Characterization And Function Analysis Of The Thermostable Nuclease Genes In Staphylococcus Aureus

Staphylococcus aureus is one of the most common and important pathogenicbacterium. Some S. aureus strains are able to produce staphylococcal enterotoxins (SEs),which are the causative agents of staphylococcal food poisoning. S. aureus is also afeared hospital pathogen, sometimes it is very virulent. This bacterium often coloniseshosts asymptomatically and cause infections affecting any organ. Methods that aim atdetection of comprehensive types of enterotoxins remain a challenging task because newserotypes of enterotoxins continue to emerge. Clinical relevance of enterotoxin-producingstrains of S. aureus is the production of a heat-resistant thermonuclease. Thermostablenuclease could be used as an indicator of S. aureus contamination. The virulencedeterminants in S.aureus are controlled by two-component regulatory systems. Theprecise regulatory mechanism is not very clear.The present study compared the biochemical characteristics, secreted exoproteinsand enterotoxin genes of different strains, developed a new and more sensitive duplexPCR assay for S. aureus and enterotoxigenic strains, investigated theromostable nucleaserelated genes and revealed two kinds of thermostable nucleases co-expression in S.aureus, evaluated the responses of nuc1 and nuc2 genes to agr and sae regulatory system.The main results from this study are as follows:1. The biochemical characteristics, secreted exoproteins and enterotoxin genes ofdifferent strains have been compared. The results showed that all the six strains couldferment glucose, sucrose and mannitol. Five strains -S2, 2611, S1', S2' and S3' couldutilize arginine. Four strains -S6, S2, S1'and S3' could hydrolyze urea. S2' could produceH2S.26111 could ferment inositol and melibiose. 2611 and S2'could ferment sorbitol,amygdalin and arabinose. Using SDS-PAGE to detect the exoproteins of S. aureus, thebands of strains-S1' and S3' on gel were found to be very similar. The other four werevarious. Strains of S6, S2, S1' and S3' could produce the most types of exotoxins andexoenzymes, suggesting the importance of control and precaution to S. aureus.Subsequently, seven synthetic oligonucleotide primers (U2/A2, U1/B2, U1/C2, U2/D2and U2/E2) were used in a multiplex PCR protocol to detect the genes encoding theStaphylococcal enterotoxins A to E simultaneously. A good correlation betweenSDS-PAGE and multiplex PCR was demonstrated.2. The rapid detection method has been developed. Firstly, four different proceduresfor extraction of genomic DNA from S.aureus were reported. Through comparison ofaverage yield and quality, a modified procedure for extraction of high-quality genomic DNA was obtained which is rapid, simple, biologically nonhazardous, and generallyapplicable to pathogenic bacteria. To further find a simple and rapid method, two pairs ofprimers targeting the sequences of the new nuc2 gene and 16S rRNA gene were designedin a duplex PCR assay for the detection of S. aureus, particularly those of theenterotoxigenic strains. After optimization of the amplification conditions by uniformdesign, two specific products were amplified in the PCR detection system: a 223-bp nuc2fragment and a 565-bp 16S rRNA fragment. Evaluation tests demonstrated that thisduplex-PCR method has high specificity for only S. aureus. The detection limit of theassay was 9.35pg/μl using S. aureus genomic DNA. The sensitivity for direct detectionof milk samples sparked with S. aureus was 10⁴～10⁵ CFU/ml, and the sensitivity was 1CFU/ml as an initial concentration when an enrichment process was used. A goodcorrelation between theromostable nuclease production and enterotoxigenicity wasdemonstrated in all the strains tested.3. The influences of different culture conditions on the thermostable nucleaseactivity and biomass of S. aureus were discussed. Studies have shown that the straincould grow in a pH range 4.0-9.6, with an optimum of 7.0. However, the activity ofnuclease was not proportionate to the biomass of S. aureus. The nuclease activity wasremarkably higher under the pH 9.0-9.6. The nuclease activity increased substantiallyduring the initial stage of growth cycle, particularly during the exponential phase. In thelate post- exponential phase and stationary phase, the activity kept stable. The nucleaseactivity could be detected with a 3.2×10³ CFU/ml S. aureus. There was no significantdifference of the trend between the activity and the biomass in different NaClconcentration, indicating that NaCl had a minor or no influence on the activity ofnuclease. The nuclease had a strong endurance to heat, which still had better activity aftertreatment by exposure to 100℃, 135 rain. All these indicated that nucleases from S.aureuswere very stable and exhibited similar stability with enterotoxins.4. Thermostable nuclease is known to be an important pathogenic