scholarly journals A screening system for carbon sources enhancing β-N-acetylglucosaminidase formation in Hypocrea atroviridis (Trichoderma atroviride)

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2003-2012 ◽  
Author(s):  
Verena Seidl ◽  
Irina S. Druzhinina ◽  
Christian P. Kubicek
Keyword(s):  
Screening Method ◽  
Carbon Sources ◽  
Transcript Abundance ◽  
Phenotype Microarray ◽  
Screening System ◽  
Trichoderma Atroviride ◽  
Wild Type ◽  
Activity Measurements ◽  
In The Wild ◽  
Biolog Phenotype Microarray

To identify carbon sources that trigger β-N-acetylglucosaminidase (NAGase) formation in Hypocrea atroviridis (anamorph Trichoderma atroviride), a screening system was designed that consists of a combination of Biolog Phenotype MicroArray plates, which contain 95 different carbon sources, and specific enzyme activity measurements using a chromogenic substrate. The results revealed growth-dependent kinetics of NAGase formation and it was shown that NAGase activities were enhanced on carbon sources sharing certain structural properties, especially on α-glucans (e.g. glycogen, dextrin and maltotriose) and oligosaccharides containing galactose. Enzyme activities were assessed in the wild-type and a H. atroviridis Δnag1 strain to investigate the influence of the two NAGases, Nag1 and Nag2, on total NAGase activity. Reduction of NAGase levels in the Δnag1 strain in comparison to the wild-type was strongly carbon-source and growth-phase dependent, indicating the distinct physiological roles of the two proteins. The transcript abundance of nag1 and nag2 was increased on carbon sources with elevated NAGase activity, indicating transcriptional regulation of these genes. The screening method for the identification of carbon sources that induce enzymes or a gene of interest, as presented in this paper, can be adapted for other purposes if appropriate enzyme or reporter assays are available.

scholarly journals Role of Acinetobacter baylyi Crc in Catabolite Repression of Enzymes for Aromatic Compound Catabolism

2009 ◽  
Vol 191 (8) ◽  
pp. 2834-2842 ◽  
Author(s):  
Tina Zimmermann ◽  
Tobias Sorg ◽  
Simone Yasmin Siehler ◽  
Ulrike Gerischer
Keyword(s):  
Catabolite Repression ◽  
Carbon Sources ◽  
Transcript Abundance ◽  
Growth Conditions ◽  
Wild Type ◽  
Acinetobacter Baylyi ◽  
Transcript Stability ◽  
Key Enzyme ◽  
In The Wild ◽  
First Time

ABSTRACT Here, we describe for the first time the Crc (catabolite repression control) protein from the soil bacterium Acinetobacter baylyi. Expression of A. baylyi crc varied according to the growth conditions. A strain with a disrupted crc gene showed the same growth as the wild type on a number of carbon sources. Carbon catabolite repression by acetate and succinate of protocatechuate 3,4-dioxygenase, the key enzyme of protocatechuate breakdown, was strongly reduced in the crc strain, whereas in the wild-type strain it underwent strong catabolite repression. This strong effect was not based on transcriptional regulation because the transcription pattern of the pca-qui operon (encoding protocatechuate 3,4-dioxygenase) did not reflect the derepression in the absence of Crc. pca-qui transcript abundance was slightly increased in the crc strain. Lack of Crc dramatically increased the mRNA stability of the pca-qui transcript (up to 14-fold), whereas two other transcripts (pobA and catA) remained unaffected. p-Hydroxybenzoate hydroxylase activity, encoded by pobA, was not significantly different in the absence of Crc, as protocatechuate 3,4-dioxygenase was. It is proposed that A. baylyi Crc is involved in the determination of the transcript stability of the pca-qui operon and thereby effects catabolite repression.

scholarly journals Mutations in Four Regulatory Genes Have Interrelated Effects on Heterocyst Maturation in Anabaena sp. Strain PCC 7120

