A major checkpoint for protein expression in Rhodobacter sphaeroides during heat stress response occurs at the level of translation

Abstract

Temperature above the physiological optimum is a stress condition frequently faced by bacteria in their natural environments. Here, we were interested in the correlation between levels of RNA and protein under heat stress. Changes in RNA and protein levels were documented in cultures of Rhodobacter sphaeroides using RNA sequencing, quantitative mass spectrometry, western blot analysis, in vivo [ 35 S] methioninelabelling and plasmid-borne reporter fusions. Changes in the transcriptome were extensive. Strikingly, the proteome remained unchanged except for very few proteins. Examples include a heat shock protein, a DUF1127 protein of unknown function and sigma factor proteins from leaderless transcripts. Insight from this study indicates that R. sphaeroides responds to heat stress by producing a broad range of transcripts while simultaneously preventing translation from nearly all of them, and that this selective production of protein depends on the untranslated region of the transcript. We conclude that measurements of transcript abundance are insufficient to understand gene regulation. Rather, translation can be an important checkpoint for protein expression under certain environmental conditions. Furthermore, during heat shock, regulation at the level of transcription might represent preparation for survival in an unpredictable environment while regulation at translation ensures production of only a few proteins.

Design–Build–Test of Synthetic Promoters for Inducible Gene Regulation in Alphaproteobacteria

“…sphaeroides, previously shown to be moderately active in R. sphaeroides during growth in defined medium . PphaP was modified with flanking Lac operators to generate PphaP-O.…”

“…To see if a promoter other than P16S could be combined with the Lac operators, we used the phaP promoter (PphaP) from R. sphaeroides, previously shown to be moderately active in R. sphaeroides during growth in defined medium. 25 PphaP was modified with flanking Lac operators to generate PphaP-O. This modification was performed on the basis of the identifiable promoter elements (see Figure S5 for alignment of promoters) and comparison to the Lac and 16S-O promoters.…”

Genetic Engineering of Agrobacterium Increases Curdlan Production through Increased Expression of the crdASC Genes

“…Previous work on this gene found that it was strongly active in the exponential growth phase of R. sphaeroides [ 52 ]. Interestingly, upon heat shock, transcription of the phaP gene increased by over 20-fold [ 53 ], making the promoter of phaP one of the most active promoters that I have ever observed. Indeed, PphaP showed very high activity in Agrobacterium strains ( Figure 4 ).…”

“…The key to engineering a higher transcription rate for the crdASC gene cluster is the knowledge of suitable promoters capable of strong transcription in agrobacterium . During investigations on transcription regulation in the alphaproteobacteria, a strong promoter named PphaP (promoter of gene phaP ) from the alphaproteobacterium rhodobacter sphaeroides was found, which is responsible for production of the phasin protein which binds to the polyhydroxyalkanoate granule [ 52 , 53 ]. I decided to test whether curdlan production could be improved using PphaP in the industrially used strain agrobacterium sp.…”

A Small RNA, UdsC, Interacts with the RpoHII mRNA and Affects the Motility and Stress Resistance of Rhodobacter sphaeroides

“…An interaction between the RpoHII 5′ UTR and UdsC was also supported by an electromobility assay. RpoHII is known as an important regulator in several stress responses of R. sphaeroides , e.g., oxidative stress, heat stress and stationary phase [ 12 , 13 , 14 , 31 , 32 , 33 ]. It activates a rather large regulon that partially overlaps with the RpoHI regulon [ 34 ].…”