Revisiting the Regulation of the Primary Scaffoldin Gene in Clostridium thermocellum

Abstract

Cellulosomes are considered to be one of the most efficient systems for the degradation of plant cell wall polysaccharides. The central cellulosome component comprises a large, noncatalytic protein subunit called scaffoldin. Multiple saccharolytic enzymes are incorporated into the scaffoldins via specific high-affinity cohesin-dockerin interactions. Recently, the regulation of genes encoding certain cellulosomal components by multiple RNA polymerase alternative σ factors has been demonstrated in () In the present report, we provide experimental evidence demonstrating that the gene, which encodes the primary cellulosomal scaffoldin, is regulated by several alternative σ factors and by the vegetative σ factor. Furthermore, we show that previously suggested transcriptional start sites (TSSs) of are actually posttranscriptional processed sites. By using comparative bioinformatic analysis, we have also identified highly conserved σ- and σ-dependent promoters upstream of the primary scaffoldin-encoding genes of other clostridia, namely, ,, , and sp. strain Bc-iso-3. Interestingly, a previously identified TSS of the primary scaffoldin CbpA gene of matches the predicted σ-dependent promoter identified in the present work rather than the previously proposed σ promoter. With the exception of , both σ and σ promoters of primary scaffoldin genes are located more than 600 nucleotides upstream of the start codon, yielding long 5'-untranslated regions (5'-UTRs). Furthermore, these 5'-UTRs have highly conserved stem-loop structures located near the start codon. We propose that these large 5'-UTRs may be involved in the regulation of both the primary scaffoldin and other cellulosomal components. Cellulosome-producing bacteria are among the most effective cellulolytic microorganisms known. This group of bacteria has biotechnological potential for the production of second-generation biofuels and other biocommodities from cellulosic wastes. The efficiency of cellulose hydrolysis is due to their cellulosomes, which arrange enzymes in close proximity on the cellulosic substrate, thereby increasing synergism among the catalytic domains. The backbone of these multienzyme nanomachines is the scaffoldin subunit, which has been the subject of study for many years. However, its genetic regulation is poorly understood. Hence, from basic and applied points of view, it is imperative to unravel the regulatory mechanisms of the scaffoldin genes. The understanding of these regulatory mechanisms can help to improve the performance of the industrially relevant strains of and related cellulosome-producing bacteria to the consolidated bioprocessing of biomass.

Regulation of biomass degradation by alternative σ factors in cellulolytic clostridia

“…Consequently, the A-tract motif of σ I -dependent promoters may also serve to avoid their recognition by ECF σ factors. This idea is further supported by the observation that none of the σ I -dependent promoters, which have been tested in our laboratory by using the B. subtilis heterologous host cell system (that is devoid of its native σ I /RsgI system), were activated by the resident B. subtilis σ factors 10 , 11 .…”

“…S1 ) are underlined. The TSS of sigI1 , sigI6 , cipA , xyn11B and xyn10Z were identified in previous works 9 , 11 , 43 . Distances between the promoter sequences and the first codon of corresponding genes are shown in the column labeled 5′-UTR (5′-untranslated region).…”

“…1B ). These genes encode the primary scaffoldin of the cellulosome, CipA 11 , 18 , 25 , and the most abundant enzyme in the cellulosome, Cel48S 6 , 26 , 27 , respectively.…”

“…To confirm the implication of the region of specificity in the stringency of σ I -dependent promoters, we searched for σ I -dependent promoters in the genome of P. cellulosolvens that resemble those of the C. thermocellum σ I3 -dependent promoters. P. cellulosolvens was chosen for further comparison, because it produces the most complex cellulosome known to date 23 , and a previous report showed that as the cellulosome becomes more elaborate, the bacterium harbors more σ I factors 11 . This observation was confirmed by our analysis which revealed that P. cellulosolvens genome encodes 16 σ I paralogues (Supplementary Table S4 ), making this bacterium an excellent model for the study of multiple σ I factors.…”

Pan-Cellulosomics of Mesophilic Clostridia: Variations on a Theme

“… Sequence conservation of the major σ A -dependent promoters upstream of the respective cip gene cluster. ( A ) The σ A (RpoD)-dependent promoter and cognate transcription start site (S1) have been experimentally identified as a major region of the C. cellulolyticum H10 cipC gene [ 52 ] and its orthologs [ 54 ]. The two T nucleotides of S1 are underlined, as well as sequences predicted to be −35, −16 and −10 elements of the cipC promoter; ( B ) aligned sequences are related to the recently identified RpoD-dependent promoter of the C. thermocellum cipA gene [ 54 ].…”