scholarly journals C-5(6) Sterol Desaturase from Tetrahymena thermophila: Gene Identification and Knockout, Sequence Analysis, and Comparison to Other C-5(6) Sterol Desaturases

2009 ◽  
Vol 8 (8) ◽  
pp. 1287-1297 ◽  
Author(s):  
Alejandro D. Nusblat ◽  
Sebastián R. Najle ◽  
Mariela L. Tomazic ◽  
Antonio D. Uttaro ◽  
Clara B. Nudel
Keyword(s):  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Tetrahymena Thermophila ◽  
Gene Replacement ◽  
Gene Identification ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Gene Coding ◽  
Transmembrane Regions

ABSTRACT The gene coding for a C-5(6) sterol desaturase in Tetrahymena thermophila, DES5A, has been identified by the knockout of the TTHERM_01194720 sequence. Macronucleus transformation was achieved by biolistic bombardment and gene replacement through phenotypic assortment, using paromomycin as the selective agent. A knockout cell line (KO270) showed a phenotype consistent with that of the DES5A deletion mutant. KO270 converted only 6% of the added sterol into the C-5 unsaturated derivative, while the wild type accumulated 10-fold larger amounts under similar conditions. The decreased desaturation activity is specific for the C-5(6) position of lathosterol and cholestanol; other desaturations, namely C-7(8) and C-22(23), were not affected. Analysis by reverse transcription-PCR reveals that DES5A is transcribed both in the presence and absence of cholestanol in wild-type cells, whereas the transcribed gene was not detected in KO270. The growth of KO270 was undistinguishable from that of the wild-type strain. Des5Ap resembles known C-5(6) sterol desaturases, displaying the three typical histidine motifs, four hydrophobic transmembrane regions, and two other highly conserved domains of unknown function. A phylogenetic analysis placed T. thermophila's enzyme and Paramecium orthologues in a cluster together with functionally characterized C-5 sterol desaturases from vertebrates, fungi, and plants, although in a different branch.

scholarly journals SdiA Aids Enterohemorrhagic Escherichia coli Carriage by Cattle Fed a Forage or Grain Diet

2013 ◽  
Vol 81 (9) ◽  
pp. 3472-3478 ◽  
Author(s):  
Haiqing Sheng ◽  
Y. N. Nguyen ◽  
Carolyn J. Hovde ◽  
Vanessa Sperandio
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Rumen Fluid ◽  
Acid Resistance ◽  
Enterohemorrhagic Escherichia Coli ◽  
Dependent Manner ◽  
Bacterial Survival ◽  
Wild Type

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) causes hemorrhagic colitis and life-threatening complications. The main reservoirs for EHEC are healthy ruminants. We reported that SdiA senses acyl homoserine lactones (AHLs) in the bovine rumen to activate expression of the glutamate acid resistance (gad) genes priming EHEC's acid resistance before they pass into the acidic abomasum. Conversely, SdiA represses expression of the locus of enterocyte effacement (LEE) genes, whose expression is not required for bacterial survival in the rumen but is necessary for efficient colonization at the rectoanal junction (RAJ) mucosa. Our previous studies show that SdiA-dependent regulation was necessary for efficient EHEC colonization of cattle fed a grain diet. Here, we compared the SdiA role in EHEC colonization of cattle fed a forage hay diet. We detected AHLs in the rumen of cattle fed a hay diet, and these AHLs activatedgadgene expression in an SdiA-dependent manner. The rumen fluid and fecal samples from hay-fed cattle were near neutrality, while the same digesta samples from grain-fed animals were acidic. Cattle fed either grain or hay and challenged with EHEC orally carried the bacteria similarly. EHEC was cleared from the rumen within days and from the RAJ mucosa after approximately one month. In competition trials, where animals were challenged with both wild-type and SdiA deletion mutant bacteria, diet did not affect the outcome that the wild-type strain was better able to persist and colonize. However, the wild-type strain had a greater advantage over the SdiA deletion mutant at the RAJ mucosa among cattle fed the grain diet.

