scholarly journals Controlling Heterogeneity and Increasing Titer from Riboswitch-RegulatedBacillus subtilisSpores for Time-Delayed Protein Expression Applications

2019 ◽  
Author(s):  
Denis Tamiev ◽  
Alyssa Lantz ◽  
Grace Vezeau ◽  
Howard Salis ◽  
Nigel F. Reuel
Keyword(s):  
Wild Type Strain ◽  
Point Mutations ◽  
Growth Curves ◽  
Oral Vaccines ◽  
Wild Type ◽  
Suspended Cell ◽  
Reporter Protein ◽  
Kinetic Rate ◽  
Fluorescent Reporter ◽  
Clonal Population

AbstractSporulated cells have potential as time-delayed expression chassis of proteins for applications such as ‘on-demand’ biologics production, whole cell biosensors, or oral vaccines. However, the desired attributes of high expression rates and low product variances are difficult to maintain from germinated spores. In this work we study the effect of an integratingvs.theta replicating plasmid in a wild-typeBacillus subtilisand two PolY mutants. The cells were engineered to produce a fluorescent reporter protein (RFP) under the control of a riboswitch activated by theophylline. This allowed for greater sensitivity to point mutations. The fluorescence and cell growth curves were fit with a custom kinetic model and a peak kinetic rate (LKPmax) was extracted for each clonal population (n = 30 for all cell, vector, and growth combinations). Plasmid based expression yields higher (8.7x) expression rates due to an increased copy number of the expression cassette (10x over integrated). The variance of LKPmaxvalues increased 2.07x after sporulation for the wild type strain. This increase in variance from sporulation is very similar to what is observed with UV exposure. This effect can be partially mitigated by the use of PolY knockouts observed in suspended cell growths and adherent biofilms.

scholarly journals Identification of a UPC2 Homolog inSaccharomyces cerevisiae and Its Involvement in Aerobic Sterol Uptake

2001 ◽  
Vol 183 (3) ◽  
pp. 830-834 ◽  
Author(s):  
Kevin V. Shianna ◽  
W. David Dotson ◽  
Shirley Tove ◽  
Leo W. Parks
Keyword(s):  
Point Mutation ◽  
Wild Type Strain ◽  
Point Mutations ◽  
Transcriptional Activator ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Reading Frame ◽  
Sterol Uptake ◽  
Major Sterol ◽  
High Level

ABSTRACT Saccharomyces cerevisiae normally will not take up sterols from the environment under aerobic conditions. A specific mutant, upc2-1, of the predicted transcriptional activator UPC2 (YDR213w) has been recognized as a strain that allows a high level of aerobic sterol uptake. Another predicted transcriptional activator, the YLR228c gene product, is highly homologous to Upc2p. In fact, at the carboxy terminus 130 of the last 139 amino acids are similar between the two proteins. Since these proteins are very similar, the effect of mutations in the YLR228c open reading frame (ORF) was compared with like alterations in UPC2. First, the YLR228c ORF was insertionally inactivated and crossed with various UPC2constructs. Deletion of YLR228c and UPC2 in combination resulted in nonviability, suggesting that the two proteins have some essential overlapping function. The upc2-1point mutation responsible for aerobic sterol uptake was duplicated in the homologous carboxy region of the YLR228c ORF using site-directed mutagenesis. This mutation on a high-copy vector resulted in an increase in sterol uptake compared to an isogenic wild-type strain. The combination of both point mutations resulted in the greatest level of aerobic sterol uptake. When the YLR228c point mutation was expressed from a low-copy vector there was little if any effect on sterol uptake. Gas chromatographic analysis of the nonsaponifiable fractions of the various strains showed that the major sterol for all YLR228c andUPC2 combinations was ergosterol, the consensus yeast sterol.

Pathogenic and virulence characterization of colonial mutants of Nocardia asteroides GUH-2

1983 ◽  
Vol 29 (9) ◽  
pp. 1126-1135 ◽  
Author(s):  
Cynthia A. Vistica ◽  
Blaine L. Beaman
Keyword(s):  
Fatty Acid ◽  
Type Strain ◽  
Wild Type Strain ◽  
Growth Curves ◽  
The Other ◽  
Nocardia Asteroides ◽  
Growth Stages ◽  
Fatty Acid Profiles ◽  
Wild Type ◽  
Colonial Morphology

