Role of glutamine synthetase in phenazine antibiotic production by Pantoea agglomerans Eh1087

2004 ◽  
Vol 50 (10) ◽  
pp. 877-881 ◽  
Author(s):  
Matthew D Galbraith ◽  
Stephen R Giddens ◽  
H Khris Mahanty ◽  
Bruce Clark
Keyword(s):  
Glutamine Synthetase ◽  
Antibiotic Production ◽  
Amino Acid Sequences ◽  
Pantoea Agglomerans ◽  
Wild Type ◽  
Isotopic Labelling ◽  
High Degree ◽  
Degree Of Similarity ◽  
Isotopic Labelling Experiments

Pantoea agglomerans strain Eh1087 produces the phenazine antibiotic D-alanylgriseoluteic acid. A glutamine auxotroph harboring an insertion in a putative glnA gene was obtained by transposon-mutagenesis of Eh1087 that produced less D-alanylgriseoluteic acid than the parental strain (strain Eh7.1). Cosmids encoding the Eh1087 glnA were isolated by their ability to complement the mutant for prototrophy. The role of the Eh1087 glnA locus was functionally confirmed by complementation of an Escherichia coli glnA mutant. Analysis of the nucleotide and deduced amino acid sequences of the Eh1087 glnA gene indicated a high degree of similarity to the glnA genes and glutamine synthetase enzymes of other Enterobacteriaceae. Isotopic labelling experiments with 15N-labelled ammonium sulfate demonstrated that wild-type Eh1087 incorporated 15N into griseoluteic acid more readily than the glnA mutant Eh7.1. We conclude that the 2 nitrogens in the phenazine nucleus originate from glutamine and the intracellular glutamine synthesized by Eh1087 is a source of the phenazine nucleus nitrogens even in glutamine-rich environments.Key words: phenazine, Pantoea, Erwinia, glutamine synthetase, biosynthesis.

scholarly journals NADPH Oxidases Are Involved in Differentiation and Pathogenicity in Botrytis cinerea

2008 ◽  
Vol 21 (6) ◽  
pp. 808-819 ◽  
Author(s):  
Nadja Segmüller ◽  
Leonie Kokkelink ◽  
Sabine Giesbert ◽  
Daniela Odinius ◽  
Jan van Kan ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Nicotinamide Adenine Dinucleotide ◽  
Amino Acid Sequences ◽  
Host Tissue ◽  
Regulatory Subunit ◽  
Phytopathogenic Fungus ◽  
Wild Type ◽  
Nadph Oxidases ◽  
Knock Out

Nicotinamide adenine dinucleotide (NADPH) oxidases have been shown to be involved in various differentiation processes in fungi. We investigated the role of two NADPH oxidases in the necrotrophic phytopathogenic fungus, Botrytis cinerea. The genes bcnoxA and bcnoxB were cloned and characterized; their deduced amino acid sequences show high homology to fungal NADPH oxidases. Analyses of single and double knock-out mutants of both NADPH oxidase genes showed that both bcnoxA and bcnoxB are involved in formation of sclerotia. Both genes have a great impact on pathogenicity: whereas bcnoxB mutants showed a retarded formation of primary lesions, probably due to an impaired formation of penetration structures, bcnoxA mutants were able to penetrate host tissue in the same way as the wild type but were much slower in colonizing the host tissue. Double mutants showed an additive effect: they were aberrant in penetration and colonization of plant tissue and, therefore, almost nonpathogenic. To study the structure of the fungal Nox complex in more detail, bcnoxR (encoding a homolog of the mammalian p67phox, a regulatory subunit of the Nox complex) was functionally characterized. The phenotype of ΔbcnoxR mutants is identical to that of ΔbcnoxAB double mutants, providing evidence that BcnoxR is involved in activation of both Bcnox enzymes.

scholarly journals An Internal Motor Kinesin Is Associated with the Golgi Apparatus and Plays a Role in Trichome Morphogenesis in Arabidopsis