2006 ◽  
Vol 188 (21) ◽  
pp. 7387-7395 ◽  
Author(s):  
Sigal Lechno-Yossef ◽  
Qing Fan ◽  
Shigeki Ehira ◽  
Naoki Sato ◽  
C. Peter Wolk
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Regulatory System ◽  
Transcript Abundance ◽  
Regulatory Genes ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
In The Wild ◽  
Pcc 7120

ABSTRACT Regulatory genes hepK, hepN, henR, and hepS are required for heterocyst maturation in Anabaena sp. strain PCC 7120. They presumptively encode two histidine kinases, a response regulator, and a serine/threonine kinase, respectively. To identify relationships between those genes, we compared global patterns of gene expression, at 14 h after nitrogen step-down, in corresponding mutants and in the wild-type strain. Heterocyst envelopes of mutants affected in any of those genes lack a homogeneous, polysaccharide layer. Those of a henR mutant also lack a glycolipid layer. patA, which encodes a positive effector of heterocyst differentiation, was up-regulated in all mutants except the hepK mutant, suggesting that patA expression may be inhibited by products related to heterocyst development. hepS and hepK were up-regulated if mutated and so appear to be negatively autoregulated. HepS and HenR regulated a common set of genes and so appear to belong to one regulatory system. Some nontranscriptional mechanism may account for the observation that henR mutants lack, and hepS mutants possess, a glycolipid layer, even though both mutations down-regulated genes involved in formation of the glycolipid layer. HepK and HepN also affected transcription of a common set of genes and therefore appear to share a regulatory pathway. However, the transcript abundance of other genes differed very significantly from expression in the wild-type strain in either the hepK or hepN mutant while differing very little from wild-type expression in the other of those two mutants. Therefore, hepK and hepN appear to participate also in separate pathways.

scholarly journals Identification and Characterization of the Dicarboxylate Uptake System DccT in Corynebacterium glutamicum

2008 ◽  
Vol 190 (19) ◽  
pp. 6458-6466 ◽  
Author(s):  
Jung-Won Youn ◽  
Elena Jolkver ◽  
Reinhard Krämer ◽  
Kay Marin ◽  
Volker F. Wendisch
Keyword(s):  
Corynebacterium Glutamicum ◽  
Tricarboxylic Acid Cycle ◽  
Carbon Sources ◽  
Mrna Levels ◽  
Uptake System ◽  
Dependent Manner ◽  
Wild Type ◽  
Prolonged Incubation ◽  
In The Wild ◽  
Biochemical Analyses

ABSTRACT Many bacteria can utilize C4-carboxylates as carbon and energy sources. However, Corynebacterium glutamicum ATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, and l-malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, and l-malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was named dccT, in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region of dccT was responsible for increased expression. The overexpression of dccT was sufficient to enable the C. glutamicum wild type to grow on succinate, fumarate, and l-malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na+ symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate, l-malate, and likely also oxaloacetate in a sodium-dependent manner.

scholarly journals Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

2000 ◽  
Vol 182 (9) ◽  
pp. 2582-2590 ◽  
Author(s):  
Valérie Dossonnet ◽  
Vicente Monedero ◽  
Monique Zagorec ◽  
Anne Galinier ◽  
Gaspar Pérez-Martínez ◽  
...  
Keyword(s):  
Catabolite Repression ◽  
Type Strain ◽  
Lactobacillus Casei ◽  
Wild Type Strain ◽  
Carbon Sources ◽  
Lag Phase ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Inducer Exclusion ◽  
In The Wild

ABSTRACT We have cloned and sequenced the Lactobacillus casei hprK gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant L. casei HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When hprK was inactivated, carbon catabolite repression of N-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the hprK mutant. However, inducer expulsion ofmethyl β-d-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative inptsH mutants altered at Ser-46 of HPr and thehprK mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.