Peptides’ involvement in Pseudomonas putida biofilm formation

2020 ◽  
Author(s):  
Riho Teras ◽  
Hanna Ainelo ◽  
Marge Puhm
Keyword(s):  
Pseudomonas Putida ◽  
Mutant Strain ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Positive Impact ◽  
Proteinase K ◽  
Wild Type ◽  
Positive Effect ◽  
Positively Charged

<p>Pseudomonas putida rapidly forms a biofilm, after which its biomass usually disperses to half its initial amount. We have observed different biofilm dynamics of P. putida in a complex medium LB and a minimal medium M9+glc+CAA and inquired about the importance of extracellular factors for the formation of P. putida biofilm.</p> <p>The proteinaceous component of LB increases the biomass of P. putida biofilm. Supplementation of M9 with tryptone but not CAA increased the biofilm biomass. Proteinase K treatment of LB medium reduced the biomass of P. putida biofilm. At the same time, growth rate or maximum OD of planktic bacteria in used media did not correlate with biofilm biomass of the same media. Thus, peptides appeared to have a positive effect on the biofilm as an extracellular factor and not as a source of C and N.</p> <p>We replaced tryptone in M9 medium with positively charged poly-L-lysine (MW. 1000-5000 Da), negatively charged poly-L-glutaminic acid (MW. 1500-5500 Da) or neutral poly-LD-alanine (MW. 3000-7000). Poly-lysine and poly-glutamic acid had a slight positive effect on the biomass of P. putida wild type strain PSm biofilm and poly-alanine did not affect the biofilm.</p> <p>We have previously shown that overexpression of fis in P. putida strain F15 increases biofilm biomass by increasing the lapA expression, the main adhesin gene of biofilm. Using media similar to that used for the wild-type strain for strain F15, we ascertained that only poly-lysine out of these three polypeptides restored the positive effect of fis-overexpression on the biofilm biomass. At the same time, the positive impact of fis-overexpression was absent in lapA deletion mutant strain, but not in lapF deletion mutant strain.</p> <p>In conclusion, the formation of P. putida biofilm depends on polypeptides in the environment. The enhancing effect of positively charged polypeptides appears to be evident in the presence of LapA, a key factor for P. putida biofilm.</p>

scholarly journals Complementation of the yeast deletion mutant DeltaNCE103 by members of the beta class of carbonic anhydrases is dependent on carbonic anhydrase activity rather than on antioxidant activity

2004 ◽  
Vol 379 (3) ◽  
pp. 609-615 ◽  
Author(s):  
Daniel CLARK ◽  
Roger S. ROWLETT ◽  
John R. COLEMAN ◽  
Daniel F. KLESSIG
Keyword(s):  
Antioxidant Activity ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Carbonic Anhydrase Activity ◽  
Site Directed Mutagenesis ◽  
Carbonic Anhydrases ◽  
Wild Type ◽  
Tobacco Chloroplast ◽  
Low Levels

In recent years, members of the β class of CAs (carbonic anhydrases) have been shown to complement ΔNCE103, a yeast strain unable to grow under aerobic conditions. The activity required for complementation of ΔNCE103 by tobacco chloroplast CA was studied by site-directed mutagenesis. E196A (Glu196→Ala), a mutated tobacco CA with low levels of CA activity, complemented ΔNCE103. To determine whether restoration of ΔNCE103 was due to residual levels of CA activity or whether it was related to previously proposed antioxidant activity of CAs [Götz, Gnann and Zimmermann (1999) Yeast 15, 855–864], additional complementation analysis was performed using human CAII, an α CA structurally unrelated to the β class of CAs to which the tobacco protein belongs. Human CAII complemented ΔNCE103, strongly arguing that CA activity is responsible for the complementation of ΔNCE103. Consistent with this conclusion, recombinant NCE103 synthesized in Escherichia coli shows CA activity, and ΔNCE103 expressing the tobacco chloroplast CA exhibits the same sensitivity to H2O2 as the wild-type strain.

scholarly journals Shigella flexneri IpaH7.8 Facilitates Escape of Virulent Bacteria from the Endocytic Vacuoles of Mouse and Human Macrophages

2000 ◽  
Vol 68 (6) ◽  
pp. 3608-3619 ◽  
Author(s):  
Carmen M. Fernandez-Prada ◽  
David L. Hoover ◽  
Ben D. Tall ◽  
Antoinette B. Hartman ◽  
June Kopelowitz ◽  
...  
Keyword(s):  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Shigella Flexneri ◽  
Human Monocyte ◽  
Avirulent Strain ◽  
Human Monocytes ◽  
Wild Type ◽  
Distribution Profile ◽  
J774 Cells

