scholarly journals SarS, a SarA Homolog Repressible by agr, Is an Activator of Protein A Synthesis in Staphylococcus aureus

2001 ◽  
Vol 69 (4) ◽  
pp. 2448-2455 ◽  
Author(s):  
Ambrose L. Cheung ◽  
Katherine Schmidt ◽  
Brian Bateman ◽  
Adhar C. Manna
Keyword(s):  
Parental Strain ◽  
Double Mutant ◽  
Specific Binding ◽  
Protein A ◽  
Acid Protein ◽  
Regulatory Loci ◽  
Parental Level ◽  
High Level ◽  
Divergent Finding ◽  
Amino Acid Protein

ABSTRACT The expression of protein A (spa) is repressed by global regulatory loci sarA and agr. Although SarA may directly bind to the spa promoter to downregulatespa expression, the mechanism by which agrrepresses spa expression is not clearly understood. In searching for SarA homologs in the partially released genome, we found a SarA homolog, encoding a 250-amino-acid protein designated SarS, upstream of the spa gene. The expression ofsarS was almost undetectable in parental strain RN6390 but was highly expressed in agr and sarA mutants, strains normally expressing high level of protein A. Interestingly, protein A expression was decreased in a sarS mutant as detected in an immunoblot but returned to near-parental levels in a complemented sarS mutant. Transcriptional fusion studies with a 158- and a 491-bp spa promoter fragment linked to the xylE reporter gene disclosed that the transcription of the spa promoter was also downregulated in thesarS mutant compared with the parental strain. Interestingly, the enhancement in spa expression in anagr mutant returned to a near-parental level in theagr sarS double mutant but not in the sarA sarSdouble mutant. Correlating with this divergent finding is the observation that enhanced sarS expression in anagr mutant was repressed by the sarA locus supplied in trans but not in a sarA mutant expressing RNAIII from a plasmid. Gel shift studies also revealed the specific binding of SarS to the 158-bp spa promoter. Taken together, these data indicated that the agr locus probably mediates spa repression by suppressing the transcription ofsarS, an activator of spa expression. However, the pathway by which the sarA locus downregulatesspa expression is sarS independent.

scholarly journals Expression of the Xylulose 5-Phosphate Phosphoketolase Gene, xpkA, from Lactobacillus pentosus MD363 Is Induced by Sugars That Are Fermented via the Phosphoketolase Pathway and Is Repressed by Glucose Mediated by CcpA and the Mannose Phosphoenolpyruvate Phosphotransferase System

2002 ◽  
Vol 68 (2) ◽  
pp. 831-837 ◽  
Author(s):  
Clara C. Posthuma ◽  
Rechien Bader ◽  
Roswitha Engelmann ◽  
Pieter W. Postma ◽  
Wolfgang Hengstenberg ◽  
...  
Keyword(s):  
Protein A ◽  
Pyruvate Decarboxylase ◽  
Thiamine Pyrophosphate ◽  
Phosphotransferase System ◽  
Knockout Mutant ◽  
Lactobacillus Pentosus ◽  
Acid Protein ◽  
Phosphoketolase Pathway ◽  
Calculated Mass ◽  
Amino Acid Protein

ABSTRACT Purification of xylulose 5-phosphate phosphoketolase (XpkA), the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and cloning and sequence analysis of the encoding gene, xpkA, from Lactobacillus pentosus MD363 are described. xpkA encodes a 788-amino-acid protein with a calculated mass of 88,705 Da. Expression of xpkA in Escherichia coli led to an increase in XpkA activity, while an xpkA knockout mutant of L. pentosus lost XpkA activity and was not able to grow on energy sources that are fermented via the PKP, indicating that xpkA encodes an enzyme with phosphoketolase activity. A database search revealed that there are high levels of similarity between XpkA and a phosphoketolase from Bifidobacterium lactis and between XpkA and a (putative) protein present in a number of evolutionarily distantly related organisms (up to 54% identical residues). Expression of xpkA in L. pentosus was induced by sugars that are fermented via the PKP and was repressed by glucose mediated by carbon catabolite protein A (CcpA) and by the mannose phosphoenolpyruvate phosphotransferase system. Most of the residues involved in correct binding of the cofactor thiamine pyrophosphate (TPP) that are conserved in transketolase, pyruvate decarboxylase, and pyruvate oxidase were also conserved at a similar position in XpkA, implying that there is a similar TPP-binding fold in XpkA.