Phenotype microarray screening of carbon sources used by Vibrio cholerae identifies genes regulated by the cAMP receptor protein

2013 ◽  
Vol 59 (7) ◽  
pp. 472-478 ◽  
Author(s):  
Baoli Chen ◽  
Weili Liang ◽  
Rui Wu ◽  
Pu Liang ◽  
Biao Kan
Keyword(s):  
Carbon Source ◽  
Vibrio Cholerae ◽  
High Throughput Screening ◽  
Carbon Sources ◽  
Experimental Studies ◽  
Receptor Protein ◽  
Phenotype Microarray ◽  
Wild Type ◽  
Rt Pcr ◽  
Mutant Strains

The cyclic AMP receptor protein (CRP) regulates genes involved in carbon source metabolism, iron uptake, and virulence in bacteria. Identifying the carbon sources utilized by bacteria that are regulated by CRP will help elucidate the CRP regulation cascade and associated responses to environmental stimuli. CRP-dependent regulation of carbon source metabolism in Vibrio cholerae is not thoroughly understood. To identify the candidate carbon sources utilized by V. cholerae that are affected by CRP, we used high-throughput screening to compare the metabolic differences between wild-type and CRP mutant strains of V. cholerae O1 El Tor. Phenotype microarray was used for primary screening of the wild-type and mutant strains, followed by minimal media growth assays and quantitative RT-PCR to validate the candidate carbon sources. In total, 24 carbon sources were subject to CRP regulation, 11 of which have not been previously reported in bacteria. The genes known to be involved in the metabolism of 4 of the carbon sources identified were verified by quantitative RT-PCR. In addition, gel shift experiments showed that CRP bound directly to VCA0053 and VC0391 promoters. Overall, this comprehensive analysis of CRP-mediated catabolite control in V. cholerae has identified new candidate carbon sources for in-depth experimental studies.

Changes in protein and gene expression during induction of the CO2-concentrating mechanism in wild-type and mutant Chlamydomonas

1991 ◽  
Vol 69 (5) ◽  
pp. 1008-1016 ◽  
Author(s):  
Martin H. Spalding ◽  
Thomas L. Winder ◽  
James C. Anderson ◽  
Anne M. Geraghty ◽  
Laura F. Marek
Keyword(s):  
Transcript Abundance ◽  
Small Subunit ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
Phosphoglycolate Phosphatase ◽  
Concentrating Mechanism ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
In The Wild ◽  
Inorganic Carbon Transport

Several changes occur in wild-type Chlamydomonas reinhardtii upon exposure to limiting CO2, including induction of several polypeptides. Polypeptide induction was previously shown to correlate with appearance of the active CO2-concentrating mechanism (CCM) of this alga. In this paper induction of polypeptides by limiting CO2 was investigated in mutants with lesions in the CCM. Mutants with lesions in the ca-1 and pmp-1 loci exhibited alterations in polypeptide induction, but it was concluded that the alterations probably do not represent their primary genetic lesions. Other changes that occur in this alga in response to limiting CO2 were also investigated. Based on a lack of significant change in the transcript abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunit genes in the wild type, it was concluded that the previously reported transient decline in synthesis of both subunits is controlled at the translational level. A transient increase in the activity of the photorespiratory enzyme phosphoglycolate phosphatase was observed in the wild type but not in a mutant, cia-5, that lacks induction of the CCM. In addition, changes in expression of genes encoding periplasmic carbonic anhydrase, a 36-kDa membrane-associated protein and a chlorophyll-binding protein occurred in the wild type but not in cia-5 in response to limiting CO2. The absence of these changes in cia-5 was attributed to a lack of either the signal itself or transduction of the signal responsible for adaptation to limiting CO2, which led to speculation that a larger range of responses are regulated by the same signal than was previously recognized. Key words: photosynthesis, photorespiration, algae, inorganic carbon transport, transcription, translation.

