scholarly journals A Naturally-Occurring Point Mutation in a Hyaluronidase Gene (hysA1) of Staphylococcus aureus UAMS-1 Results in Reduced Enzymatic Activity

2021 ◽  
Author(s):  
Haijing Hu ◽  
Huanli Liu ◽  
Ohgew Kweon ◽  
Mark E. Hart
Keyword(s):  
Staphylococcus Aureus ◽  
Hyaluronic Acid ◽  
Enzymatic Activity ◽  
Base Change ◽  
Wild Type ◽  
Animal Tissues ◽  
Plasmid Copy ◽  
Naturally Occurring ◽  
Single Base Change ◽  
High Degree

Hyaluronic acid is a high molecular weight polysaccharide that is widely distributed in animal tissues. Bacterial hyaluronidases degrade hyaluronic acid as secreted enzymes and have been shown to contribute to infection. <i>Staphylococcus aureus</i> UAMS-1 is a clinical isolate that codes for two hyaluronidases (<i>hysA1</i> and <i>hysA2</i>). Previous research has shown the presence of a full-length HysA1 protein from the <i>S. aureus</i> UAMS-1 strain with no evidence of enzymatic activity. A single base change resulting in an E480G amino acid change was identified in the <i>S. aureus</i> UAMS-1 <i>hysA1</i> gene when compared to the <i>S. aureus</i> Sanger 252 <i>hysA1</i> gene. A plasmid copy of the <i>S. aureus</i> Sanger 252 <i>hysA1 </i>gene transduced into a <i>hysA2 </i>deletion mutant strain of <i>S. aureus</i> UAMS-1 restored near wild type levels of enzymatic activity. Homology modeling and structural analysis suggested that Glu-480 in the HysA1 is critically responsible for maintaining the structural and functional ensemble of the catalytic and tunnel-forming residues, which are essential for enzyme activity. A high degree of relatedness among several Gram-positive bacterial hyaluronidases indicate the loss of enzymatic activity of HysA1 in the <i>S. aureus</i> UAMS-1 strain is most likely caused by the mutation identified in our study.

scholarly journals Differential expression of five tRNA(UAGTrp) amber suppressors in Caenorhabditis elegans.

1988 ◽  
Vol 8 (9) ◽  
pp. 3627-3635 ◽  
Author(s):  
K Kondo ◽  
J Hodgkin ◽  
R H Waterston
Keyword(s):  
Nervous System ◽  
Caenorhabditis Elegans ◽  
Gene Family ◽  
Development Stage ◽  
Base Change ◽  
Wild Type ◽  
Specific Manner ◽  
Flanking Sequences ◽  
Single Base Change ◽  
The Individual

Caenorhabditis elegans has 12 tRNA(UGGTrp) genes as defined by Southern analysis. In order to evaluate the function of the individual members of this multigene family, we sought to recover amber (UAG)-suppressing mutations from reversion experiments with animals carrying amber mutations in a nervous system-affecting gene (unc-13) or a sex-determining gene (tra-3). Revertants were analyzed by Southern blot, exploiting the fact that the CCA to CTA change at the anticodon creates a new XbaI site. Five different members of the tRNATrp gene family were identified as suppressors: sup-7 X, sup-5 III, sup-24 IV, sup-28 X, and sup-29 IV. All five suppressor genes were sequenced and found to encode identical tRNA(UAGTrp) molecules with a single base change (CCA to CTA) at the anticodon compared with their wild-type counterparts. The flanking sequences had only limited homology. The relative expression of these five genes was determined by measuring the efficiencies of suppressers against amber mutations in genes affecting the nervous system, hypodermis, muscle, and sex determination. The results of these cross-suppression tests showed that the five members of the tRNA(Trp) gene family were differentially regulated in a tissue- or development stage-specific manner.

scholarly journals Sortase mutants with improved protein thermostability and enzymatic activity obtained by consensus design

2019 ◽  
Vol 32 (12) ◽  
pp. 555-564
Author(s):  
Magdalena Wójcik ◽  
Susana Vázquez Torres ◽  
Wim J Quax ◽  
Ykelien L Boersma
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Enzymatic Activity ◽  
Sortase A ◽  
Wild Type ◽  
Protein Thermostability ◽  
Consensus Analysis ◽  
Gram Positive Bacteria ◽  
Covalently Linked

Abstract Staphylococcus aureus sortase A (SaSrtA) is an enzyme that anchors proteins to the cell surface of Gram-positive bacteria. During the transpeptidation reaction performed by SaSrtA, proteins containing an N-terminal glycine can be covalently linked to another protein with a C-terminal LPXTG motif (X being any amino acid). Since the sortase reaction can be performed in vitro as well, it has found many applications in biotechnology. Although sortase-mediated ligation has many advantages, SaSrtA is limited by its low enzymatic activity and dependence on Ca2+. In our study, we evaluated the thermodynamic stability of the SaSrtA wild type and found the enzyme to be stable. We applied consensus analysis to further improve the enzyme’s stability while at the same time enhancing the enzyme’s activity. As a result, we found thermodynamically improved, more active and Ca2+-independent mutants. We envision that these new variants can be applied in conjugation reactions in low Ca2+ environments.