ABSTRACT The behavior of Shigella flexneri ipaH mutants was studied in human monocyte-derived macrophages (HMDM), in 1-day-old human monocytes, and in J774 mouse macrophage cell line. In HMDM, strain pWR700, an ipaH 7.8 deletion mutant ofS. flexneri 2a strain 2457T, behaved like the wild-type strain 2457T. This strain caused rapid host cell death by oncosis, and few bacterial CFU were recovered after incubation in the presence of gentamicin as previously described for 2457T-infected HMDM. However, analysis of bacterial compartmentalization within endocytic vacuoles with gentamicin and chloroquine indicated that more pWR700 than 2457T was present within the endocytic vacuoles of HMDM, suggesting thatipaH 7.8 deletion mutant transited more slowly from the vacuoles to the cytoplasm. In contrast to findings with HMDM, CFU recovered from pWR700-infected mouse J774 cells were 2 to 3 logs higher than CFU from 2457T-infected J774 cells. These values exceeded CFU recovered after infection of J774 cells with plasmid-cured avirulent strain M4243A1. Incubation with gentamicin and chloroquine clearly showed that pWR700 within J774 cells was mostly present within the endocytic vacuoles. This distribution pattern was similar to that seen with M4243A1 and contrasted with the pattern seen with 2457T. Complementation of pWR700 with a recombinant clone expressingipaH 7.8 restored the intracellular distribution of bacteria to that seen with the wild-type strain. Strains with deletions in ipaH 4.5 oripaH 9.8, however, behaved like 2457T in both HMDM and J774 cells. The distribution profile of pWR700 in 1-day-old monocytes was similar to that seen in J774 cells. Like infected J774 cells, 1-day-old human monocytes demonstrated apoptosis upon infection with virulent Shigella. These results suggest that a role of the ipaH 7.8 gene product is to facilitate the escape of the virulent bacteria from the phagocytic vacuole of monocytes and macrophages.

scholarly journals Functional Characterization of Calcineurin-Responsive Transcription Factors Fg01341 and Fg01350 in Fusarium graminearum

2020 ◽  
Vol 11 ◽  
Author(s):  
Xiangxiang Zhang ◽  
Shulin Cao ◽  
Wei Li ◽  
Haiyan Sun ◽  
Yuanyu Deng ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Functional Characterization ◽  
Hyphal Growth ◽  
Basic Medium ◽  
Dependent Manner ◽  
Wild Type ◽  
Sexual Production

Ca2 +/calmodulin-dependent phosphatase calcineurin is one of the important regulators of intracellular calcium homeostasis and has been investigated extensively in Saccharomyces cerevisiae. However, only a few reports have explored the function of the Crz1 homolog in filamentous fungi, especially in Fusarium graminearum. In this study, we identified Fg01341 as a potential ortholog of yeast Crz1. Fg01341 could interact with calcineurin and initiate nuclear transport in a calcineurin-dependent manner. The ΔFg01341 mutant exhibited normal hyphal growth on basic medium and conidia formation, but sexual reproduction was partially blocked. Pathogenicity assays showed that the virulence of the ΔFg01341 mutant in flowering wheat heads and corn silks dramatically decreased and was thus consistent with the reduction in deoxynivalenol production. Unexpectedly, the sensitivity to osmotic stress of the deletion mutant and that of the wild-type strain did not present any differences. The deletion mutant showed higher sensitivity to tebuconazole than the wild-type strain. Results also showed that the transcription factor Fg01350 might be the calcineurin target and was independent of Crz1. Furthermore, ΔFg01350 showed defects in hyphal growth, sexual production, virulence, and deoxynivalenol production. Collectively, the results indicate that these two proteins functionally redundant and that the calcineurin–Crz1-independent pathway is particularly important in F. graminearum.

scholarly journals Characterization of emeA, anorA Homolog and Multidrug Resistance Efflux Pump, inEnterococcus faecalis

