Staphylococcal nuclease A (SNA) may be used to produce bacterial ghosts for further inactivation of host bacteria and elimination of residual genetic materials. It is still controversial if SNA without signal peptide can be secreted to extracellular matrix and if fusion with other peptide is required for its function in the cytoplasm of host bacteria. To clarify this dispute, a series of temperature-inducible plasmids carrying SNA alone or SNA fused with partial sequences of λ phage cro gene (cSNA) or Mycobacterium tuberculosis urease gene (uSNA) were constructed and evaluated in Escherichia coli. Results show that the percentages of inactivated E. coli by SNA, cSNA and uSNA after 4 h of induction were 99.9%, 99.8% and 74.2%, respectively. Moreover, SNA and cSNA in the cytoplasm of host bacteria were initially detectable after 30 min of induction, whereas uSNA was after 1 h. In comparison, SNA and cSNA in culture supernatant were initially detectable 1 h later, whereas uSNA was 2 h later. The nuclease activity in the cytoplasm or supernatant was ranked as follows: SNA > cSNA > uSNA, and the activity in the supernatant was significantly lower than that in the cytoplasm. Furthermore, host genomic DNA was degraded by SNA or cSNA after 2 h of induction but not by uSNA even throughout the whole experiment. In conclusion, this study indicates that SNA, cSNA and uSNA expressed in host bacteria all have nuclease activity, the enzymes can be released to culture media, and fusion with exogenous peptide negatively reduces the nuclease activity of SNA.
Bacteriolysis ; Bacteriophage lambda ; DNA ; chemistry ; Escherichia coli ; Genetic Vectors ; Micrococcal Nuclease ; chemistry ; Plasmids ; Protein Sorting Signals

BACKGROUND: Staphylococcus aureus remains one of the most frequently encountered bacterial pathogens and is responsible for a variety of mild to life- threatening infections. There is a substantial body of evidence that individuals who are asymptomatic nasal carriers of S. aureus are at increased risk of developing serious staphylococcal infections. Approximately 20% to 30% of health care workers at any given time are also nasal carriers of S. aureus. A subset of these may spread the organism to patients by direct contact transmission. CHROMagar Staph aureus (CSA) is a new chromogenic medium for identification of S. aureus on the basis of colony pigmentation. METHODS: The abilities of CSA, thermostable nuclease (DNase), and mannitol salt agar (MSA) to identify S. aureus isolates (n=70) and discriminate between S. aureus and coagluase-negative staphylococci (CoNS; n=8) were compared. RESULTS: CSA proved to be more sensitive and specific than DNase and MSA, allowing a reliable, simple, and rapid method for the identification of S. aureus isolates. All CoNS encountered in this study could be easily differentiated from S. aureus on the medium. The supplementation with 4 microgram/mL of oxacillin allowed simple identification of methicillin resistance in hospital acquired S. aureus strains which show multiple drug resistance profiles. CONCLUSION: CSA proved to be simple and reliable method for the identification of nasal carriers of S. aureus of health care workers.
Agar ; Delivery of Health Care ; Deoxyribonucleases ; Drug Resistance, Multiple ; Humans ; Mannitol ; Methicillin Resistance* ; Methicillin-Resistant Staphylococcus aureus* ; Micrococcal Nuclease ; Oxacillin ; Pigmentation ; Staphylococcal Infections ; Staphylococcus aureus

