTV3) relA and hpt transposon insertion mutants that were impaired in growth after attachment to a model surface. Both mutants were unable to accumulate (p)ppGpp in response to amino acid starvation, whereas the wild-type strain accumulated (p)ppGpp within 30 min of stress induction. The induction of transcription of the relA gene after adhesion was demonstrated, suggesting that the ability to mount a stringent response and undergo physiological adaptation to nutrient deprivation is essential for the subsequent growth of the adhered bacteria. The absence of (p)ppGpp in the hpt mutant, which is blocked in the purine salvage pathway, is curious and suggests that a functional purine salvage pathway is required for the biosynthesis of (p)ppGpp. Both mutants were avirulent in a murine model of listeriosis, indicating an essential role for the stringent response in the survival and growth of L. monocytogenes in the host. Taken as a whole, this study provides new information on the role of the stringent response and the physiological adaptation of L. monocytogenes for biofilm growth and pathogenesis.

scholarly journals The PhlA Hemolysin from the Entomopathogenic Bacterium Photorhabdusluminescens Belongs to the Two-Partner Secretion Family of Hemolysins

2002 ◽  
Vol 184 (14) ◽  
pp. 3871-3878 ◽  
Author(s):  
Julien Brillard ◽  
Eric Duchaud ◽  
Noël Boemare ◽  
Frank Kunst ◽  
Alain Givaudan
Keyword(s):  
Hemolytic Activity ◽  
Pathogenic Bacteria ◽  
Fluorescent Protein ◽  
Gene Encoding ◽  
Rich Media ◽  
The Family ◽  
Insect Interactions ◽  
Green Fluorescent

ABSTRACT Photorhabdus is an entomopathogenic bacterium symbiotically associated with nematodes of the family Heterorhabditidae. Bacterial hemolysins found in numerous pathogenic bacteria are often virulence factors. We describe here the nucleotide sequence and the molecular characterization of the Photorhabdus luminescens phlBA operon, a locus encoding a hemolysin which shows similarities to the Serratia type of hemolysins. It belongs to the two-partner secretion (TPS) family of proteins. In low-iron conditions, a transcriptional induction of the phlBA operon was observed by using the chloramphenicol acetyltransferase reporter gene, causing an increase in PhlA hemolytic activity compared to iron-rich media. A spontaneous phase variant of P. luminescens was deregulated in phlBA transcription. The phlA mutant constructed by allelic exchange remained highly pathogenic after injection in the lepidopteran Spodoptera littoralis, indicating that PhlA hemolysin is not a major virulence determinant. Using the gene encoding green fluorescent protein as a reporter, phlBA transcription was observed in hemolymph before insect death. We therefore discuss the possible role of PhlA hemolytic activity in the bacterium-nematode-insect interactions.

scholarly journals Role of Ornibactin Biosynthesis in the Virulence ofBurkholderia cepacia: Characterization of pvdA, the Gene Encoding l-OrnithineN5-Oxygenase

1999 ◽  
Vol 67 (9) ◽  
pp. 4443-4455 ◽  
Author(s):  
P. A. Sokol ◽  
P. Darling ◽  
D. E. Woods ◽  
E. Mahenthiralingam ◽  
C. Kooi
Keyword(s):  
Burkholderia Cepacia ◽  
Respiratory Infections ◽  
Cosmid Library ◽  
Gene Encoding ◽  
Transposon Insertion ◽  
Peptide Synthetase ◽  
Transposon Insertions ◽  
Siderophore Activity

ABSTRACT Burkholderia cepacia is a frequent cause of respiratory infections in cystic fibrosis patients. B. cepacia has been shown to produce at least four siderophores which may play a role in the virulence of this organism. To characterize genes involved in the synthesis of siderophores, Tn5-OT182 mutants were isolated in strain K56-2, which produces two siderophores, salicylic acid (SA) and ornibactins. Two mutants were characterized that did not produce zones on Chrome Azurol S agar in a commonly used assay to detect siderophore activity. These mutants were determined to produce sevenfold more SA than K56-2 yet did not produce detectable amounts of ornibactins. These mutants, designated I117 and T10, had a transposon insertion in genes with significant homology to pyoverdine biosynthesis genes of Pseudomonas aeruginosa. I117 contained an insertion in a pvdA homolog, the gene for the enzymel-ornithine N 5-oxygenase, which catalyzes the hydroxylation of l-ornithine. Ornibactin synthesis in this mutant was partially restored when the precursorl-N 5-OH-Orn was added to the culture medium. T10 contained an insertion in a pvdDhomolog, which is a peptide synthetase involved in pyoverdine synthesis. β-Galactosidase activity was iron regulated in both I117 and T10, suggesting that the transposon was inserted downstream of an iron-regulated promoter. Tn5-OT182 contains alacZ gene that is expressed when inserted downstream of an active promoter. Both I117 and T10 were deficient in uptake of iron complexed to either ornibactins or SA, suggesting that transposon insertions in ornibactin biosynthesis genes also affected other components of the iron transport mechanism. The B. cepacia pvdA homolog was approximately 47% identical and 59% similar tol-ornithine N 5-oxygenase fromP. aeruginosa. Three clones were identified from a K56-2 cosmid library that partially restored ornibactin production, SA production, and SA uptake to parental levels but did not affect the rate of 59Fe-ornibactin uptake in I117. A chromosomalpvdA deletion mutant was constructed that had a phenotype similar to that of I117 except that it did not hyperproduce SA. ThepvdA mutants were less virulent than the parent strain in chronic and acute models of respiratory infection. A functionalpvdA gene appears to be required for effective colonization and persistence in B. cepacia lung infections.

Digital x-ray mapping of nanometer-size supported bimetallic catalysts

1988 ◽  
Vol 46 ◽  
pp. 530-531
Author(s):  
L. T. Germinario
Keyword(s):  
Background Radiation ◽  
Metal Cluster ◽  
Single Element ◽  
Beam Diameter ◽  
X Rays ◽  
X Ray ◽  
Major Interest ◽  
Induced Changes

Understanding the role of metal cluster composition in determining catalytic selectivity and activity is of major interest in heterogeneous catalysis. The electron microscope is well established as a powerful tool for ultrastructural and compositional characterization of support and catalyst. Because the spatial resolution of x-ray microanalysis is defined by the smallest beam diameter into which the required number of electrons can be focused, the dedicated STEM with FEG is the instrument of choice. The main sources of errors in energy dispersive x-ray analysis (EDS) are: (1) beam-induced changes in specimen composition, (2) specimen drift, (3) instrumental factors which produce background radiation, and (4) basic statistical limitations which result in the detection of a finite number of x-ray photons. Digital beam techniques have been described for supported single-element metal clusters with spatial resolutions of about 10 nm. However, the detection of spurious characteristic x-rays away from catalyst particles produced images requiring several image processing steps.

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