2001 ◽  
Vol 45 (12) ◽  
pp. 3574-3579 ◽  
Author(s):  
Brandie M. Jonas ◽  
Barbara E. Murray ◽  
George M. Weinstock
Keyword(s):  
Drug Resistance ◽  
Multidrug Resistance ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Wild Type ◽  
Type Gene ◽  
Encoding Genes

ABSTRACT We hypothesized that multidrug resistance efflux pumps (MDRs) may be contributing to the drug resistance of enterococci. We recently identified potential MDR-encoding genes in the Enterococcus faecalis V583 genome. Among the putative MDRs, we found a gene that encodes a NorA homolog and have characterized this enterococcal MDR in the present study. A mutant from which the enterococcal NorA homolog has been deleted has reduced resistance to several NorA substrates. Complementation of the deletion mutant with the wild-type gene verified the involvement of this enterococcal gene in resistance to ethidium bromide (EtBr) and norfloxacin. Known MDR inhibitors (reserpine, lansoprazole, and verapamil) inhibit the efflux of EtBr and norfloxacin in wild-type strain OG1RF. A fluorescence assay with EtBr allowed us to quantitate the efflux capability of the enterococcal NorA pump. On the basis of these results, we have named this enterococcal gene emeA (enterococcal multidrug resistance efflux).

scholarly journals Identification and Characterization of hmr19 Gene Encoding a Multidrug Resistance Efflux Protein from Streptomyces hygroscopicus subsp. yingchengensis Strain 10–22

2004 ◽  
Vol 36 (8) ◽  
pp. 519-528 ◽  
Author(s):  
Lei Qin ◽  
Heng-An Wang ◽  
Zhong-Qin Wu ◽  
Xiao-Feng Zhang ◽  
Mei-Lei Jin ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sequence Similarity ◽  
Gene Replacement ◽  
Major Facilitator Superfamily ◽  
Streptomyces Hygroscopicus ◽  
Wild Type ◽  
Gene Encoding ◽  
Major Facilitator

Abstract The hmr19 gene was cloned from Streptomyces hygroscopicus subsp. yingchengensis strain 10–22, a bacterium strain producing agricultural antibiotics. Sequence similarity comparison indicates that hmr19 gene may encode a predicted protein with 14 putative transmembrane α-helical spanners, belonging to the drug:H+ antiporter-2 family of the major facilitator superfamily. The expression of hmr19 in the mycelium of strain 10-22 was detected by Western blotting analysis. Gene replacement technology was employed to construct an hmr19 disruption mutant. The growth inhibition test against different antibiotics indicated that the mutant strain was 5–20 fold more susceptible to tetracycline, vancomycin and mitomycin C than the parental wild type strain. The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-22. While with the addition of an energy uncoupler, carbonyl cyanide m-chlorophenylhydrazone, the characteristics of the accumulation of [3H]tetracycline in these two strains were almost the same. It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.

scholarly journals Telithromycin Resistance in Streptococcus pneumoniae Is Conferred by a Deletion in the Leader Sequence of erm(B) That Increases rRNA Methylation

2007 ◽  
Vol 52 (2) ◽  
pp. 435-440 ◽  
Author(s):  
Nicole Wolter ◽  
Anthony M. Smith ◽  
David J. Farrell ◽  
John Blackman Northwood ◽  
Stephen Douthwaite ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Leader Sequence ◽  
Lag Phase ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Resistant Phenotype ◽  
Type Gene ◽  
Rrna Methylation

ABSTRACT A telithromycin-resistant clinical isolate of Streptococcus pneumoniae (strain P1501016) has been found to contain a version of erm(B) that is altered by a 136-bp deletion in the leader sequence. By allele replacement mutagenesis, a second strain of S. pneumoniae (PC13) with a wild-type erm(B) gene was transformed to the telithromycin-resistant phenotype by introduction of the mutant erm(B) gene. Whereas the wild-type PC13 strain showed slight telithromycin resistance only after induction by erythromycin (telithromycin MIC increased from 0.06 to 0.5 μg/ml), the transformed PC13 strain is constitutively resistant (MIC of 16 μg/ml). Expression of erm(B) was quantified by real-time reverse transcription-PCR in the presence of erythromycin or telithromycin; erm(B) expression was significantly higher in the transformed PC13 strain than the wild-type strain. Furthermore, the transformed strain had significantly higher levels of ribosomal methylation in the absence as well as in the presence of the antibiotics. Growth studies showed that the transformed PC13 strain had a shorter lag phase than the wild-type strain in the presence of erythromycin. Telithromycin resistance is conclusively shown to be conferred by the mutant erm(B) gene that is expressed at a constitutively higher level than the inducible wild-type gene. Elevated erm(B) expression results in a higher level of rRNA methylation that presumably hinders telithromycin binding to the ribosome.

