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Title: TIMING OF INDUCTION OF OSMOTICALLY CONTROLLED GENES IN SALMONELLA ENTERICA SEROVAR TYPHIMURIUM DETERMINED WITH QUANTITATIVE REAL TIME RT-PCR

Author
item BALAJI, BOOVARAGHAN - PURDUE UNIVERSITY
item O'CONNOR, KATHLEEN - PURDUE UNIVERSITY
item Anderson, Joseph
item CSONKA, LASZLO - PURDUE UNIVERSITY

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/6/2005
Publication Date: 12/1/2005
Citation: Balaji, B., O'Connor, K., Anderson, J.M., Csonka, L.N. 2005. Timing of induction of osmotically controlled genes in Salmonella enterica serovar typhimurium determined with quantitative real time RT-PCR. Applied and Environmental Microbiology. 71:8273-8283.

Interpretive Summary: The ability of cells to adjust and live when exposed to various types of abiotic stress, such as osmotic stress due to high salt or sugar concentrations, has been studied for many years. This research has many applications in plant research because soil salinity is increasing in many areas of the world due to irrigation. Bacteria have been ideal systems to examine cellular responses to osmotic stress. We studied the response of Salmonella enterica to salt and sugar stress by determining the expression pattern of six genes in four different groups of bacterial genes called operons. Our results indicated that these stress genes can be divided into at least three groups: rpoS, proV, and proP, which are induced early after the addition of salt; kdp which is induced somewhat later; and otsB and ompC, which are induced last. Because these groups of genes have different threshold and peak times of induction, they might respond to different regulatory signals. These differences in expression and regulation do not follow two previously proposed models. It is evident that additional research is required to identify the regulatory signals that control the two principal operons (proU and kdp) which respond to osmotic stress. The gene induction timeline proven in this study will be used by scientists to identify the regulatory factors required for cells to adjust to osmotic stress.

Technical Abstract: The timing of the osmotic induction of the proU and kdp operons was analyzed in S. enterica serovar Typhimurium with Quantitative Real Time RT-PCR. Induction of the proU operon was detectable within 4 min after a challenge with 0.3 M NaCl and reached a peak of 600-fold induction at 14 min. Induction of kdp was not detectable until 9 min and it peaked at 670-fold induction at 20 min. We also determined the kinetics of induction of other osmotically controlled genes after shock with 0.3 M NaCl: proP and rpoS were induced 4 to 5 min and otsB and ompC were induced 10 to 12 min after the shock. In cells that were shocked with 0.6 M sucrose, proU was induced at 3 min after the osmotic shift and reached a maximum of 590-fold induction at 8 min, similar to that seen after shock with 0.3 M NaCl. In contrast, the kdp mRNA exhibited only a 4-fold increase above basal level upon 0.6 M sucrose shock and then decreased to the pre-induction level by 30 min. Our observations cannot be explained by the 'turgor control' model of osmoregulation (Epstein. W. 1968. FEMS Microbiol. Rev. 39, 73-79), because that model proposed that the kdp operon would be induced before the proU operon after any osmotic upshift. These data are also incompatible with the 'ionic strength' model (Poolman et al., 2002. Molec. Microbiol. 44, 889-902), because it predicted that all osmotically controlled operons would be induced simultaneously by an osmotic upshift.