Regulation of virulence gene expression in Streptococcus pyogenes: determinants of differential mRNA decay

Abstract

Differential mRNA stability is an important mechanism for regulation of virulence factors in Streptococcus pyogenes (group A streptococcus, GAS), a serious and prevalent human pathogen. We have described 2 Classes of mRNA in GAS that are distinguishable by 1) stability in the stationary phase of growth, 2) kinetics of decay in exponential phase, and 3) effect of depletion of RNases J1 and J2 and polynucleotide phosphorylase (PNPase) on decay in exponential phase. We discuss features of the structure of an mRNA that appear to be important for determining the Class to which it belongs and present a model to explain differential mRNA decay.

Figure 1

Models for exponential phase mRNA decay in GAS. (A) The 5′ end of Class I mRNA (shown in blue) makes it a good substrate for RNase J1 and/or RNase J2 (blue pacman). Cleavage is most likely initiated by endonucleolytic activity of these RNases, which produces two type of products. One has an accessible 3′-end and is subject to digestion by 3′-to-5′ exonucleases (red pacman) and the other has a 5′-monophosphate which makes it a good substrate for the 5′-to-3′ exonucleolytic activity of the J RNases. (B) Class II mRNA (shown in red) is less sensitive to the J RNases, so cleavage of these mRNAs does not begin until Class I messages are depleted, freeing RNase J1 and J2 to bind to Class II mRNAs. Cleavage of Class II mRNAs is initiated endonucleolytically by the J RNases, releasing products similar to those produced from Class I mRNAs. These products are degraded 5′-to-3′ by RNase J1 and J2 and 3′-to-5′ by the 3′-to-5′ exonucleases, predominantly PNPase.

Figure 2

Decay rates of chimeric messages. The sag transcript is represented in red and the has transcript in blue. All transcripts are produced from Psag. The 5′-UTR begins with the transcription start, includes the ribosome binding site and ends just upstream of the translation initiation codon. The next segment starts with the translation initiation codon (AUG for sag and GUG for has), ends with the translation stop codon and comprises sagA for sag or hasABC for has. The 3′-UTR starts immediately after the translation stop codon and includes the predicted transcription pause site for sag or transcription terminator for has (lollipop). The messages were assembled using overlapping PCR and cloned under the Psag promoter (bent arrow) into plasmid pJRS9508 to generate plasmids: (1) pJRS2187; (2) pJRS1293, (3) pEU7745; (4) pEU7748; (5) pEU7753; (6) pEU7754. The chimeric constructs were expressed in the GAS strain JRS1288 (Δcov Δsag MGAS315) as described. GAS was grown to 1 hour into stationary phase in Todd Hewitt broth with pH adjusted to 7.5 with 100 mM HEPES, transcription was stopped by addition of rifampicin to the culture and RNA was isolated from cells collected at four to six consequent time points after rifampicin addition as described. The amounts for sag and has messages in each RNA sample were determined by Q-RT-PCR (four technical replicates for each sample) with primers designed to detect the middle part of the translated portion for sagA or hasA mRNA. RNA isolated from at least two independent cultures was used for Q-RT-PCR for constructs 1 to 5 and from a single culture for construct 6. Decay rates were confirmed using northern blot analysis, for constructs in lines 1, 2 and 4.