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Transcriptional induction and expression of the endoglucanase celA gene from a ruminal Clostridium sp. ("C. longisporum").

ABSTRACT: Northern (RNA) blot analysis of RNA from Clostridium sp. revealed induction of transcription of the celA gene when barley beta-glucan was used as carbon source, while no celA mRNA was detected after growth on cellobiose. Western blots (immunoblots), prepared by using a rabbit antiserum raised against CelA protein purified from Escherichia coli, revealed the extracellular location of CelA in Clostridium sp. Despite the absence of detectable celA mRNA, significant quantities of CelA were detected in the culture supernatant during growth on cellobiose. This finding indicated a low constitutive expression of celA. A 6.7-fold increase in the total beta-glucanase specific activity in the extracellular fraction was observed during growth on beta-glucan. The transcriptional start site of celA was mapped by extension and was found to be the same in Clostridium sp. and in E. coli expressing the cloned celA gene. A consensus E. coli -10 promoter region (AATAAT), but not a -35 promoter region, could be identified. Two direct repeats (TATTGAATTTAT) separated by 15 nucleotides flank the region where the consensus -35 promoter regions would have been. The size of the celA mRNA transcript corresponded with the size of the open reading frame. A potential stem-loop structure was found 18 nucleotides downstream of the 3' stop codon, which could be responsible for termination of transcription.

SUBMITTER: Mittendorf V

PROVIDER: S-EPMC177248 | biostudies-other | 1995 Aug

REPOSITORIES: biostudies-other

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Transcriptional induction and expression of the endoglucanase celA gene from a ruminal Clostridium sp. ("C. longisporum").

Mittendorf V V Thomson J A JA

Journal of bacteriology 19950801 16

Northern (RNA) blot analysis of RNA from Clostridium sp. revealed induction of transcription of the celA gene when barley beta-glucan was used as carbon source, while no celA mRNA was detected after growth on cellobiose. Western blots (immunoblots), prepared by using a rabbit antiserum raised against CelA protein purified from Escherichia coli, revealed the extracellular location of CelA in Clostridium sp. Despite the absence of detectable celA mRNA, significant quantities of CelA were detected ...[more]

PMID: 7642509

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Project description:Subacute ruminal acidosis (SARA) negatively impacts the dairy industry by decreasing dry matter intake, milk production, profitability, and increasing culling rate and death loss. Six ruminally cannulated, lactating Holstein cows were used in a replicated incomplete Latin square design to determine the effects of SARA induction on the ruminal microbiome and epithelium. Experimental periods were 10 days with days 1-3 for ad libitum intake of control diet, followed by 50% feed restriction on day 4, and ad libitum access on day 5 to the basal diet or the basal diet with an additional 10% of a 50:50 wheat/barley pellet. Based on subsequent ruminal pH, cows were grouped (SARA grouping; SG) as Non-SARA or SARA based on time <5.6 pH (0 and 3.4 h, respectively). Ruminal samples were collected on days 1 and 6 of each period prior to feeding and separated into liquid and solid fractions. Microbial DNA was extracted for bacterial analysis using 16S rRNA gene paired-end sequencing on the MiSeq Illumina platform and quantitative PCR (qPCR). Ruminal epithelium biopsies were taken on days 1 and 6 before feeding. Quantitative RT-PCR was used to determine gene expression in rumen epithelium. Bray-Curtis similarity indicated samples within the liquid fraction separated by day and coincided with an increased relative abundance of genera Prevotella, Ruminococcus, Streptococcus, and Lactobacillus on day 6 (P < 0.06). Although Firmicutes was the predominant phyla in the solid fraction, a SG × day interaction (P < 0.01) indicated a decrease on day 6 for SARA cows. In contrast, phylum Bacteroidetes increased on day 6 (P < 0.01) for SARA cows driven by greater genera Prevotella and YRC22 (P < 0.01). Streptococcus bovis and Succinivibrio dextrinosolvens populations tended to increase on day 6 but were not affected by SG. In ruminal epithelium, CLDN1 and CLDN4 expression increased on day 6 (P < 0.03) 24 h after SARA induction and a tendency for a SG × day interaction (P < 0.10) was observed for CLDN4. Overall, results indicate more rapid adaptation to an induced bout of SARA in the solid fraction ruminal microbiome compared with ruminal epithelium.

Dataset Information

Transcriptional induction and expression of the endoglucanase celA gene from a ruminal Clostridium sp. ("C. longisporum").

Publications

Transcriptional induction and expression of the endoglucanase celA gene from a ruminal Clostridium sp. ("C. longisporum").

Similar Datasets

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