2005 ◽  
Vol 16 (2) ◽  
pp. 811-823 ◽  
Author(s):  
Ling Lu ◽  
Yuh-Ru Julie Lee ◽  
Ruiqin Pan ◽  
Julin N. Maloof ◽  
Bo Liu
Keyword(s):  
Golgi Apparatus ◽  
Cell Cortex ◽  
Wild Type ◽  
Internal Motor ◽  
Kinesin Superfamily ◽  
High Degree ◽  
Mutant Cells ◽  
Transport Molecules ◽  
Degree Of Similarity ◽  
Sequence Similarities

Members of the kinesin superfamily are microtubule-based motor proteins that transport molecules/organelles along microtubules. We have identified similar internal motor kinesins, Kinesin-13A, from the cotton Gossypium hirsutum and Arabidopsis thaliana. Their motor domains share high degree of similarity with those of internal motor kinesins of animals and protists in the MCAK/Kinesin13 subfamily. However, no significant sequence similarities were detected in sequences outside the motor domain. In Arabidopsis plants carrying the T-DNA knockout kinesin-13a-1 and kinesin-13a-2 mutations at the Kinesin-13A locus, >70% leaf trichomes had four branches, whereas wild-type trichomes had three. Immunofluorescent results showed that AtKinesin-13A and GhKinesin-13A localized to entire Golgi stacks. In both wild-type and kinesin-13a mutant cells, the Golgi stacks were frequently associated with microtubules and with actin microfilaments. Aggregation/clustering of Golgi stacks was often observed in the kinesin-13a mutant trichomes and other epidermal cells. This suggested that the distribution of the Golgi apparatus in cell cortex might require microtubules and Kinesin-13A, and the organization of Golgi stacks could play a regulatory role in trichome morphogenesis. Our results also indicate that plant kinesins in the MCAK/Kinesin-13 subfamily have evolved to take on different tasks than their animal counterparts.

scholarly journals Characterization of a Thermoacidophilic l-Arabinose Isomerase from Alicyclobacillus acidocaldarius: Role of Lys-269 in pH Optimum

2005 ◽  
Vol 71 (12) ◽  
pp. 7888-7896 ◽  
Author(s):  
Sang-Jae Lee ◽  
Dong-Woo Lee ◽  
Eun-Ah Choe ◽  
Young-Ho Hong ◽  
Seong-Bo Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Bacillus Halodurans ◽  
Wild Type ◽  
Ph Optimum ◽  
Calculated Molecular Mass ◽  
Alicyclobacillus Acidocaldarius ◽  
Arabinose Isomerase

ABSTRACT The araA gene encoding l-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65°C under the assay conditions used, and it required divalent cations such as Mn2+, Co2+, and Mg2+ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent Km values of the recombinant AAAI for l-arabinose and d-galactose were 48.0 mM (V max, 35.5 U/mg) and 129 mM (V max, 7.5 U/mg), respectively, at pH 6 and 65°C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (k cat/Km ) of each mutant at different pHs was significantly affected by an increase or decrease in V max. From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.

Microfungi associated with Abies needles and Betula leaf litter in a subalpine coniferous forest

2007 ◽  
Vol 53 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Takashi Osono ◽  
Hiroshi Takeda
Keyword(s):  
Leaf Litter ◽  
Trichoderma Viride ◽  
Coniferous Forest ◽  
Subalpine Forest ◽  
Decomposition Processes ◽  
Volutella Ciliata ◽  
High Degree ◽  
The Impact ◽  
Degree Of Similarity

We investigated microfungal assemblages on leaf litter within a subalpine forest in central Japan and their variation with season, litter depth, and litter species. Microfungal assemblages were compared for Abies needles and Betula leaf litter collected from litter and fermentation layers of the forest floor during the growing season in spring, summer, and autumn. A total of 35 and 42 species were isolated from Abies needles and Betula leaf litter, respectively. The observed variation in microfungal assemblages was primarily attributable to seasonal differences. The frequencies of Trichoderma viride, Volutella ciliata, Mucor sp., and Umbelopsis ramanniana increased in summer, leading to a high degree of similarity of microfungal assemblages in different litter depths and litter species. Microfungal assemblages on Abies needles in spring and autumn and those on Betula leaves in spring were characterized by Trichoderma viride, V. ciliata, Thysanophora penicillioides, Trichoderma polysporum, and (or) Mortierella alpina. Microfungal assemblages on Betula leaves in autumn were characterized by the absence of these species and the occurrence of Cladosporium cladosporioides. The results were discussed with an emphasis on the role of microfungi in decomposition processes and the impact on fungi of predicted future increases in global temperature.Key words: birch, decomposition, diversity, fir, global warming.

