scholarly journals Homologous and heterologous expression and maturation processing of extracellular glutamyl endopeptidase of Staphylococcus epidermidis

2008 ◽  
Vol 389 (9) ◽  
Author(s):  
Yuko Ohara-Nemoto ◽  
Toshio Ono ◽  
Yu Shimoyama ◽  
Shigenobu Kimura ◽  
Takayuki K. Nemoto
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Amino Acid ◽  
Heterologous Expression ◽  
Virulence Factors ◽  
Staphylococcus Epidermidis ◽  
Essential Amino Acid ◽  
Glutamyl Endopeptidase ◽  
Molecular Masses ◽  
Mature Protease

AbstractThe extracellular serine endopeptidase GluSE (EC 3.4.21.19) is considered to be one of the virulence factors ofStaphylococcus epidermidis. The present study investigated maturation processing of native GluSE and that heterologously expressed inEscherichia coli.In addition to the 28-kDa mature protease, small amounts of proenzymes with molecular masses of 32, 30, and 29 kDa were identified in the extracellular and cell wall-associated fractions. We defined the pre (M1-A27)- and pro (K28-S66)-segments, and found that processing at the E32-S33and D48-I49bonds was responsible for production of the 30- and 29-kDa intermediates, respectively. The full-length form of C-terminally His-tagged GluSE was purified as three proenzymes equivalent to the native ones. These molecules possessing an entire or a part of the pro-segment were proteolytically latent and converted to a mature 28-kDa form by thermolysin cleavage at the S66-V67bond. Mutation of the essential amino acid S235suggested auto-proteolytic production of the 30- and 29-kDa intermediates. Furthermore, an undecapeptide (I56-S66) of the truncated pro-segment not only functions as an inhibitor of the protease but also facilitates thermolysin processing. These findings could offer clues to the molecular mechanism involved in the regulation of proteolytic activity of pathogenic proteases secreted fromS. epidermidis.

Heterologous expression and characterization of l-amino acid deaminases from Proteus mirabilis in Escherichia coli

2008 ◽  
Vol 136 ◽  
pp. S300 ◽  
Author(s):  
Jin-Oh Baek ◽  
Jeong-Woo Seo ◽  
Ohsuk Kwon ◽  
Su-Il Seong ◽  
Ik-Hwan Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Heterologous Expression ◽  
Proteus Mirabilis

scholarly journals Staphylococcus epidermidis MSCRAMM SesJ Is Encoded in Composite Islands

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Srishtee Arora ◽  
Xiqi Li ◽  
Andrew Hillhouse ◽  
Kranti Konganti ◽  
Sara V. Little ◽  
...  
Keyword(s):  
Cell Wall ◽  
Antibiotic Resistance ◽  
Virulence Factors ◽  
Staphylococcus Epidermidis ◽  
Core Genome ◽  
Clinical Isolates ◽  
Modification System ◽  
Content Type ◽  
Genes Encoding ◽  
Staphylococcal Cassette Chromosome