The pathogenicities in mice (comparing LD50 determinations) of two mutant strains and one wild-type strain of Nocardia asteroides GUH-2, each possessing a colonial morphology distinct from the other, were compared at respective stages of growth. Despite the three strains' colinear growth curves and similar physiological properties, unique patterns of pathogenicity emerged for each strain upon analysis. Ultrastructural and fatty acid profiles of cultures at the various growth stages were monitored. The mutant strain SCII-A1 was consistently less virulent than the other strains N. asteroides GUH-2 (SCII-P and SCII-C). Further, its fatty acid profiles as well as the shape and consistency of its colonies differed greatly from those of the wild-type strain. The fatty acid composition and the colonial morphology of strain SCII-C more closely resembled those of the parent, although its virulence was both greater than (before 28 h of growth) and less than the parent's depending upon the specific stage of growth. The comparative degrees of cellular fragmentation and complexity, as determined by scanning and transmission electron microscopy, were found to coincide with changes in relative degrees of pathogenicity.

scholarly journals Fitness Cost of Rifampin Resistance in Neisseria meningitidis:In VitroStudy of Mechanisms Associated withrpoBH553Y Mutation

2015 ◽  
Vol 59 (12) ◽  
pp. 7637-7649 ◽  
Author(s):  
Roberta Colicchio ◽  
Chiara Pagliuca ◽  
Gabiria Pastore ◽  
Annunziata Gaetana Cicatiello ◽  
Caterina Pagliarulo ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Capsular Polysaccharide ◽  
Culture Media ◽  
Point Mutations ◽  
Clinical Isolates ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Rifampin Resistance

ABSTRACTRifampin chemoprophylaxis againstNeisseria meningitidisinfections led to the onset of rifampin resistance in clinical isolates harboring point mutations in therpoBgene, coding for the RNA polymerase β chain. These resistant strains are rare in medical practice, suggesting their decreased fitness in the human host. In this study, we isolated rifampin-resistantrpoBmutants from hypervirulent serogroup C strain 93/4286 and analyzed their different properties, including the ability to grow/survive in different culture media and in differentiated THP-1 human monocytes and to compete with the wild-type strainin vitro. Our results demonstrate that differentrpoBmutations (H553Y, H553R, and S549F) may have different effects, ranging from low- to high-cost effects, on bacterial fitnessin vitro. Moreover, we found that the S549F mutation confers temperature sensitivity, possibly explaining why it is observed very rarely in clinical isolates. Comparative high-throughput RNA sequencing analysis of bacteria grown in chemically defined medium demonstrated that the low-cost H553Y substitution resulted in global transcriptional changes that functionally mimic the stringent response. Interestingly, many virulence-associated genes, including those coding for meningococcal type IV pili, porin A, adhesins/invasins, IgA protease, two-partner secretion system HrpA/HrpB, enzymes involved in resistance to oxidative injury, lipooligosaccharide sialylation, and capsular polysaccharide biosynthesis, were downregulated in the H553Y mutant compared to their level of expression in the wild-type strain. These data might account for the reduced capacity of this mutant to grow/survive in differentiated THP-1 cells and explain the rarity of H553Y mutants among clinical isolates.

scholarly journals Isolation of Rifampin-Resistant Mutants ofListeria monocytogenes and Their Characterization byrpoB Gene Sequencing, Temperature Sensitivity for Growth, and Interaction with an Epithelial Cell Line

1999 ◽  
Vol 37 (9) ◽  
pp. 2913-2919 ◽  
Author(s):  
Robert Morse ◽  
Karen O’Hanlon ◽  
Mumtaz Virji ◽  
Matthew D. Collins
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Point Mutations ◽  
Gene Sequencing ◽  
Epithelial Cell Line ◽  
Wild Type ◽  
Partial Sequencing ◽  
Resistant Mutants ◽  
Oflisteria Monocytogenes ◽  
Rna Polymerase Holoenzyme

The sequence of the rpoB gene from Listeria monocytogenes was determined. Rifampin-resistant (Rifr) mutants arising from L. monocytogenes cultures exposed to rifampin were isolated, and by partial sequencing of their rpoB genes, seven different point mutations affecting five different amino acids (473Asp→Asn or Gly, 479Gly→Asp, 483His→Tyr or Leu, 528Ile→Phe, and 530Ser→Tyr), which led to MICs of 0.5 to 100 μg/ml for the organisms, were determined. These mutants showed various deficiencies for growth at 42°C, with only one being comparable to the wild-type strain. The interaction of these Rifr mutants with human Caco-2 cells was examined by using an immunofluorescence technique. Three mutants failed to interact, while three showed a reduced interaction compared to that of the wild type. It is believed that these pleiotropic phenotypes have arisen as a result of mutations within the DNA-dependent RNA polymerase holoenzyme.