scholarly journals Loss of Meningococcal PilU Delays Microcolony Formation and Attenuates VirulenceIn Vivo

2012 ◽  
Vol 80 (7) ◽  
pp. 2538-2547 ◽  
Author(s):  
Jens Eriksson ◽  
Olaspers Sara Eriksson ◽  
Ann-Beth Jonsson
Keyword(s):  
Dna Uptake ◽  
Wild Type ◽  
Content Type ◽  
Type Iv ◽  
Hexameric Atpase ◽  
Normal Human ◽  
Bacterial Aggregates ◽  
High Degree ◽  
Type Infection

ABSTRACTNeisseria meningitidisis a major cause of sepsis and bacterial meningitis worldwide. This bacterium expresses type IV pili (Tfp), which mediate important virulence traits such as the formation of bacterial aggregates, host cell adhesion, twitching motility, and DNA uptake. The meningococcal PilT protein is a hexameric ATPase that mediates pilus retraction. The PilU protein is produced from thepilT-pilUoperon and shares a high degree of homology with PilT. The function of PilT in Tfp biology has been studied extensively, whereas the role of PilU remains poorly understood. Here we show thatpilUmutants have delayed microcolony formation on host epithelial cells compared to the wild type, indicating that bacterium-bacterium interactions are affected. In normal human serum, thepilUmutant survived at a higher rate than that for wild-type bacteria. However, in a murine model of disease, mice infected with thepilTmutant demonstrated significantly reduced bacterial blood counts and survived at a higher rate than that for mice infected with the wild type. Infection of mice with thepilUmutant resulted in a trend of lower bacteremia, and still a significant increase in survival, than that of the wild type. In conclusion, these data suggest that PilU promotes timely microcolony formation and that both PilU and PilT are required for full bacterial virulence.

scholarly journals Role of the Haemophilus ducreyi Ton System in Internalization of Heme from Hemoglobin

1998 ◽  
Vol 66 (1) ◽  
pp. 151-160 ◽  
Author(s):  
Christopher Elkins ◽  
Pat A. Totten ◽  
Bonnie Olsen ◽  
Christopher E. Thomas
Keyword(s):  
Outer Membrane Proteins ◽  
Amino Acid Sequences ◽  
Haemophilus Ducreyi ◽  
Wild Type ◽  
E Coli ◽  
Allelic Replacement ◽  
Low Levels ◽  
High Concentrations ◽  
Isogenic Mutants

ABSTRACT By cloning into Escherichia coli and construction of isogenic mutants of Haemophilus ducreyi, we showed that the hemoglobin receptor (HgbA) is TonB dependent. An E. coli hemA tonB mutant expressing H. ducreyi hgbA grew on low levels of hemoglobin as a source of heme only when an intact H. ducreyi Ton system plasmid was present. In contrast, growth on heme by the E. coli hemA tonB mutant expressinghgbA was observed only at high concentrations of heme, was TonB independent, and demonstrated that H. ducreyi HgbA was not sufficient to function as a typical TonB-dependent heme receptor inE. coli. Allelic replacement of the wild-type H. ducreyi exbB, exbD, and tonB loci with the exbB, exbD, and tonB deletion resulted in an H. ducreyi isogenic mutant unable to utilize hemoglobin but able to utilize hemin at the same levels as the parent strain to fulfill its heme requirement. This finding confirms the TonB dependence of HgbA-mediated hemoglobin utilization and suggests that uptake of hemin in H. ducreyi is TonB independent. Additionally, the H. ducreyi Ton system mutant synthesized increased amounts of HgbA and other heme-regulated outer membrane proteins, consistent with derepression of these proteins due to lower intracellular heme and/or iron concentrations in the mutant. Sequencing of the Ton system genes revealed that the arrangement of the genes wasexbB exbD tonB. The proximity and structure of these genes suggested that they are transcribed as an operon. This arrangement, as well as the DNA and deduced amino acid sequences of these H. ducreyi genes, was most similar to those from other pasteurellae.