ABSTRACT Staphylococcus epidermidis is a leading cause of nosocomial infections in patients with a compromised immune system and/or an implanted medical device. Seventy to 90% of S. epidermidis clinical isolates are methicillin resistant and carry the mecA gene, present in a mobile genetic element (MGE) called the staphylococcal cassette chromosome mec (SCCmec) element. Along with the presence of antibiotic and heavy metal resistance genes, MGEs can also contain genes encoding secreted or cell wall-anchored virulence factors. In our earlier studies of S. epidermidis clinical isolates, we discovered S. epidermidis surface protein J (SesJ), a prototype of a recently discovered subfamily of the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) group. MSCRAMMs are major virulence factors of pathogenic Gram-positive bacteria. Here, we report that the sesJ gene is always accompanied by two glycosyltransferase genes, gtfA and gtfB, and is present in two MGEs, called the arginine catabolic mobile element (ACME) and the staphylococcal cassette chromosome (SCC) element. The presence of the sesJ gene was associated with the left-hand direct repeat DR_B or DR_E. When inserted via DR_E, the sesJ gene was encoded in the SCC element. When inserted via DR_B, the sesJ gene was accompanied by the genes for the type 1 restriction modification system and was encoded in the ACME. Additionally, the SCC element and ACME carry different isoforms of the SesJ protein. To date, the genes encoding MSCRAMMs have been seen to be located in the bacterial core genome. Here, we report the presence of an MSCRAMM in an MGE in S. epidermidis clinical isolates. IMPORTANCE S. epidermidis is an opportunistic bacterium that has established itself as a successful nosocomial pathogen. The modern era of novel therapeutics and medical devices has extended the longevity of human life, but at the same time, we also witness the evolution of pathogens to adapt to newly available niches in the host. Increasing antibiotic resistance among pathogens provides an example of such pathogen adaptation. With limited opportunities to modify the core genome, most of the adaptation occurs by acquiring new genes, such as virulence factors and antibiotic resistance determinants present in MGEs. In this study, we describe that the sesJ gene, encoding a recently discovered cell wall-anchored protein in S. epidermidis, is present in both ACME and the SCC element. The presence of virulence factors in MGEs can influence the virulence potential of a specific strain. Therefore, it is critical to study the virulence factors found in MGEs in emerging pathogenic bacteria or strains to understand the mechanisms used by these bacteria to cause infections.

scholarly journals Role of Glutamine Synthetase in Nitrogen Metabolite Repression in Aspergillus nidulans

2001 ◽  
Vol 183 (20) ◽  
pp. 5826-5833 ◽  
Author(s):  
Soula Margelis ◽  
Cletus D'Souza ◽  
Anna J. Small ◽  
Michael J. Hynes ◽  
Thomas H. Adams ◽  
...  
Keyword(s):  
Amino Acid ◽  
Glutamine Synthetase ◽  
Heterologous Expression ◽  
Aspergillus Nidulans ◽  
Essential Amino Acid ◽  
Gene Encoding ◽  
Nitrogen Metabolite Repression ◽  
Link Type ◽  
Wide Range

ABSTRACT Glutamine synthetase (GS), EC 6.3.1.2 , is a central enzyme in the assimilation of nitrogen and the biosynthesis of glutamine. We have isolated the Aspergillus nidulans glnA gene encoding GS and have shown that glnA encodes a highly expressed but not highly regulated mRNA. Inactivation of glnA results in an absolute glutamine requirement, indicating that GS is responsible for the synthesis of this essential amino acid. Even when supplemented with high levels of glutamine, strains lacking a functionalglnA gene have an inhibited morphology, and a wide range of compounds have been shown to interfere with repair of the glutamine auxotrophy. Heterologous expression of the prokaryotic Anabaena glnA gene from the A. nidulans alcA promoter allowed full complementation of the A. nidulans glnAΔ mutation. However, the A. nidulans fluG gene, which encodes a protein with similarity to prokaryotic GS, did not replace A. nidulans glnA function when similarly expressed. Our studies with theglnAΔ mutant confirm that glutamine, and not GS, is the key effector of nitrogen metabolite repression. Additionally, ammonium and its immediate product glutamate may also act directly to signal nitrogen sufficiency.

scholarly journals In silico analysis of virulence associated genes in genomes of Escherichia coli strains causing colibacillosis in poultry

2017 ◽  
Vol 61 (4) ◽  
pp. 421-426 ◽  
Author(s):  
Joanna Kołsut ◽  
Paulina Borówka ◽  
Błażej Marciniak ◽  
Ewelina Wójcik ◽  
Arkadiusz Wojtasik ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Virulence Factors ◽  
De Novo ◽  
Protein Sequences ◽  
In Silico Analysis ◽  
Amino Acid Sequences ◽  
Common Disease ◽  
Bacterial Genomes ◽  
E Coli