factor andcommonly presumed to be one compound which has the same genetic origin. However,two ORFs in S. aureus genomes were predicted to encode nucleases. One is encoded thestaphylococcal nuclease (SNase) without a gene name (now termed nuc1); the other isencoded a thermonuclease (TNase) with a name of nuc (now termed nuc2). The completecoding sequences of the nuc1 and nuc2 genes were separately cloned into the prokaryoticexpression vectors pET-17b and pET-28a and the resulting vectors (pET17-nuc1,pET17-nuc2, pET28-nuc1 and pET28-nuc2) were conformed by restriction enzymeanalysis. Thermostable nuclease activity was detected in the induced cultures of all fourkinds of transformants in the toluidine blue-DNA assays. All these showed the nuc2 gene could possibly express a new nuclease. Multisequence alignment of the amino acids ofthe recombinant SNase and TNase was further performed and the results showed that twoproteins had a much higher similarity. The three-dimensional structures of the twohomologous proteins were further predicted and the result showed that the two proteinshad similar structure. The results also showed that SNase A (149 aa) was the part ofSNase.5. To further investigate the function of nuc2 in S. aureus, homologousrecombination was applied to construct the nuc1deletion mutant. Fragments of upstreamand downstream of nuc1gene were amplified from S. aureus RN4220 and cloned into thepolylinker region of plasmid pUC19, flanking the selected marker-erm resistance geneseparating from p646. This resulted in the construction of the pUCâ–³nuc1plasmid.Subsequently, the 4.5kb fragment was partially digested from pUCâ–³nuc1and cloned intothe shuttle plasmid pBT₂. The resulting plasmid, pBT₂â–³nuc1 was successfullyconstructed. In order to obtain optimal transformation efficiency, the influence of variousparameters on plasmid transformation by electroporation of S. aureus RN4220 wasinvestigated. With the optimized procedure, the recombinant plasmid pBT₂â–³nuc1 wasintroduced into S. aureus RN4220. Using the resistant marker for selection, Em-resistant,Cm-sensitive colonies indicated successful replacement recombination. Therecombination rate was about 2% (7/345) with seven double cross-over mutants obtained.A nuc1-deleted mutant of S. aureus strain RN4220 (termed RNâ–³nuc1) was successfullyconstructed and conformed by PCR and RT-PCR. Toluidine blue-DNA assay revealedthat the nuc1-deleted mutant still exhibited thermostable nuclease activity, but theactivity was significantly reduced when compared with that of wild type strain. Thenucleases secreted by parent and mutant strains still had functional activity after 121℃,30 min. Our findings revealed that two kinds of thermostable nucleases, encoded by twodifferent genes (termed nuc1 and nuc2 respectively), coexisted in S. aureus. SDS-PAGErevealed that the protein expression patterns of the wild-type and mutant strains werevery similar, with exception that two bands near 14.4 kDa (about 16.8kDa and 12.2kDa)were missed and an additional band of approximately 9 kDa was appeared in the mutantstrain. To determine whether the decreased thermonuclease activity of the mutant strainRNâ–³nuc1 was due to the deletion of nuclgene, a complementation experiment wascarried out. A cinstructed recombinant plasmid pBT₂NUC1-C, carrying thenuc1-expressing cassette derived from S. aureus strain S6, was electroporated into themutant strain. The decreased thermostable nuclease activity was complemented by thetransformation of the recombinant plasmid pBT₂ NUC1-C which could expressstaphylococcal nuclease of S. aureus S6 origin. 6. The sae and agr operons are the key regulators in S. aureus. However, therelationship between the sae, agr and other structural genes needs to further study.Nuclease is known to be an important pathogenic factor unique to Staphylococcus aureus.This enzyme could convert organic compounds and macromolecules of host tissues intonutrients required for bacterial growth and contribute to selffuelling and invasiveness ofS. aureus. To date, its regulatory mechanism is still not very dear. In this assay, plasmidpBT₂â–³sae was constructed and this homologous recombination vector was transformedinto S. aureus RN4220 by electroporation, S.aureus RNâ–³sae deletion mutant wassuccessfully selected by homologous recombination at 40℃and confirmed by PCR andRT-PCR. Subsequently, tohidine blue-DNA assay of the different strains-RNâ–³sae,RN4220, SH1000 682, SH1000 1046 and RN6911 revealed that, although severaldistinct regulatory circuits can affect theromostable nuclease expression, sae appears toplay a crucial role in this context. SDS-PAGE revealed that the protein expressionpatterns of the wild-type strain RN4220 and mutant strain RNâ–³sae had visible difference.The most striking difference in the sae-mutant samples containing exoproteins were thedecrease ofα-hemolysin,β-hemolysin and protein A.