scholarly journals Off-target expression of Cre-dependent adeno-associated viruses in wild type C57BL/6J mice

2021 ◽  
Author(s):  
Justin J. Botterill ◽  
Abdessattar Khlaifia ◽  
Brandon J. Walters ◽  
Mark A. Brimble ◽  
Helen E. Scharfman ◽  
...  
Keyword(s):  
Contextual Fear Conditioning ◽  
Ease Of Use ◽  
Designer Drugs ◽  
Detectable Effect ◽  
Specific Cell ◽  
Wild Type ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Specific Targeting ◽  
Viral Expression

AbstractAdeno-associated viruses (AAVs) are a commonly used tool in neuroscience to efficiently label, trace, and/or manipulate neuronal populations. Highly specific targeting can be achieved through recombinase-dependent AAVs in combination with transgenic rodent lines that express Cre-recombinase in specific cell types. Visualization of viral expression is typically achieved through fluorescent reporter proteins (e.g., GFP or mCherry) packaged within the AAV genome. Although non-amplified fluorescence is usually sufficient to observe viral expression, immunohistochemical amplification of the fluorescent reporter is routinely used to improve viral visualization. In the present study, Cre-dependent AAVs were injected into the hippocampus and cortex of wild-type C57BL/6J mice. While we observed weak but consistent non-amplified off-target DIO expression in C57BL/6J mice, antibody amplification of the GFP or mCherry reporter revealed extensive Cre-independent viral expression. Off-target expression of DIO constructs in wild-type C57BL/6J mice occurred independent of vendor, AAV serotype or promoter. We also evaluated whether Cre-independent expression had functional effects via Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). The DREADD agonist C21 had no effect on contextual fear conditioning or cFos expression in DIO-hM3Dq-mCherry+ cells of C57BL/6J mice. Taken together, our results indicate that DIO constructs have considerable off-target expression in wild type subjects. Our findings are particularly important for the design of experiments featuring sensitive systems and/or quantitative measurements that could be negatively impacted by off-target expression.Significance StatementAdeno-associated viruses (AAV) are widely used in neuroscience because of their safety and ease of use. Combined with specific promoters, Cre/loxP, and stereotaxic injections, highly specific targeting of cells and circuits within the brain can be achieved. In the present study we injected Cre-dependent AAVs into wild-type C57BL/6J mice and found considerable Cre-independent viral expression of AAVs encoding mCherry, GFP, or hM3Dq following immunohistochemical amplification of the fluorescent reporter protein. Importantly, we observed no functional effects of the Cre-independent expression in the hippocampus, as C21 had no detectable effect on DIO-hM3Dq-mCherry infected neurons in C57BL/6J mice. Given the widespread use of DIO rAAVs by the neuroscience community, our data supports careful consideration when using DIO constructs in control animals.

scholarly journals The putative phosphate transporter PitB (PP1373) is involved in tellurite uptake in Pseudomonas putida KT2440

2020 ◽  
Author(s):  
Rafael Montenegro ◽  
Sofía Vieto ◽  
Daniela Wicki-Emmenegger ◽  
Felipe Vásquez-Castro ◽  
Carolina Coronado-Ruiz ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Type Strain ◽  
Wild Type Strain ◽  
Electronic Waste ◽  
Chemical Species ◽  
Growth Curves ◽  
Phosphate Transporter ◽  
Transport Systems ◽  
Wild Type ◽  
Pseudomonas Putida Kt2440

AbstractTellurium oxyanions are chemical species with great toxicity; their presence in the environment has increased because of mining industries and photovoltaic and electronic waste. Recovery strategies based on microorganisms for this metalloid are of interest, but further studies of the transport systems and enzymes responsible for implementing tellurium transformations are required because many mechanisms remain unknown. Here, we investigated the involvement in tellurite uptake of the putative phosphate transporter PitB (PP1373) in soil bacterium Pseudomonas putida KT2440. For this purpose, through a method based on the CRISPR/Cas9 system, we generated a strain deficient in pitB gene and characterized its phenotype on exposing it to varied concentrations of tellurite. Growth curves and Transmission Electronic Microscopy experiments of wild type and ΔpitB showed that both strains were able to internalize tellurite into the cytoplasm and reduce the oxyanion to black nano-sized and rod-shaped tellurium particles, however, ΔpitB strain showed an increased resistance to the tellurite toxic effects. At a concentration of 100 uM tellurite, where the biomass formation of wild type strain decreased by half, we observed a greater ability of ΔpitB to reduce this oxyanion with respect to wild type strain (~38% vs ~16%), which is related by the greater biomass production of ΔpitB and not by a greater consumption of tellurite per cell. The phenotype of the mutant was restored on over-expressing pitB in trans. In summary, our results indicate that PitB is one of several transporters responsible for tellurite uptake in P. putida KT2440.