scholarly journals GzSNF1 Is Required for Normal Sexual and Asexual Development in the Ascomycete Gibberella zeae

2008 ◽  
Vol 8 (1) ◽  
pp. 116-127 ◽  
Author(s):  
Seung-Ho Lee ◽  
Jungkwan Lee ◽  
Seunghoon Lee ◽  
Eun-Hee Park ◽  
Ki-Woo Kim ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Catalytic Domain ◽  
Carbon Sources ◽  
Gene Replacement ◽  
Gibberella Zeae ◽  
Asexual Development ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Genes Encoding

ABSTRACT The sucrose nonfermenting 1 (SNF1) protein kinase of yeast plays a central role in the transcription of glucose-repressible genes in response to glucose starvation. In this study, we deleted an ortholog of SNF1 from Gibberella zeae to characterize its functions by using a gene replacement strategy. The mycelial growth of deletion mutants (ΔGzSNF1) was reduced by 21 to 74% on diverse carbon sources. The virulence of ΔGzSNF1 mutants on barley decreased, and the expression of genes encoding cell-wall-degrading enzymes was reduced. The most distinct phenotypic changes were in sexual and asexual development. ΔGzSNF1 mutants produced 30% fewer perithecia, which matured more slowly, and asci that contained one to eight abnormally shaped ascospores. Mutants in which only the GzSNF1 catalytic domain was deleted had the same phenotype changes as the ΔGzSNF1 strains, but the phenotype was less extreme in the mutants with the regulatory domain deleted. In outcrosses between the ΔGzSNF1 mutants, each perithecium contained ∼70% of the abnormal ascospores, and ∼50% of the asci showed unexpected segregation patterns in a single locus tested. The asexual spores of the ΔGzSNF1 mutants were shorter and had fewer septa than those of the wild-type strain. The germination and nucleation of both ascospores and conidia were delayed in ΔGzSNF1 mutants in comparison with those of the wild-type strain. GzSNF1 expression and localization depended on the developmental stage of the fungus. These results suggest that GzSNF1 is critical for normal sexual and asexual development in addition to virulence and the utilization of alternative carbon sources.

Deletion of HAPS_2096 Increases Sensitivity to Cecropin B in Haemophilus parasuis

2015 ◽  
Vol 25 (4) ◽  
pp. 284-291
Author(s):  
Fanjie Chen ◽  
Han Hu ◽  
Zhonghua Li ◽  
Jiacheng Huang ◽  
Xuwang Cai ◽  
...  
Keyword(s):  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Sublethal Concentration ◽  
Haemophilus Parasuis ◽  
Wild Type ◽  
Microscopy Observation ◽  
Cecropin B ◽  
In The Wild ◽  
Substrate Binding Protein

Cecropin B (CB) is a very effective natural antimicrobial peptide that has shown great potential for future antimicrobial drug development. HAPS_2096 is a <i>Haemophilus parasuis</i> gene that encodes the periplasmic substrate-binding protein of an ATP-binding cassette-type amino acid transporter. In this research, we constructed and verified an HAPS_2096 deletion mutant and a complementary HAPS_2096 mutant of <i>H. parasuis</i> JS0135. A bactericidal assay revealed that the HAPS_2096 deletion mutant was significantly more sensitive than the wild-type strain to 0.25-0.5 µg/ml CB. However, the gene complementation alleviated the CB sensitivity of the mutant. Immunoelectron microscopy observation following a 30-min treatment with a sublethal concentration of CB (0.25 μg/ml) revealed more extensive morphological damage in the mutant strain than in the wild-type strain. Hence, our results suggest that the HAPS_2096 gene contributes to <i>H. parasuis</i> resistance to CB.

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