Differential expression of five tRNA(UAGTrp) amber suppressors in Caenorhabditis elegans

1988 ◽  
Vol 8 (9) ◽  
pp. 3627-3635 ◽  
Author(s):  
K Kondo ◽  
J Hodgkin ◽  
R H Waterston
Keyword(s):  
Nervous System ◽  
Caenorhabditis Elegans ◽  
Gene Family ◽  
Development Stage ◽  
Base Change ◽  
Wild Type ◽  
Specific Manner ◽  
Flanking Sequences ◽  
Single Base Change ◽  
The Individual

Caenorhabditis elegans has 12 tRNA(UGGTrp) genes as defined by Southern analysis. In order to evaluate the function of the individual members of this multigene family, we sought to recover amber (UAG)-suppressing mutations from reversion experiments with animals carrying amber mutations in a nervous system-affecting gene (unc-13) or a sex-determining gene (tra-3). Revertants were analyzed by Southern blot, exploiting the fact that the CCA to CTA change at the anticodon creates a new XbaI site. Five different members of the tRNATrp gene family were identified as suppressors: sup-7 X, sup-5 III, sup-24 IV, sup-28 X, and sup-29 IV. All five suppressor genes were sequenced and found to encode identical tRNA(UAGTrp) molecules with a single base change (CCA to CTA) at the anticodon compared with their wild-type counterparts. The flanking sequences had only limited homology. The relative expression of these five genes was determined by measuring the efficiencies of suppressers against amber mutations in genes affecting the nervous system, hypodermis, muscle, and sex determination. The results of these cross-suppression tests showed that the five members of the tRNA(Trp) gene family were differentially regulated in a tissue- or development stage-specific manner.

scholarly journals Correlation of Acetate Catabolism and Growth Yield in Staphylococcus aureus: Implications for Host-Pathogen Interactions

2003 ◽  
Vol 71 (8) ◽  
pp. 4724-4732 ◽  
Author(s):  
Greg A. Somerville ◽  
Battouli Saïd-Salim ◽  
Jaala M. Wickman ◽  
Sandra J. Raffel ◽  
Barry N. Kreiswirth ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Culture Medium ◽  
Growth Yield ◽  
Tca Cycle ◽  
Wild Type ◽  
Gene Encoding ◽  
Lower Growth ◽  
Naturally Occurring ◽  
Derivative Strain ◽  
Surprising Discovery

ABSTRACT Recently, we reported that the prototypical Staphylococcus aureus strain RN6390 (a derivative of NCTC 8325) had significantly reduced aconitase activity relative to a diverse group of S. aureus isolates, leading to the hypothesis that strain RN6390 has impaired tricarboxylic acid (TCA) cycle-mediated acetate catabolism. Analysis of the culture supernatant from RN6390 confirmed that acetate was incompletely catabolized, suggesting that the ability to catabolize acetate can be lost by S. aureus. To test this hypothesis, we examined the carbon catabolism of the S. aureus strains whose genome sequences are publicly available. All strains catabolized glucose and excreted acetate into the culture medium. However, strains NCTC 8325 and N315 failed to catabolize acetate during the postexponential growth phase, resulting in significantly lower growth yields relative to strains that catabolized acetate. Strains NCTC 8325 and RN6390 contained an 11-bp deletion in rsbU, the gene encoding a positive regulator of the alternative sigma factor σB encoded by sigB. An isogenic derivative strain of RN6390 containing the wild-type rsbU gene had significantly increased acetate catabolism, demonstrating that σB is required for acetate catabolism. Taken together, the data suggest that naturally occurring mutations can alter the ability of S. aureus to catabolize acetate, a surprising discovery, as TCA cycle function has been demonstrated to be involved in the virulence, survival, and persistence of several pathogenic organisms. Additionally, these mutations decrease the fitness of S. aureus by reducing the number of progeny placed into subsequent generations, suggesting that in certain situations a decreased growth yield is advantageous.

scholarly journals Novel Polymorphisms in mec Genes and a New mec Complex Type in Methicillin-Resistant Staphylococcus aureus Isolates Obtained in Rural Wisconsin

2004 ◽  
Vol 48 (8) ◽  
pp. 3080-3085 ◽  
Author(s):  
Sanjay K. Shukla ◽  
Srinivas V. Ramaswamy ◽  
Jennifer Conradt ◽  
Mary E. Stemper ◽  
Robert Reich ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Wild Type ◽  
Promoter Mutation ◽  
Complex Type ◽  
Methicillin Resistant ◽  
Type Iv ◽  
Wide Range ◽  
Complete Sequencing ◽  
High Degree