scholarly journals ssgA Is Essential for Sporulation ofStreptomyces coelicolor A3(2) and Affects Hyphal Development by Stimulating Septum Formation

2000 ◽  
Vol 182 (20) ◽  
pp. 5653-5662 ◽  
Author(s):  
Gilles P. van Wezel ◽  
Jannes van der Meulen ◽  
Shinichi Kawamoto ◽  
Ruud G. M. Luiten ◽  
Henk K. Koerten ◽  
...  
Keyword(s):  
Cell Division ◽  
Morphological Changes ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Wild Type ◽  
Septum Formation ◽  
Transmission Electron ◽  
In The Wild ◽  
Regulator Genes

ABSTRACT The role of ssgA in cell division and development of streptomycetes was analyzed. An ssgA null mutant ofStreptomyces coelicolor produced aerial hyphae but failed to sporulate, and ssgA can therefore be regarded as a novelwhi gene. In addition to the morphological changes, antibiotic production was also disturbed, with strongly reduced actinorhodin production. These defects could be complemented by plasmid-borne ssgA. In the wild-type strain, transcription of ssgA was induced by nutritional shift-down and was shown to be linked to that of the upstream-located gene ssgR, which belongs to the family of iclR-type transcriptional regulator genes. Analysis of mycelium harvested from liquid-grown cultures by transmission electron microscopy showed that septum formation had strongly increased in ssgA-overexpressing strains in comparison to wild-type S. coelicolor and that spore-like compartments were produced at high frequency. Furthermore, the hyphae were significantly wider and contained irregular and often extremely thick septa. These data underline the important role forssgA in Streptomyces cell division.

scholarly journals Robust Utilization of Phospholipase-Generated Metabolites, Glycerophosphodiesters, by Candida albicans: Role of the CaGit1 Permease

2011 ◽  
Vol 10 (12) ◽  
pp. 1618-1627 ◽  
Author(s):  
Andrew C. Bishop ◽  
Tao Sun ◽  
Mitchell E. Johnson ◽  
Vincent M. Bruno ◽  
Jana Patton-Vogt
Keyword(s):  
Candida Albicans ◽  
Insertional Mutagenesis ◽  
Single Gene ◽  
Open Reading Frames ◽  
Content Type ◽  
Phosphate Source ◽  
High Degree ◽  
Degree Of Similarity ◽  
Reading Frames

ABSTRACTGlycerophosphodiesters are the products of phospholipase-mediated deacylation of phospholipids. InSaccharomyces cerevisiae, a single gene,GIT1, encodes a permease responsible for importing glycerophosphodiesters, such as glycerophosphoinositol and glycerophosphocholine, into the cell. In contrast, theCandida albicansgenome contains four open reading frames (ORFs) with a high degree of similarity toS. cerevisiaeGIT1(ScGIT1) Here, we report thatC. albicansutilizes glycerophosphoinositol (GroPIns) and glycerophosphocholine (GroPCho) as sources of phosphate at both mildly acidic and physiological pHs. Insertional mutagenesis ofC. albicansGIT1(CaGIT1) (orf19.34), the ORF most similar toScGit1, abolished the ability of cells to use GroPIns as a phosphate source at acidic pH and to transport [3H]GroPIns at acidic and physiological pHs, while reintegration of aGIT1allele into the genome restored those functions. Several lines of evidence, including the detection of internal [3H]GroPIns, indicated that GroPIns is transported intact through CaGit1. GroPIns transport was shown to conform to Michaelis-Menten kinetics, with an apparentKmof 28 ± 6 μM. Notably, uptake of label from [3H]GroPCho was found to be roughly 50-fold greater than uptake of label from [3H]GroPIns and roughly 500-fold greater than the equivalent activity inS. cerevisiae.Insertional mutagenesis ofCaGIT1had no effect on the utilization of GroPCho as a phosphate source or on the uptake of label from [3H]GroPCho. Growth under low-phosphate conditions was shown to increase label uptake from both [3H]GroPIns and [3H]GroPCho. Screening of a transcription factor deletion set identifiedCaPHO4as required for the utilization of GroPIns, but not GroPCho, as a phosphate source.

