Prophage-Encoded Staphylococcal Enterotoxin A: Regulation of Production in Staphylococcus aureus Strains Representing Different Sea Regions

Abstract

The present study investigates the nature of the link between the staphylococcal enterotoxin A (SEA) gene and the lifecycle of Siphoviridae bacteriophages, including the origin of strain variation regarding SEA production after prophage induction. Five strains representing three different genetic lines of the sea region were studied under optimal and prophage-induced growth conditions and the Siphoviridae lifecycle was followed through the phage replicative form copies and transcripts of the lysogenic repressor, cro. The role of SOS response on prophage induction was addressed through recA transcription in a recA-disruption mutant. Prophage induction was found to increase the abundance of the phage replicative form, the sea gene copies and transcripts and enhance SEA production. Sequence analysis of the sea regions revealed that observed strain variances were related to strain capacity for prophage induction, rather than sequence differences in the sea region. The impact of SOS response activation on the phage lifecycle was demonstrated by the absence of phage replicative form copies in the recA-disruption mutant after prophage induction. From this study it emerges that all aspects of SEA-producing strain, the Siphoviridae phage and the food environment must be considered when evaluating SEA-related hazards.

Keywords: Siphoviridae; Staphylococcus aureus; enterotoxin A; prophage; staphylococcal food poisoning.

Figure 1

Schematic representation of the circular form of the sea-carrying Siphoviridae bacteriophage genome. The sites of integration to the bacterial chromosome are indicated as attR and attL and the nucleotide sequence of the respective cohesive ends is shown above. The primers designed for the specific detection and monitoring of the RF (circular form of the phage genome) are noted as RF_f and RF_r. The sea gene is indicated by the white arrow.

Figure 2

Staphylococcus aureus (S. aureus) strain Sa17 grown in Brain Heart Infusion (BHI) under control and Mitomycin C (MMC) induced conditions. Average values including standard deviations (^┬, _┴) of three independent experiments are presented. In the MMC induced culture, the point of induction at 4 h is indicated with an arrow. Time is represented in the X-axis in hours (h). (A) Ratio of RF and sea copy levels (Y-axis) as calculated based on Cq values and according to the equation ratio = (E _target) ^{ΔCq target(control-sample)}. Bars represent RF copy levels; dark grey bars designate RF levels for the control culture while light grey bars for the MMC induced culture. The sea₁ gene copy levels are represented by symbols. The levels for the control culture are designated the symbol (▲), while the levels for the MMC induced culture are designated the symbol (×); (B) Ratio (left Y-axis) of total sea₁ transcript represented in dark grey bars for the control culture and light grey bars for the MMC induced, and cro transcripts represented by the symbol (×). Ratio of long sea₁ transcript (right Y-axis) is designated the symbol (♦). The cro and long sea₁ transcripts were only detected in the MMC induced culture.

Figure 3

S. aureus strain Sa48 grown in BHI under control and MMC induced conditions. Average values including standard deviations (^┬, _┴) of three technical replicates are presented. In the MMC induced culture, the point of induction at 4 h is indicated with an arrow. Time is represented in the X-axis in hours (h). (A) Ratio of RF and sea copy levels (Y-axis) as calculated based on Cq values and according to the equation ratio = (E _target) ^{ΔCq target(control-sample)}. Bars represent RF copy levels; dark grey bars designate RF levels for the control culture while light grey bars for the MMC induced culture. The sea₁ gene copy levels are represented by symbols. The levels for the control culture are designated the symbol (▲), while the levels for the MMC induced culture are designated the symbol (×) (B). Ratio (left Y-axis) of total sea₁ transcript represented in dark grey bars for the control culture and light grey bars for the MMC induced, and cro transcripts represented by the symbol (■) for the control culture and (×) for the induced. Ratio of long sea₁ transcript (right Y-axis) is designated the symbol (♦). The long sea₁ transcript was only detected in the MMC induced culture.

Figure 3

S. aureus strain Sa48 grown in BHI under control and MMC induced conditions. Average values including standard deviations (^┬, _┴) of three technical replicates are presented. In the MMC induced culture, the point of induction at 4 h is indicated with an arrow. Time is represented in the X-axis in hours (h). (A) Ratio of RF and sea copy levels (Y-axis) as calculated based on Cq values and according to the equation ratio = (E _target) ^{ΔCq target(control-sample)}. Bars represent RF copy levels; dark grey bars designate RF levels for the control culture while light grey bars for the MMC induced culture. The sea₁ gene copy levels are represented by symbols. The levels for the control culture are designated the symbol (▲), while the levels for the MMC induced culture are designated the symbol (×) (B). Ratio (left Y-axis) of total sea₁ transcript represented in dark grey bars for the control culture and light grey bars for the MMC induced, and cro transcripts represented by the symbol (■) for the control culture and (×) for the induced. Ratio of long sea₁ transcript (right Y-axis) is designated the symbol (♦). The long sea₁ transcript was only detected in the MMC induced culture.

Figure 4

S. aureus strain, Sa21 grown in BHI under control and MMC induced conditions. Average values including standard deviations (^┬, _┴) of three independent experiments are presented. In MMC induced culture, the point of induction at 4 h is indicated with an arrow. Time is represented in the X-axis in hours (h). (A) Ratio of RF and sea copy levels (Y-axis) as calculated based on Cq values and according to the equation ratio = (E _target) ^{ΔCq target(control-sample)}. Bars represent RF copy levels; dark grey bars designate RF levels for the control culture while light grey bars for the MMC induced culture. The sea₁ gene copy levels are represented by symbols. The levels for the control culture are designated the symbol (▲), while the levels for the MMC induced culture are designated the symbol (×); (B) Ratio of total sea₁ transcript represented in dark grey bars for the control culture and light grey bars for the MMC induced. The cro and long sea₁ transcripts were not detected in either the control or MMC induced cultures.

Figure 5

S. aureus strain Mu50 grown in BHI under control and MMC induced conditions. Average values including standard deviations (^┬, _┴) of three technical replicates are presented. In MMC induced culture the point of induction at 4 h is indicated with an arrow. Time is represented in the X-axis in hours (h). (A) Ratio of RF and sea copy levels (Y-axis) as calculated based on Cq values and according to the equation ratio = (E _target) ^{ΔCq target(control-sample)}. Bars represent RF copy levels; dark grey bars designate RF levels for the control culture while light grey bars for the MMC induced culture. The sea₁ gene copy levels are represented by symbols. The levels for the control culture are designated the symbol (▲), while the levels for the MMC induced culture are designated the symbol (×); (B) Ratio of total sea₁ transcript represented in dark grey bars for the control culture and light grey bars for the MMC induced. The cro and long sea₁ transcripts were not detected in either the control or MMC induced cultures.

Figure 6

The sea gene region (3.6 kb long) of 11 S. aureus strains. The black arrow represents the sea gene while the grey arrows represent genes in the surrounding regions. The endogenous sea promoter is marked as P₁ and the latent promoter as P₂. Observed sequence differences are represented with dotted lines.

Figure 7

Detection of RF by PCR and agarose gel electrophoresis in S. aureus strains Sa17 wild type and Sa17 recA-disruption mutant grown in BHI under induced and controlled conditions: (a) control Sa17 wild type; (b) induced Sa17 wild type; (c) control Sa17 recA-disruption mutant; and (d) induced Sa17 recA-disruption mutant. Positive and negative control are denoted + and −, respectively. DNA ladder mix is included to verify the RF product size.