ABSTRACT We determined allelic polymorphisms in the mec complexes of 524 methicillin-resistant Staphylococcus aureus isolates by partial or complete sequencing of three mec genes, mecA, mecI, and mecR1. The isolates had been collected over a 10-year period from patients living in rural Wisconsin, where the use of antibiotics was expected to be lower than in the bigger cities. Of the 18 mutation types identified, 16 had not been described previously. The five most common mutations were a mutation 7 nucleotides (nt) upstream from the start site (G→T) in the mecA promoter (34.7%), an E246G change encoded by mecA (2.2%), a cytosine insertion at codon 257 in mecA (13.2%), an N121K change encoded by mecI (7.8%), and a major mecI-mecR1 deletion designated as a class B1 mec complex deletion type (25.4%). There was a high degree of conservation of the amino acid sequence of MecR1. Strains with the same mutations had variable resistance to oxacillin, and the median MIC for isolates that harbored the 7-nt-upstream mutation was lower than that for strains harboring other mutations. Our data suggest that the mecA promoter mutation plays a more important role in determining methicillin resistance than mutations in mecI and mecA do. Eighty-five percent of the tested isolates (n = 148) with the mecA promoter mutation and the class B1 mec complex deletion belonged to the same major clonal group, identified as MCG-2, and harbored the type IV staphylococcal cassette chromosome mec DNA. There was also a wide range of oxacillin MICs for strains with wild-type mecA, mecI, and mecR1 sequences, suggesting that the genetic backgrounds of clinical strains are significant in determining susceptibility to methicillin.

NATURE OF THE DETERMINANT OF TETRACYCLINE RESISTANCE IN STAPHYLOCOCCUS AUREUS

1967 ◽  
Vol 13 (8) ◽  
pp. 1079-1086 ◽  
Author(s):  
S. S. Kasatiya ◽  
Jack N. Baldwin
Keyword(s):  
Staphylococcus Aureus ◽  
Low Dose ◽  
Tetracycline Resistance ◽  
Wild Type ◽  
Naturally Occurring ◽  
Resistant Strains

Ninety-one tetracycline-sensitive mutants were isolated from naturally occurring tetracycline-resistant strains of Staphylococcus aureus. Only two mutants reverted to the typical wild type. All of the sensitive mutants tested were competent recipients of the tetracycline marker. In 150 crosses between non-reverting mutants, no tetracycline-resistant recombinants were observed. Transduction frequencies obtained with ultraviolet irradiated phage lysates indicated that the tetracycline determinant is an extrachromosomal gene resistant to the low dose of ultraviolet used. The penicillinase determinant may be either on the chromosome or in the cytoplasm, depending on the strain tested.

scholarly journals Comparison of the Antibacterial Properties of Phage Endolysins SAL-1 and LysK

2011 ◽  
Vol 55 (4) ◽  
pp. 1764-1767 ◽  
Author(s):  
Soo Youn Jun ◽  
Gi Mo Jung ◽  
Jee-Soo Son ◽  
Seong Jun Yoon ◽  
Yun-Jaie Choi ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Enzymatic Activity ◽  
Bacterial Cell ◽  
Sequence Similarity ◽  
Antibacterial Properties ◽  
Amino Acid Sequence Similarity ◽  
High Degree

ABSTRACTIn spite of the high degree of amino acid sequence similarity between the newly discovered phage endolysin SAL-1 and the phage endolysin LysK, SAL-1 has an approximately 2-fold-lower MIC against severalStaphylococcus aureusstrains and higher bacterial cell-wall-hydrolyzing activity than LysK. The amino acid residue change contributing the most to this enhanced enzymatic activity is a change from glutamic acid to glutamine at the 114th residue.

Evaluation of flow-cytometry for the monitoring of infectious human rotavirus in water

1997 ◽  
Vol 35 (11-12) ◽  
pp. 451-453
Author(s):  
F. X. Abad ◽  
A. Bosch ◽  
J. Comas ◽  
D. Villalba ◽  
R. M. Pintó
Keyword(s):  
Flow Cytometry ◽  
Water Samples ◽  
Wild Type ◽  
Cell Monolayers ◽  
Naturally Occurring ◽  
Human Rotavirus ◽  
Faecal Samples

A method has been developed for the detection of infectious human rotavirus (HRV), based on infection of MA104 and CaCo-2 cell monolayers and ulterior flow cytometry. The sensitivity of the flow cytometry procedure for the cell-adapted HRV enabled the detection of 200 and 2 MPNCU in MA104 and CaCo-2 cells, respectively. Flow cytometry performed five days after infection of CaCo-2 enabled the detection of naturally occurring wild-type HRV in faecal samples and concentrated water samples.

Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 661-668
Author(s):  
Mandy Kim ◽  
Erika Wolff ◽  
Tiffany Huang ◽  
Lilit Garibyan ◽  
Ashlee M Earl ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Deinococcus Radiodurans ◽  
Genetic System ◽  
Base Change ◽  
Base Substitution ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
Radiation Induced ◽  
Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced &gt;250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

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