Emergence of phenotypic variants upon mismatch repair disruption in Pseudomonas aeruginosa

Microbiology ◽  
2004 ◽  
Vol 150 (5) ◽  
pp. 1327-1338 ◽  
Author(s):  
Andrea M. Smania ◽  
Ignacio Segura ◽  
Roberto J. Pezza ◽  
Cecilia Becerra ◽  
Inés Albesa ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
High Frequency ◽  
Type Strain ◽  
Wild Type Strain ◽  
Point Mutations ◽  
Repair System ◽  
Wild Type ◽  
Antibiotic Resistant ◽  
Prolonged Incubation ◽  
Resistant Mutants

MutS is part of the bacterial mismatch repair system that corrects point mutations and small insertions/deletions that fail to be proof-read by DNA polymerase activity. In this work it is shown that the disruption of the P. aeruginosa mutS gene generates the emergence of diverse colony morphologies in contrast with its parental wild-type strain that displayed monomorphic colonies. Interestingly, two of the mutS morphotypes emerged at a high frequency and in a reproducible way and were selected for subsequent characterization. One of them displayed a nearly wild-type morphology while the other notably showed, compared with the wild-type strain, increased production of pyocyanin and pyoverdin, lower excretion of LasB protease and novel motility characteristics, mainly related to swarming. Furthermore, it was reproducibly observed that, after prolonged incubation in liquid culture, the pigmented variant consistently emerged from the mutS wild-type-like variant displaying a reproducible event. It is also shown that these P. aeruginosa mutS morphotypes not only displayed an increase in the frequency of antibiotic-resistant mutants, as described for clinical P. aeruginosa mutator isolates, but also generated mutants whose antibiotic-resistant levels were higher than those measured from spontaneous resistant mutants derived from wild-type cells. It was also found that both morphotypes showed a decreased cytotoxic capacity compared to the wild-type strain, leading to the emergence of invasive variants. By using mutated versions of a tetracycline resistance gene, the mutS mutant showed a 70-fold increase in the reversion frequency of a +1 frameshift mutation with respect to its parental wild-type strain, allowing the suggestion that the phenotypical diversity generated in the mutS population could be produced in part by frameshift mutations. Finally, since morphotypical diversification has also been described in clinical isolates, the possibility that this mutS diversification was related to the high frequency hypermutability observed in P. aeruginosa CF isolates is discussed.

scholarly journals Genetic Analysis of Mutant Strains of Saccharomyces cerevisiae with Defects in Mannoprotein Synthesis

Proceedings ◽  
2020 ◽  
Vol 70 (1) ◽  
pp. 18
Author(s):  
Patricia Gil ◽  
Alberto Martínez ◽  
Elena Palencia ◽  
Rocío Velázquez ◽  
Manuel Ramírez ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Point Mutations ◽  
Random Mutagenesis ◽  
Selection Method ◽  
Wild Type ◽  
Truncated Protein ◽  
Mutant Strains

Mannan defective (mnn) mutants have constituted a fundamental tool in the study of the structure and biosynthesis of mannoproteins in Saccharomyces cerevisiae. They were isolated by the group of Dr. C.E. Ballou by random mutagenesis, and a selection method using specific antibodies obtained against the wild-type strain. Initially, the mutants were characterized biochemically, and in subsequent years the genes in which they were mutated were identified. All of them encode membrane proteins that catalyze the transfer of mannoses to N-oligosaccharides, sometimes isolated or as part of complexes made up of several proteins. However, the specific mutation of each of these mutants has only been identified in the case of mnn3. In this work, we have completed the characterization of the mutants by sequencing the mutated genes in each of them. As expected, they are point mutations that involve the change of one amino acid for another in the mutated protein, or for a stop signal, resulting in a truncated protein.

Recovery of Recombinant Rotavirus Expressing Fluorescent Reporter Protein

2019 ◽  
Author(s):  
Asha Philip ◽  
Jin Dai ◽  
Sarah Katen ◽  
John Patton
Keyword(s):  
Reporter Protein ◽  
Fluorescent Reporter

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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