AbstractIntroduction: Colibacillosis – the most common disease of poultry, is caused mainly by avian pathogenic Escherichia coli (APEC). However, thus far, no pattern to the molecular basis of the pathogenicity of these bacteria has been established beyond dispute. In this study, genomes of APEC were investigated to ascribe importance and explore the distribution of 16 genes recognised as their virulence factors.Material and Methods: A total of 14 pathogenic for poultry E. coli strains were isolated, and their DNA was sequenced, assembled de novo, and annotated. Amino acid sequences from these bacteria and an additional 16 freely available APEC amino acid sequences were analysed with the DIFFIND tool to define their virulence factors.Results: The DIFFIND tool enabled quick, reliable, and convenient assessment of the differences between compared amino acid sequences from bacterial genomes. The presence of 16 protein sequences indicated as pathogenicity factors in poultry resulted in the generation of a heatmap which categorises genomes in terms of the existence and similarity of the analysed protein sequences.Conclusion: The proposed method of detection of virulence factors using the capabilities of the DIFFIND tool may be useful in the analysis of similarities of E. coli and other sequences deriving from bacteria. Phylogenetic analysis resulted in reliable segregation of 30 APEC strains into five main clusters containing various virulence associated genes (VAGs).

scholarly journals CO-FERMENTION OF MAIZE (Zea mays L.) AND AFRICAN BREAD FRUIT SEED (Treculia africana Dec'ne) FOR THE PRODUCTION OF IMPROVED PAP (OGI)

2020 ◽  
Vol 7 ◽  
pp. 1
Author(s):  
Funmilayo Mujidat Oyeyipo ◽  
Olugbenga Samson Taiwo ◽  
Yemisi Dorcas Obafemi
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Protein Content ◽  
Essential Amino Acid ◽  
Biochemical Tests ◽  
Rotational Viscometer ◽  
Fermentation Time ◽  
African Breadfruit ◽  
Amino Acid Contents

Cereals such as maize are generally having low essential amino acid contents which may cause protein malnutrition. The high essential amino acid in African breadfruit could make it suitable for supplementation of grains/cereals. The microbiological, sensory and nutritional evaluation of cofermented maize (70%) /African breadfruit seed (30%) was carried out. Characterization of isolates were by macroscopic, microscopic and biochemical tests. Standard Methods according to AOAC (1990) were used for proximate compositions and pH determination. Viscosity was measured using Rotational Viscometer. The pH of the co-fermented sample decreased from 6.2±0.01 to 4.2±0.01. Microbial isolates included Coryne bacterium spp., Lactobacillus spp., Escherichia coli, Staphylococcus aureus, Aspergillus niger and Saccharomyces cerevisae in fermenting samples. Escherichia coli was inhibited with increase in fermentation time. Higher microbial counts occurred in maize-African breadfruit ogi (MABO) than maize ogi (MOGI) during primary and secondary -1fermentations. The viscosity of the co-fermented sample was 1.990±0.12 pas . Proximate composition shows that MOGI had lower crude protein content (2.14±0.2) than MABO (9.83±0.3). In contrast, MOGI had higher crude carbohydrate (82.12±0.1 vs 72.59±0.2). Ash, moisture and lipid contents were not significantly different in both samples. Sensory evaluation revealed that MABO recorded higher scores (7±0.02 and 7±0.01) in taste and aroma as against (6±0.01 and 6±0.02) for MOGI. Although consumers accepted both samples, overall, MOGI was more acceptable. MABO oflour kept well and retained their original viscosity after 27 days of storage at (30±2C). MABO gave a dual advantage in that protein content was increased and enteropathogens were inhibited and is therefore recommended for consumption in areas where protein intake is inadequate and food safety is an issue.

scholarly journals THE NATURE OF THE COLICIN K RECEPTOR OF ESCHERICHIA COLI CULLEN

1971 ◽  
Vol 133 (3) ◽  
pp. 534-553 ◽  
Author(s):  
Hans Ulrich Weltzien ◽  
Margeris A. Jesaitis
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Biological Activity ◽  
Amino Acid ◽  
Cell Walls ◽  
Cytoplasmic Membrane ◽  
Proteolytic Enzymes ◽  
E Coli ◽  
Chemical Reagents ◽  
Specific Receptors

