Regulation of Expression of Cellulosomal Cellulase and ...

Abstract

The study focuses on the regulation of gene expression for cellulases and hemicellulases in Clostridium cellulovorans at the mRNA level, examining cells grown under various culture conditions. Results indicate a fundamental expression pattern and relative expression levels based on the sugar composition of the growth media, including poly-, di-, and monomeric sugars. When cells were cultured in media containing cellobiose or cellulose, there was concerted expression of cellulase genes (cbpA and engE) along with the hemicellulase gene (xynA). Notably, growing cells with cellulose, xylan, and pectin resulted in pronounced expression of many genes, including cbpA-exgS, engH, hbpA, manA, engM, engE, xynA, and pelA. Moderate levels of expression for cbpA, engH, manA, engE, and xynA were observed when cellobiose or fructose served as carbon sources. Conversely, very low mRNA levels were detected for cbpA, manA, engE, and xynA in cells grown on lactose, mannose, and locust bean gum, with negligible expression in cultures fed on glucose, galactose, maltose, or sucrose. Throughout all conditions assessed, the cbpA-exgS and engE genes displayed the highest expression frequencies, while xynA and pelA expression was more robustly induced in xylan- or pectin-supplemented media, respectively. The experiments confirm hypotheses surrounding the coordinate expression of certain cellulases and hemicellulases, illustrating a catabolite repression mechanism that governs cellulase expression in rapidly growing cells and highlighting the influence of hemicelluloses on cellulose utilization.

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Plant cell walls comprise the primary components of cellulose, hemicellulose, and lignin. Cellulose is the predominant element, with hemicelluloses following closely behind. Cellulose is a collection of long polymers formed from β-1,4-linked glucose units, establishing a crystal-like architecture, while hemicelluloses maintain a more variable structure. Various types of hemicelluloses include xylan (composed of β-1,4-linked xylose units), glucomannans (see β-1,4-linked glucose and mannose units), as well as arabinans and galactans derived from arabinose and galactose, respectively. Cellulolytic bacteria produce an array of enzymes, collectively known as cellulosomes, that facilitate the hydrolysis of crystalline cellulose and hemicelluloses into oligosaccharides, ultimately yielding monosaccharides.

Clostridium cellulovorans is classified as an anaerobic, mesophilic, and sporulating bacterium recognized for its high efficiency in cellulolytic activity. The cellulases and hemicellulases—collectively referred to as (hemi-)cellulases—produced by C. cellulovorans have been extensively studied. A variety of cellulases—including family 5 and 9 endoglucanases and a family 48 exoglucanase—along with a mannanase, a xylanase, and a pectate lyase have been characterized. A group of genes coding for cellulosomal subunits, including cbpA (for a scaffolding protein), exgS (for exoglucanase), engH, engK, and engM (continued for endoglucanases), hbpA (for a hydrophilic domain and cohesin), and manA (for mannanase), have been cloned and sequenced. Additionally, the unlinked genes engE (endoglucanase), xynA (xylanase), and pelA (pectate lyase) fall under this same category of cellulosomal enzymes.

Understanding the regulation of different hydrolytic enzymes by cellulolytic microorganisms is essential as plant polysaccharides constitute the most abundant renewable biomass, playing a vital role in carbon cycling within nature. While protein-level analysis of cellulase gene expression in C. cellulovorans has been previously explored, few studies have delved into the regulatory mechanisms governing (hemi-)cellulase expression. Hence, significant queries remain unanswered concerning whether expression across different (hemi-)cellulase genes is coordinated through a shared regulatory mechanism and whether a baseline level of constitutive expression exists under all conditions. Preliminary evidence implies that the synthesis of cellulosome components is constitutive when cells are grown in glucose-rich environments. Comprehensive investigations into true induction or repression mechanisms are lacking. Consequently, this paper addresses issues associated with (hemi-)cellulase gene expression in C. cellulovorans.

MATERIALS AND METHODS

Bacterial Strain and Growth Conditions

C. cellulovorans ATCC served as the origin for genomic DNA and total RNA. The organism was maintained in strictly anaerobic conditions at 37°C within round-bottom flasks containing a previously established medium, incorporating either di- or monomeric sugars (including fructose, glucose, mannose, galactose, lactose, maltose, sucrose, and cellobiose at 0.5%, wt/vol) or polymeric sugars (such as microcrystalline cellulose [Avicel], locust bean gum, xylan, and pectin at 1%, wt/vol). Procurement of Avicel was through FMC Corporation, while locust bean gum, xylan (from birch wood), and pectin (from apples) were sourced from Sigma.

Bacterial Protein Determination

Cell mass was evaluated in cultures cultivated with substrates such as cellobiose, cellulose, locust bean gum, pectin, and xylan based on the bacterial protein estimation method described by Bensadoun and Weinstein. A 500-µl aliquot underwent centrifugation for 10 minutes at 13,000 g. The pellets were subsequently washed with sodium phosphate buffer (50 mM, pH 7.0) and incubated in sodium deoxycholate (2%) for 20 minutes. The mixture was treated with trichloroacetic acid (24%) and then centrifuged. The concentration of protein was measured using the BCA Compat-Able protein assay kit (Pierce), leveraging bovine serum albumin as the standard.

