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The widely conserved protein CsrA (carbon storage regulator A) globally regulates bacterial gene expression at the post-transcriptional level. In many species, CsrA activity is governed by untranslated sRNAs, CsrB and CsrC in Escherichia coli, which bind to multiple CsrA dimers, sequestering them from lower affinity mRNA targets. Both the synthesis and turnover of CsrB/C are regulated. Their turnover requires the housekeeping endonuclease RNase E and is activated by the presence of a preferred carbon source via the binding of EIIA(Glc) of the glucose transport system to the GGDEF-EAL domain protein CsrD. We demonstrate that the CsrB 3' segment contains the features necessary for CsrD-mediated decay. RNase E cleavage in an unstructured segment located immediately upstream from the intrinsic terminator is necessary for subsequent degradation to occur. CsrA stabilizes CsrB against RNase E cleavage by binding to two canonical sites adjacent to the necessary cleavage site, while CsrD acts by overcoming CsrA-mediated protection. Our genetic, biochemical and structural studies establish a molecular framework for sRNA turnover by the CsrD-RNase E pathway. We propose that CsrD evolution was driven by the selective advantage of decoupling Csr sRNA decay from CsrA binding, connecting it instead to the availability of a preferred carbon source.

beta-1,6-N-Acetylglucosaminidase (dispersin B), which cleaves poly-ss-(1,6)-linked N-acetylglucosamine, is encoded by dspB of Aggregatibacter actinomycetemcomitans. To enhance the production of dispersin B, we engineered dspB to transcribe mRNAs devoid of the trinucleotide ACA. Transcription and translation levels of ACA-less and wild-type dspB expressed in Escherichia coli (E. coli) under T5 and T7 promoters were analyzed by real-time RT-PCR and protein quantification, respectively. The ACA-less dspB mRNA level was significantly higher (P < 0.01) and produced 77.6 and 34.9% more dispersin B than wild-type dspB expressed under T7 and T5 promoters, respectively. Dispersin B expression under T7 promoter caused a 98-99.5% drop in the glyceraldehyde-3-phosphate dehydrogenase (gapA) mRNA level, which was not observed with T5 promoter. Fusion of green fluorescent protein (GFP) with dispersin B allowed rapid quantification of dispersin B production by measuring fluorescence intensity in culture broth. Although the cultures containing 0.1% glucose showed sustained increase in dispersin B-GFP production until 12 h, no significant increase in dispersin B activity was observed beyond 4 and 6 h after induction when expressed under T7 and T5 promoters, respectively. This study demonstrates the effectiveness of ACA-less mRNA and the advantage of GFP tagging for enhanced dispersin B production and quantification, which could be adapted for improving the production of other commercially important proteins in E. coli.

Alginase-secreting bacteria associated with actively growing tissues of the marine Phaeophyta speciesSargassum fluitans andS. natans have been isolated and evaluated for their ability to degrade alginate (ALG), carboxymethylcellulose, and agar. Of seven isolates selected for their ability to grow on 2% agar containing 1% sodium alginate, none were able to grow on either 2% agar or 2% agar supplemented with 0.1% carboxymethylcellulose. Two of these with fermentative potential, i.e., ALG-A and ALG-G, showed selective activities with respect to their ability to degrade native alginate and/or take up the products resulting from alginate degradation. The ALG-A isolate was able to rapidly degrade native alginate with the generation of a stable polymer fraction and small oligouronides, most of which were dissimilated for growth. The ALG-G isolate was able to completely degrade native alginate with the accumulation of significant quantities of unsaturated dimeric and trimeric oligouronides. A limit polymer was generated from the action of a polymannuronan-specific extracellular alginate lyase purified from exponential cultures of the ALG-A organism. This product proved to be an effective substrate for the alginate lyase activity obtained from the medium of exponential phase cultures of the ALG-G isolate, and upon incubation with concentrated and dialyzed ALG-G medium was converted to the products that were observed to accumulate in the medium of the ALG-G isolate grown on native alginate. These organisms represent examples of the microflora associated with actively growingSargassum tissues, each with a selective ability to degrade and dissimilate the biomass of the marine brown algae.