Phenotypic characterization and virulence of a sae−agr− mutant of Staphylococcus aureus

1996 ◽  
Vol 42 (2) ◽  
pp. 120-123 ◽  
Author(s):  
Ana T. Giraudo ◽  
Horacio Rampone ◽  
Aldo Calzolari ◽  
Rosa Nagel
Keyword(s):  
Staphylococcus Aureus ◽  
Parental Strain ◽  
Intraperitoneal Injection ◽  
Double Mutant ◽  
Protein A ◽  
Synergistic Interaction ◽  
Extracellular Protein ◽  
Phenotypic Characterization ◽  
Single Mutant

A sae::Tn551 agr::tetM double mutant was constructed and characterized. The production of several exoproteins (e.g., β-hemolysin, DNase, and proteases) by this mutant was determined and found to be lower than the already diminished production of either isogenic single mutant sae− or agr−. The double mutant also showed, like the agr− mutant, null production of α- and δ-hemolysins and diminished levels of lipase. The reduced levels of many exoproteins in the double mutant as compared with their already diminished levels in either single mutant suggest that there is an additive or synergistic interaction between the two mutations involved, sae− and agr−. However, inactivation of both loci, sae and agr, had a different effect on the two exoproteins that are up regulated in the agr− mutant; thus, coagulase dropped to levels close to the null levels of the sae− parental strain, while extracellular protein A displayed the high levels characteristic of the agr− single mutant. The virulence of the sae−agr− double mutant, determined by intraperitoneal injection in mice, was found to be significantly diminished as compared with that of the sae+agr+ parental strain or the sae−agr+ single mutant.Key words: Staphylococcus aureus, exoprotein expression, sae− mutant, agr− mutant.

Sequence and nuclear localization of the Saccharomyces cerevisiae HAP2 protein, a transcriptional activator

1987 ◽  
Vol 7 (2) ◽  
pp. 578-585
Author(s):  
J L Pinkham ◽  
J T Olesen ◽  
L P Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Fusion Gene ◽  
Protein A ◽  
Acid Protein ◽  
Genes Encoding ◽  
Beta Galactosidase ◽  
Amino Acid Protein ◽  
Operator Site ◽  
Direct Activator

Activation of the CYC1 upstream activation site (UAS2) and other Saccharomyces cerevisiae genes encoding respiratory functions requires the products of the regulatory loci HAP2 and HAP3. We present here the DNA sequence of the yeast HAP2 gene and an initial investigation into the function of its product. The DNA sequence indicated that HAP2 encoded a 265-amino-acid protein whose carboxyl third was highly basic. Also found in the sequence was a polyglutamine tract spanning residues 120 to 133. Several experiments described herein suggest that HAP2 encodes a direct activator of transcription. First, a bifunctional HAP2-beta-galactosidase fusion gene was localized to the yeast nucleus. Second, a lexA-HAP2 fusion gene was capable of activating transcription when bound to a lexA operator site. The additional requirement for the HAP3 product in activation is discussed.

Overexpression, purification, and analysis of the c1 repressor protein of Pseudomonas aeruginosa bacteriophage D3

1997 ◽  
Vol 43 (3) ◽  
pp. 220-226 ◽  
Author(s):  
Mark A. Farinha ◽  
Andrew M. Kropinski
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sequence Analysis ◽  
Sequence Data ◽  
Specific Binding ◽  
Protein Product ◽  
Repressor Protein ◽  
Acid Protein ◽  
Bacteriophage Genome ◽  
Protein Sequence Data ◽  
Amino Acid Protein

A 3.1-kb region of the bacteriophage D3 genome which contains the immunity functions has recently been sequenced (GenBank accession No. L22692). Sequence analysis indicated the presence of a putative repressor gene (c1) whose protein product functions to maintain the bacteriophage genome as a stably integrated prophage in the chromosome of Pseudomonas aeruginosa. A plasmid was constructed that overexpresses repressor C1 protein under control of Ptac in Escherichia coli. C1 protein was subsequently purified and characterized as a 223 amino acid protein with specific binding affinity for 14-base imperfect palindromic operator sequences located on the genome of bacteriophage D3. N-terminal protein sequence data obtained from automated Edman degradation (16 cycles) of purified repressor protein were identical to the predicted sequence based on DNA sequence analysis of the c1 open reading frame.Key words: promoter, repressor, operator, lambdoid phage, Pseudomonas aeruginosa.

scholarly journals Purification, Characterization, and Molecular Analysis of Thermostable Cellulases CelA and CelB fromThermotoga neapolitana

1998 ◽  
Vol 64 (12) ◽  
pp. 4774-4781 ◽  
Author(s):  
Jin-Duck Bok ◽  
Dinesh A. Yernool ◽  
Douglas E. Eveleigh
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Genomic Library ◽  
Cellulose Hydrolysis ◽  
Thermotoga Neapolitana ◽  
Glycosyl Hydrolases ◽  
Transglycosylation Activity ◽  
Acid Protein ◽  
High Level ◽  
Amino Acid Protein