scholarly journals Molecular Cloning and Spatiotemporal Expression of Prostaglandin F Synthase and Microsomal Prostaglandin E Synthase-1 in Porcine Endometrium

Endocrinology ◽  
2006 ◽  
Vol 147 (1) ◽  
pp. 210-221 ◽  
Author(s):  
Agnieszka Waclawik ◽  
Adolfo Rivero-Muller ◽  
Agnieszka Blitek ◽  
Monika M. Kaczmarek ◽  
Leon J. S. Brokken ◽  
...  
Keyword(s):  
Estrous Cycle ◽  
Early Pregnancy ◽  
Amino Acid Sequences ◽  
Prostaglandin E ◽  
Prostaglandin E Synthase ◽  
Spatiotemporal Expression ◽  
Supportive Role ◽  
Prostaglandin F ◽  
High Degree

Endometrial prostaglandins (PGs) and the PGE2/PGF2α ratio play an important role in regulating the estrous cycle and establishment of pregnancy. The enzymes downstream of cyclooxygenase-2 may determine the PGE2/PGF2α ratio in the porcine uterus. Thus, we have cloned porcine PGF synthase (PGFS) and microsomal PGE synthase-1 (mPGES-1) and characterized their expression in porcine endometrium during the estrous cycle and early pregnancy. PGFS and mPGES-1 amino acid sequences possessed a high degree (>67% and >77%, respectively) of identity with the other mammalian homologs. There was little modulation of mPGES-1 throughout the estrous cycle; however, PGFS expression was highly up-regulated in endometrium around the time of luteolysis. During early pregnancy, PGFS at the protein level showed a time-dependent increase (low on d 10–13, intermediate on d 14–23, and high on d 24–25). In pregnancy, expression of mPGES-1 was intermediate on d 10–11 and low on d 14–17 and then increased after d 22, reaching the maximum on d 24–25. Immunohistochemistry showed localization of PGFS and mPGES-1 proteins mainly in luminal and glandular epithelium. Concluding, the spatiotemporal expression of PGFS throughout the estrous cycle indicates an involvement of PGFS in regulating luteolysis in the pig. The comparison of endometrial PGFS and mPGES-1 expression on d 10–13 of the estrous cycle and pregnancy suggest a supportive role of these enzymes in determining the increase of uterine PGE2/PGF2α ratio during maternal recognition of pregnancy. Moreover, high expression of both PG synthases after initiation of implantation may indicate their significant role in placentation.

scholarly journals Ovine herpesvirus-2 glycoprotein B sequences from tissues of ruminant malignant catarrhal fever cases and healthy sheep are highly conserved

2001 ◽  
Vol 82 (11) ◽  
pp. 2785-2790 ◽  
Author(s):  
Magdalena Dunowska ◽  
Geoffrey J. Letchworth ◽  
James K. Collins ◽  
James C. DeMartini
Keyword(s):  
Severe Disease ◽  
Amino Acid Sequences ◽  
Glycoprotein B ◽  
Malignant Catarrhal Fever ◽  
Ruminant Species ◽  
Target Dna ◽  
Healthy Sheep ◽  
High Degree ◽  
Different Sources ◽  
Degree Of Similarity

Ovine herpesvirus-2 (OHV-2) infection has been associated with malignant catarrhal fever (MCF) in susceptible ruminants. In order to further investigate whether OHV-2 is an aetiological agent for sheep-associated (SA) MCF in cattle and bison, the entire sequences of OHV-2 glycoprotein B (gB) from different sources of viral DNA were compared. Target DNA was derived from tissues of bovine and bison cases of SA-MCF, from a lymphoblastoid cell line established from another bovine case of SA-MCF, and from a healthy sheep. The divergence between deduced amino acid sequences of OHV-2 gB ranged from 0·5 to 1·2%. The high degree of similarity between gB sequences from a healthy sheep and clinical cases of SA-MCF in cattle and bison suggests that OHV-2 is an ovine virus that is occasionally transmitted to other ruminant species, in which it can cause severe disease.

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