Leader peptidase is a novel serine protease in Escherichia coli, which catalyzes the cleavage of amino-terminal signal sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Recently, the x-ray crystal structure of signal peptidase-inhibitor complex showed that Asp 280, a highly conserved consensus sequence of E. coli leader peptidase is the closest charged residue in the vicinity of two catalytic dyad, Ser 90 and Lys 145, and it is likely held in place by a salt bridge to Arg 282. Possible roles of Asp 280 and Arg 282 in the structure-catalytic function relationship were investigated by the site-directed mutagenesis of Asp 280 substituted with alanine, glutamic acid, glycine, or asparagine and of Arg 282 with methionine. All of mutants purified with nickel affinity chromatography were inactive using in vitro assay. It is surprising to find complete lose of activity by an extension of one carbon units in the mutant where Asp 280 is substituted with glutamic acid. These results suggest that Asp 280 and Arg 282 are in a sequence which constitutes catalytic crevice of leader peptidase and are essential for maintaining the conformation of catalytic pocket.
Aspartic Acid/chemistry* ; Bacterial Outer Membrane Proteins/metabolism ; Blotting, Western ; Escherichia coli/enzymology* ; Escherichia coli/chemistry ; Micrococcal Nuclease/metabolism ; Mutagenesis, Site-Directed ; Oligonucleotides ; Protein Precursors/metabolism ; Serine Endopeptidases/metabolism* ; Serine Endopeptidases/genetics ; Serine Endopeptidases/chemistry* ; Structure-Activity Relationship

One pair of primers was designed based on the sequence encoding capsid protein C of classical swine fever virus (CSFV). The C gene fragment was amplified by RT-PCR and PCR products were inserted into eukaryotic expression vector pcDNA-SN containing staphylococcal nuclease (SN) gene resulting in recombinant plasmid pcDNA-C-SN. 48h after transfection of the recombinant into porcine kidney (PK)-15 cells using liposome, the expression of fusion protein was identified through RT-PCR, Western blot and indirect immunofluorescence, and nuclease activity was detected by in vitro DNA digestion assay. The results showed that fusion protein of C-SN was expressed stably in PK-15 cells, and could be identified by rabbit polyclonal antibody against CSFV capsid protein and had good nuclease activity to cleave DNA. Meanwhile, the expressed fusion protein of C-SN in the transfected cells could effectively inhibit the proliferation of CSFV, reducing the infection rate by 10(2)-10(3) times. Our findings laid a foundation for further application of capsid-targeted antiviral strategies for CSFV.
Animals ; Capsid Proteins ; genetics ; metabolism ; Cell Line ; Classical Swine Fever ; virology ; Classical swine fever virus ; genetics ; metabolism ; Gene Expression ; Genetic Engineering ; Micrococcal Nuclease ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Swine

The present study was carried out to genotypically characterize Staphylococcus aureus (S. aureus) isolated from bovine mastitis cases. A total of 37 strains of S. aureus were isolated during processing of 552 milk samples from 140 cows. The S. aureus strains were characterized phenotypically, and were further characterized genotypically by polymerase chain reaction using oligonucleotide primers that amplified genes encoding coagulase (coa), clumping factor (clfA), thermonuclease (nuc), enterotoxin A (entA), and the gene segments encoding the immunoglobulin G binding region and the X region of protein A gene spa. All of the isolates yielded an amplicon with a size of approximately 1,042 bp of the clfA gene. The amplification of the polymorphic spa gene segment encoding the immunoglobulin G binding region was observed in 34 isolates and X-region binding was detected in 26 isolates. Amplification of the coa gene yielded three different products in 20, 10, and 7 isolates. The amplification of the thermonuclease gene, nuc, was observed in 36 out of 37 isolates. All of the samples were negative for the entA gene. The phenotypic and genotypic findings of the present strategies might provide an understanding of the distribution of the prevalent S. aureus clones among bovine mastitis isolates, and might aid in the development of steps to control S. aureus infections in dairy herds.
Animals ; Bacterial Proteins/chemistry/genetics ; Cattle ; Coagulase/chemistry/genetics ; DNA, Bacterial/chemistry/genetics ; Endonucleases/chemistry/genetics ; Female ; Mastitis, Bovine/*microbiology ; Micrococcal Nuclease/chemistry/genetics ; Milk/microbiology ; Polymerase Chain Reaction/veterinary ; Staphylococcal Infections/microbiology/*veterinary ; Staphylococcus aureus/*genetics/pathogenicity ; Virulence Factors/chemistry/*genetics