Transcriptional Activation of the Staphylococcus aureus putP Gene by Low-Proline-High Osmotic Conditions and during Infection of Murine and Human Tissues

Abstract

Staphylococcus aureus can grow virtually anywhere in the human body but needs to import proline through low-and high-affinity proline transporters to survive. This study examined the regulation of the S. aureus putP gene, which encodes a high-affinity proline permease. putP::lacZ and putP::lux transcriptional fusions were constructed and integrated into the genomes of several S. aureus strains. Enzyme activity was measured after growth in media with various osmolyte concentrations. As osmolarity rose, putP expression increased, with a plateau at 2 M for NaCl in strain LL3-1. Proline concentrations as low as 17.4 M activated expression of the putP gene. The putP::lux fusion was also integrated into the genomes of S. aureus strains that were either SigB inactive (LL3-1, 8325-4, and SH1003) or SigB active (Newman and SH1000). SigB inactive strains showed increased putP gene expression as NaCl concentrations rose, whereas SigB active strains displayed a dramatic decrease in putP expression, suggesting that the alternative sigma factor B plays a negative role in putP regulation. Mice inoculated with S. aureus strains containing the putP::lux fusion exhibited up to a 715-fold increase in putP expression, although levels in the various murine organs differed. Moreover, urine from human patients infected with S. aureus showed elevated putP levels by use of a PCR procedure, whereas blood and some abscess material had no significant increase. Thus, putP is transcriptionally activated by a lowproline and high osmotic environment both in growth media and in murine or human clinical specimens.

The Effect of Rotating Magnetic Field on Enterotoxin Genes Expression in Staphylococcus Aureus Strains

“…Ideally, the HKGs used in relative RT-qPCR studies are controls equally expressed under different conditions. Several HKGs have already been reported in S. aureus gene expression studies, including ftsZ [29], pta [30], hu [31], and gyrB [32]. However, previous studies have shown that the expression of the particular HKGs can be differentially regulated, depending on experimental conditions [33,34].…”

Tool for Quantification of Staphylococcal Enterotoxin Gene Expression in Cheese

“…However, during growth in cheese, transcriptional stability of the reference genes differed substantially, indicating that expression has to be validated under each experimental condition (6). Each of the three genes chosen to normalize our RT-qPCR data during S. aureus growth in cheese (ftsZ, pta, and gyrB) had already been used separately in previous studies (37,41,50). We used the geometric mean of expression results using all three genes to normalize expression of our gene of interest (47).…”

“…Ideally, reference genes used in relative RT-qPCR studies are controls equally expressed under different conditions. Several housekeeping genes have already been used in S. aureus gene expression studies as single internal reference genes, including ftsZ (41), pta (37), hu (7,17,39), and gyrB (19,20,46,50). However, previous studies have shown that the expression of many housekeeping genes is differentially regulated, depending on experimental conditions (14,44).…”

Role for dnaK locus in tolerance of multiple stresses in Staphylococcus aureus

“…A mixture of wild-type and dnaK mutant cells, containing a total of 5610 8 c.f.u. ml 21 , was prepared (76/24 % mixture of mutant/wild-type) in 1 % TSB, and 0.2 ml of this suspension was injected into the peritoneal cavity of Swiss white Hla(ICR)CVF female mice (16-20 g) (Hilltop Lab Animals Inc.) with a 26 gauge needle fitted to a 1 ml syringe, following published procedures with appropriate modifications (Schwan et al, 2006). At 4, 8 and 30 h, the mice were euthanized by CO 2 asphyxiation.…”