Cell walls of E. coli strains B and Cullen contain specific receptors for colicin K and for the T2, T6, and C16 phages. The receptors for the bacteriocin and the T6 virus are located in the outer layers of the cell wall of these microorganisms and are absent in their cytoplasmic membrane. The receptors for colicin K, phage T2, and the T6 and C16 viruses differ in their stability toward enzymes and chemical reagents. Their specificity must therefore be determined by different chemical groupings. The colicin K receptor is inactivated by certain proteolytic enzymes and by reagents which combine with tryptophan. It is concluded therefore that proteins or peptides containing this amino acid are essential for biological activity of the receptor.

scholarly journals The preparation of ribosomal ribonucleic acid from whole bacteria

1968 ◽  
Vol 106 (4) ◽  
pp. 897-903 ◽  
Author(s):  
H. K. Robinson ◽  
H. E. Wade
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Pseudomonas Fluorescens ◽  
Ribosomal Rna ◽  
Staphylococcus Epidermidis ◽  
Ribonucleic Acid ◽  
Potassium Acetate ◽  
Sedimentation Pattern ◽  
Simple Method ◽  
Ribosomal Ribonucleic Acid

1. A simple method for the preparation of ribonuclease-free ribosomal RNA is described in which ribonuclease-deficient bacteria are treated with acetone and the RNA is extracted with phenol and purified by precipitating it with potassium acetate. The treatment with acetone appears to render the cell wall permeable to RNA but not to DNA during the extraction with phenol. The method thus avoids the need to disrupt the bacteria and greatly simplifies the subsequent purification. 2. The method has been used successfully with ribonuclease-deficient strains of Escherichia coli, Pseudomonas fluorescens and Staphylococcus epidermidis. The recovered purified RNA accounts for about 70% of the total ribosomal RNA and shows the normal sedimentation pattern of the 16s and 23s components in the analytical centrifuge.

Properties of cell wall peptidoglycan synthesized by amino acid deprived relA mutants of Escherichia coli

1984 ◽  
Vol 30 (10) ◽  
pp. 1239-1246 ◽  
Author(s):  
Désirée Vanderwel ◽  
Edward E. Ishiguro
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Amino Acid ◽  
Free Form ◽  
Amino Acid Deprivation ◽  
Peptidoglycan Synthesis ◽  
Cell Wall Peptidoglycan ◽  
Covalently Linked

Cell wall peptidoglycan synthesis in Escherichia coli is under stringent control. During amino acid deprivation, peptidoglycan synthesis is inhibited in re1A+ bacteria but not in re1A mutants. The relaxed synthesis of peptidoglycan by amino acid deprived re1A bacteria was inhibited by Several β-lactam antibiotics at concentrations which inhibited cell elongation in growing cultures suggesting that the transpeptidase activity of penicillin-binding protein (PBP-1B) was involved in this process. Structural studies on the peptidoglycan also indicated the involvement of transpeptidation in relaxed peptidoglycan synthesis. The peptidoglycan synthesized during amino acid deprivation was cross-linked to the existing cell wall peptidoglycan, and the degree of cross-linkage was the same as that of peptidoglycan synthesized by growing control cells. The relaxed synthesis of peptidoglycan was also inhibited by moenomycin, an inhibitor of the in vitro transglycosylase activities of PBPs, but the interpretation of this result depends on whether the transglycosylases are the sole targets of moenomycin in vivo. Most of the peptidoglycan lipoprotein synthesized by histidine-deprived re1A+ bacteria was in the free form as previously reported, possibly because of the restriction in peptidoglycan synthesis. In support of this proposal, most of the lipoprotein synthesized during histidine deprivation of re1A mutants was found to be covalently linked to peptidoglycan. Nevertheless, the peptidoglycan synthesized by amino acid deprived re1A bacteria was apparently deficient in bound lipoprotein as compared with peptidoglycan synthesized by normal growing control bacteria suggesting that the rate of lipoprotein synthesis during amino acid deprivation may be limiting.

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