Freeze-substituted fungal cells of Nectria haematococca: A comparison of the macroconidial walls of the adhesion-competent wild type with an adhesion-reduced mutant

1990 ◽  
Vol 48 (3) ◽  
pp. 688-689
Author(s):  
Thecan Caesar-Ton That ◽  
Lynn Epstein
Keyword(s):  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Wild Type ◽  
Nectria Haematococca ◽  
Fruit Extract ◽  
Dialysis Tubing ◽  
Tube Emergence ◽  
Contrast Microscopy ◽  
In The Wild ◽  
Freeze Damage

Nectria haematococca mating population I (anamorph, Fusarium solani) macroconidia attach to its host (squash) and non-host surfaces prior to germ tube emergence. The macroconidia become adhesive after a brief period of protein synthesis. Recently, Hickman et al. (1989) isolated N. haematococca adhesion-reduced mutants. Using freeze substitution, we compared the development of the macroconidial wall in the wild type in comparison to one of the mutants, LEI.Macroconidia were harvested at 1C, washed by centrifugation, resuspended in a dilute zucchini fruit extract and incubated from 0 - 5 h. During the incubation period, wild type macroconidia attached to uncoated dialysis tubing. Mutant macroconidia did not attach and were collected on poly-L-lysine coated dialysis tubing just prior to freezing. Conidia on the tubing were frozen in liquid propane at 191 - 193C, substituted in acetone with 2% OsO4 and 0.05% uranyl acetate, washed with acetone, and flat-embedded in Epon-Araldite. Using phase contrast microscopy at 1000X, cells without freeze damage were selected, remounted, sectioned and post-stained sequentially with 1% Ba(MnO4)2 2% uranyl acetate and Reynold’s lead citrate. At least 30 cells/treatment were examined.

Application of Phenotype Microarray for Profiling Carbon Sources Utilization between Biofilm and Non-Biofilm of Pseudomonas aeruginosa from Clinical Isolates

2020 ◽  
Vol 21 (14) ◽  
pp. 1539-1550
Author(s):  
Nur S. Ismail ◽  
Suresh K. Subbiah ◽  
Niazlin M. Taib
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Carbon Sources ◽  
Anaerobic Respiration ◽  
Regulatory System ◽  
Negative Control ◽  
High Morbidity ◽  
Metabolic Profiles ◽  
Phenotype Microarray ◽  
Diverse Environment ◽  
Phenotype Microarrays

Background: This is the fastest work in obtaining the metabolic profiles of Pseudomonas aeruginosa in order to combat the infection diseases which leads to high morbidity and mortality rates. Pseudomonas aeruginosa is a high versatility of gram-negative bacteria that can undergo aerobic and anaerobic respiration. Capabilities in deploying different carbon sources, energy metabolism and regulatory system, ensure the survival of this microorganism in the diverse environment condition. Determination of differences in carbon sources utilization among biofilm and non-biofilm of Pseudomonas aeruginosa provides a platform in understanding the metabolic activity of the microorganism. Methods: The study was carried out from September 2017 to February 2019. Four archive isolates forming strong and intermediate biofilm and non-biofilms producer were subcultured from archive isolates. ATCC 27853 P. aeruginosa was used as a negative control or non-biofilm producing microorganism. Biofilm formation was confirmed by Crystal Violet Assay (CVA) and Congo Red Agar (CRA). Metabolic profiles of the biofilm and non-biofilms isolates were determined by phenotype microarrays (Biolog Omnilog). Results and Discussion: In this study, Pseudomonas aeruginosa biofilm isolates utilized uridine, L-threonine and L-serine while non-biofilm utilized adenosine, inosine, monomethyl, sorbic acid and succinamic acid. Conclusion: The outcome of this result will be used for future studies to improve detection or inhibit the growth of P. aeruginosa biofilm and non-biofilm respectively.

scholarly journals Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

2007 ◽  
Vol 28 (3) ◽  
pp. 897-906 ◽  
Author(s):  
Thomas J. Pohl ◽  
Jac A. Nickoloff
Keyword(s):  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Homology Search ◽  
Wild Type ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
In The Wild ◽  
Break Induced Replication

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

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