ABSTRACT Two thermostable endocellulases, CelA and CelB, were purified fromThermotoga neapolitana. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95°C, while CelB (molecular mass, 30 kDa; pI 4.1) has a broader optimal pH range (pH 6.0 to 6.6) at 106°C. Both enzymes are characterized by a high level of activity (high V max value and low apparentKm value) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With p-nitrophenyl cellobioside theV max values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the Km values are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. The Ki values for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; the Ki values for CelB are 0.2 M for glucose and 1.16 mM for cellobiose. CelB preferentially cleaves larger cellooligomers, producing cellobiose as the end product; it also exhibits significant transglycosylation activity. This enzyme is highly thermostable and has half-lives of 130 min at 106°C and 26 min at 110°C. A single clone encoding thecelA and celB genes was identified by screening a T. neapolitana genomic library in Escherichia coli. The celA gene encodes a 257-amino-acid protein, while celB encodes a 274-amino-acid protein. Both proteins belong to family 12 of the glycosyl hydrolases, and the two proteins are 60% similar to each other. Northern blots of T. neapolitana mRNA revealed that celA andcelB are monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.

scholarly journals Sequence and nuclear localization of the Saccharomyces cerevisiae HAP2 protein, a transcriptional activator.

1987 ◽  
Vol 7 (2) ◽  
pp. 578-585 ◽  
Author(s):  
J L Pinkham ◽  
J T Olesen ◽  
L P Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Fusion Gene ◽  
Protein A ◽  
Acid Protein ◽  
Genes Encoding ◽  
Beta Galactosidase ◽  
Amino Acid Protein ◽  
Operator Site ◽  
Direct Activator

Activation of the CYC1 upstream activation site (UAS2) and other Saccharomyces cerevisiae genes encoding respiratory functions requires the products of the regulatory loci HAP2 and HAP3. We present here the DNA sequence of the yeast HAP2 gene and an initial investigation into the function of its product. The DNA sequence indicated that HAP2 encoded a 265-amino-acid protein whose carboxyl third was highly basic. Also found in the sequence was a polyglutamine tract spanning residues 120 to 133. Several experiments described herein suggest that HAP2 encodes a direct activator of transcription. First, a bifunctional HAP2-beta-galactosidase fusion gene was localized to the yeast nucleus. Second, a lexA-HAP2 fusion gene was capable of activating transcription when bound to a lexA operator site. The additional requirement for the HAP3 product in activation is discussed.

scholarly journals Novel β-Lactamase Genes from Two Environmental Isolates of Vibrio harveyi

2000 ◽  
Vol 44 (5) ◽  
pp. 1309-1314 ◽  
Author(s):  
Jeanette W. P. Teo ◽  
Antonius Suwanto ◽  
Chit Laa Poh
Keyword(s):  
Amino Acid ◽  
Vibrio Harveyi ◽  
Amino Acid Sequences ◽  
Gene Probe ◽  
Reading Frame ◽  
Class A ◽  
Acid Protein ◽  
Low Levels ◽  
High Level ◽  
Amino Acid Protein

ABSTRACT Two ampicillin-resistant (Ampr) isolates ofVibrio harveyi, W3B and HB3, were obtained from the coastal waters of the Indonesian island of Java. Strain W3B was isolated from marine water near a shrimp farm in North Java while HB3 was from pristine seawater in South Java. In this study, novel β-lactamase genes from W3B (bla VHW-1) and HB3 (bla VHH-1) were cloned and their nucleotide sequences were determined. An open reading frame (ORF) of 870 bp encoding a deduced protein of 290 amino acids (VHW-1) was revealed for the bla gene of strain W3B while an ORF of 849 bp encoding a 283-amino-acid protein (VHH-1) was deduced forbla VHH-1. At the DNA level, genes for VHW-1 and VHH-1 have a 97% homology, while at the protein level they have a 91% homology of amino acid sequences. Neither gene sequence showed homology to any other β-lactamases in the databases. The deduced proteins were found to be class A β-lactamases bearing low levels of homology (<50%) to other β-lactamases of the same class. The highest level of identity was obtained with β-lactamases from Pseudomonas aeruginosa, i.e., PSE-1, PSE-4, and CARB-3, and Vibrio cholerae CARB-6. Our study showed that both strains W3B and HB3 possess an endogenous plasmid of approximately 60 kb in size. However, Southern hybridization analysis employingbla VHW-1 as a gene probe demonstrated that thebla gene was not located in the plasmid. A total of nine ampicillin-resistant V. harveyi strains, including W3B and HB3, were examined by pulsed-field gel electrophoresis ofNotI-digested genomic DNA. Despite a high level of intrastrain genetic diversity, thebla VHW-1 probe hybridized only to an 80- or 160-kb NotI genomic fragment in different isolates.

scholarly journals Single Chain Variable Fragment Fused to Maltose Binding Protein: A Modular Nanocarrier Platform for the Targeted Delivery of Antitumorals

2021 ◽  
Author(s):  
Francisco J. Reche-Perez ◽  
Simona Plesselova ◽  
Eduardo De los Reyes-Berbel ◽  
Mariano Ortega-Muñoz ◽  
F. Javier Lopez-Jaramillo ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Targeted Delivery ◽  
Specific Binding ◽  
Protein A ◽  
Antibody Fragments ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Binding Properties ◽  
Single Chain Variable Fragments ◽  
Selective Delivery

The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due...

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