Nucleic Acid Isolation

Genomic DNA was isolated from C. cellulovorans utilizing a genomic DNA purification kit (Promega), following the manufacturer’s protocol. For total RNA extraction from C. cellulovorans cultures, an RNeasy kit (QIAGEN) was utilized, which included a treatment step with RNAlater RNA stabilization reagent (Ambion) and RNase-free DNase (Promega), adhering to the manufacturers' instructions.

Northern Blot Analysis

RNA samples (up to 20 µg) were denatured within an RNA sample buffer and then separated through 1% agarose gels embedded in MOPS buffer containing formaldehyde. DNA probes were created through PCR, sourced from specific oligonucleotides based on the C. cellulovorans sequence. Probes were nonradioactively labeled via random priming using digoxigenin (DIG) High Prime. Subsequent hybridizations were performed to determine the presence of specific transcripts on the blots using a DIG luminescent detection kit (Roche).

TABLE 1.

PCR primers were employed for amplifying reverse transcripts and for synthesizing gene-specific probes. Explore more about Yulin HB™.

RNA Slot Blot Analysis

Appropriate concentrations of total RNAs were obtained, mixed with a twofold volume of RNA sample buffer, and denatured before being applied to a positively charged nylon membrane using a Hybri-slot apparatus. The membrane was subsequently baked under vacuum conditions. Filters were hybridized using specific probes similarly to the Northern blot analysis methods previously outlined.

RT-PCR Analysis

Reverse transcriptase (RT) reactions were conducted using total RNA with a commercially available reverse transcription system, slightly amended from the recommended protocol. The mixtures were incubated and subsequently processed to yield cDNA products, which were amplified through PCR protocol involving 2.5 µl of RT reaction mixture.

DISCUSSION

This research explores the expression patterns of (hemi-)cellulase genes, utilizing mRNA isolated from continuous cultures across various time points. The findings illustrate the general and specific regulatory mechanisms influencing the expression of (hemi-)cellulase genes in C. cellulovorans, reflecting relative expression levels under differing conditions, such as various carbon sources and growth phases. Notably, cellulose and cellobiose prompted transcription for most (hemi-)cellulase genes (i.e. cbpA-exgS-engH-engK, manA, engE, and xynA). The transcriptional timeline for each gene remained largely uniform across instances. This represents the inaugural report addressing the transcriptional coordination of (hemi-)cellulase gene expression in a clostridial (hemi-)cellulolytic framework. It was particularly evident that the regulation of the noncellulosomal cellulase gene, engF, resembled that of other cellulosomal cellulase genes, though distinct time courses were visible through Northern blot analyses.

High expression levels for most genes were evident with polysaccharide substrates, with moderate expression levels detected in media enriched with cellobiose and fructose. Conversely, minimal levels of (hemi-)cellulase mRNAs were derived from cultures sustained on lactose, mannose, and locust bean gum, with almost negligible expression observed on media comprising glucose, galactose, maltose, and sucrose. These observations present a comprehensive overview concerning (hemi-)cellulase expression potential amid varying carbon sources. The consensus was that cellulase expression would not manifest in carbon sources favoring rapid growth but would be stimulated in conjunction with polysaccharides presenting challenges to degradation. Remarkably, cbpA-exgS and engE expressions were consistently strong under every condition tested. Furthermore, the relative transcript levels corresponded closely with the specific proteins exhibited in the culture medium.

Evidence suggests that (hemi-)cellulase genes in C. cellulovorans exhibit low constitutive expression yet demonstrate elevated expression levels in the presence of certain polysaccharides such as cellulose. In contrast, it has been noted that basal constitutive levels of certain cellulosomal proteins were produced when cells were sustained on glucose or cellobiose, demonstrating a complex regulatory relationship. Conclusively, while specific carbon sources can promote high expression levels for certain genes, others may exhibit minimal expression, indicating variability subject to the carbon source present. The strongest induction beneficially correlated with cellulose, which suggests the influence of moieties like cellobiose or derivatives produced within the cell.

Inductive and repressive behaviors concerning cellulases have been documented, especially with regard to mixed substrates comprised of both cellulosic and hemicellulosic sugars. Historically, it was understood that initial growth phases relied on hemicellulose consumption, transitioning to a second phase dedicated to cellulase system development. This insight posits that degradation products of select hemicelluloses potentially repress cellulolytic action at elevated concentrations, fading once their levels diminish. This further underscores the interrelated regulatory mechanisms governing (hemi-)cellulases within this bacterium.

The observed propensity for certain di- or monosaccharides such as fructose, lactose, and cellobiose to stimulate (hemi-)cellulase gene expression aligns with findings regarding other cellulolytic bacteria, yet C. cellulovorans often failed to express (hemi-)cellulases in the presence of easily metabolized sugars like glucose. Therefore, a catabolite repression type mechanism likely governs the expression of diverse genes coding for distinct extracellular hydrolases alongside the scaffolding protein.

Acknowledgments

The authors extend gratitude to Helen Chan for her adept technical assistance and contributions to media preparation.

This research received partial funding from the Research Institute of Innovative Technology for the Earth (RITE) through the Japanese Ministry of Economy, Trade, and Industry (METI), and was supported by grant DE-DDF03-92ER from the U.S. Department of Energy.

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