Elucidation of the gene transfer mechanism of staphylococcal
enterotoxin AYuko SHIMAMURA
School of Food and Nutritional Sciences, University of Shizuoka

Research aims
Staphylococcus aureus produces enterotoxin A (SEA), causing not only food poisoning but
also a zoonosis whose toxin gene is transferred by bacteriophage. In order to prevent foodpoisoning and disease caused by S. aureus, we used another strain of S. aureus to inhibit thetransfer of SEA gene by phage. We demonstrated that the SEA-producing strain (No.29) waslysed and the SEA gene was transferred to the non-SEA producing strain (No.77) when No.
29 was in contact with colonies of No.77 (Fig. 1). However, the mechanisms of lysis induction
and the adsorption of the phage to host bacteria have notbeen clarified. In this study, we aimed to examine thefactors affecting the lysis of the SEA-producing strain andthe mechanism of SEA gene transfer.
Fig. 1: Lysis of S. aureus No.29
Methods
S. aureus No.29 (SEA+; No.29) and S. aureus No. 77 (SEA-; No.77) were used in this study.
Growth conditions including growth stage, temperature, medium, MgCl2 and NaCl
concentrations, and pH were examined to assess if they affected the transfer of the SEA gene.
Thereafter, metabolome analysis using LC-MS/MS was performed on No.29 cells when they
were co-cultured with No.77 in a dialysis culture flask. Furthermore, based on the reference
genome base sequence of the known S. aureus NCTC8325 (accession number NC-0077795)1),
genomic resequencing of No.29, No.77, and No.77-L22 (strain receiving SEA gene of No.29)
was performed using a next-generation sequencer.

Results
Factors promoting lysis colony formation of S. aureus No.29 were examined. Optimaltemperature and incubation of seed-culture was at 37 °C for 6 h. When the NaCl concentrationand pH value in the medium were high, No.29 lysed easily. In contrast, No.29 did not lyse
when MgCl2 was added. These results suggested that formation of lysis colonies tends to occur
in an environment suitable for the growth of bacteria. Principal component analysis (PCA)
showed that flavin adenine dinucleotide (FAD) levels were significantly decreased in No.29
co-cultured with No.77 in a dialysis culture flask for 10 h compared to levels in No.29 withouta co-culture (Fig. 2). In contrast, N-acetylglucosamine 6-phosphate (GlcNAc-6-P) andphosphatidylglycerol (PG) levels significantly increased in No.29 co-cultured with No.77 (Fig.
3). There was no difference in the content of N-acetylmuramic acid (MurNAc) which is acomponent of the S. aureus cell wall. These results suggest that the self-lytic enzyme of No.29
was activated by co-culture with No.77. Single Nucleotide Polymorphism (SNP) analysis wasperformed by mapping to the reference genome sequence S. aureus NCTC 8325 (accessionnumber NC-0077795). The transfer of the SEA phage derived from No.29 into No.77 caused
the addition of 1,103 SNPs. In addition, the insertion position of the SEA gene of No.77-L22
was between 2,037,774 bases and 2,038,315 bases.
Conclusion
In this study, we examined the mechanisms of lysis of an SEA producing strain (strainNo.29) and the transfer of the SEA gene. We observed that the formation of lysis colonies
occurred in an environment suitable for the growth of bacteria, and activated the self-lytic
enzyme of No.29 by co-culture with No.77. Strains No.29, No.77. and No.77-L22 wereresequenced using a next-generation sequencer. We are currently analyzing the insertion
position of the SEA gene phage cassette of No.77-L22. Based on the obtained results, we plan
to identify genes involved in SEA gene transfer. Further research is needed to clarify themechanisms of lysis of the SEA-producing strain as well as SEA gene transfer.
References1) Bæk, K. T., Frees, D., Renzoni, A., Barras, C., Rodriguez, N., Manzano, C., and Kelley,W. L. (2013). Genetic variation in the Staphylococcus aureus 8325 strain lineage revealedby whole-genome sequencing. PLoS One, 8(9): e77122.

No.29 co-cultured with No.77

E01

E02

No.29

E03

B02

B03

B01Fig. 2: Clustering analysis of co-cultivation-resolved intracellular metabolite values.
Co-cultivation-resolved intracellular metabolite values were visualized using a heatmap. Green colorations display lower than average concentrations whereas redcolorations represent higher than average concentrations of intracellular metabolites.
E01-E03: Co-culture with S. aureus No.29 and S. aureus No.77 in dialysis culture
flask, B01-03: A single cultured S. aureus No.29.

(A)

(B)
No.29 co-cultured with No.77
FAD
PC2（18.3%）

PC2（18.3%）

GlcNAc-6-P

No.29
PC1（56.5%）

Phosphatidylglycerol

PC1（56.5%）

Fig. 3: (A) Score and (B) loading plots of the two component principal component analysis
model developed using identified intracellular metabolites from S. aureus No.29.
(A) B01-B03: intracellular metabolites of S. aureus No.29, which increased by cocultivation, E01-E03: intracellular metabolites of S. aureus No.29, which decreased
by co-cultivation.