Agr Quorum Sensing influences the Wood-Ljungdahl pathway in Clostridium autoethanogenum

Acetogenic bacteria are capable of fermenting CO2 and carbon monoxide containing waste-gases into a range of platform chemicals and fuels. Despite major advances in genetic engineering and improving these biocatalysts, several important physiological functions remain elusive. Among these is quorum sensing, a bacterial communication mechanism known to coordinate gene expression in response to cell population density. Two putative agr systems have been identified in the genome of Clostridium autoethanogenum suggesting bacterial communication via autoinducing signal molecules. Signal molecule-encoding agrD1 and agrD2 genes were targeted for in-frame deletion. During heterotrophic growth on fructose as a carbon and energy source, single deletions of either gene did not produce an observable phenotype. However, when both genes were simultaneously inactivated, final product concentrations in the double mutant shifted to a 1.5:1 ratio of ethanol:acetate, compared to a 0.2:1 ratio observed in the wild type control, making ethanol the dominant fermentation product. Moreover, CO2 re-assimilation was also notably reduced in both hetero- and autotrophic growth conditions. These findings were supported through comparative proteomics, which showed lower expression of carbon monoxide dehydrogenase, formate dehydrogenase A and hydrogenases in the ∆agrD1∆agrD2 double mutant, but higher levels of putative alcohol and aldehyde dehydrogenases and bacterial micro-compartment proteins. These findings suggest that Agr quorum sensing, and by inference, cell density play a role in carbon resource management and use of the Wood-Ljungdahl pathway as an electron sink.

Effects of Dietary Inclusion of Clostridium autoethanogenum Protein on the Growth Performance and Liver Health of Largemouth Bass (Micropterus salmoides)

Clostridium autoethanogenum protein (CAP) is a novel protein source for aqua-feeds. The present study aimed to investigate the effects of dietary CAP on growth performance, immunity, and liver health status of juvenile largemouth bass (Micropterus salmoides). Four isonitrogenous and isolipid experimental diets were formulated to replace 0% (D1, control), 25% (D2), 50% (D3), and 75% (D4) of fish meal by CAP. Fish (15.05 ± 0.08 g) were randomly fed one of four experimental diets for 8 weeks. The results showed that weight gain (WG), specific growth rate (SGR), feeding rate (FR), viscerosomatic index (VSI), and hepatosomatic index (HSI) of the D4 group were significantly lower than D1, D2, and D3 groups (P < 0.05). With the increase of substitution level, the total antioxidant capacity (T-AOC) of liver tissue was significantly decreased, while the plasma alkaline phosphatase (AKP) activity was significantly increased (P < 0.05). Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were significantly higher in D3 and D4 groups than in D1 and D2 groups (P < 0.05). Replacing 50 or 75% fish meal by CAP significantly induced the transcription level of apoptosis-promoting genes (bcl-2-associated death protein [bad] and bcl-2-assoxicated × protein; bag [bax]), anti-apoptosis-related genes (tumor protein 53 [p53] and b-cell lymphoma-2 [bcl-2]), and the apoptotic Caenorhabditis elegans (C. elegans) death gene-3 like caspases (cysteine-aspartic proteases-3 [caspase-3], cysteine-aspartic proteases-8 [caspase-8], cysteine-aspartic proteases-9 [caspase-9], and cysteine-aspartic proteases-10 [caspase-10]) in liver, while suppressed the gene expression of the inflammatory factors [interleukin-1β (il-1β), interleukin-8 (il-8), and tumor necrosis factor, tnf ] in head kidney. At the same time, dietary inclusion of CAP elevated the protein expression of bcl-2, autophagy microtubule-associated protein light chain 3A/B (LC3A/B-I), and LC3A/B-II by inhibiting the phosphorylation of the mammalian target of rapamycin (mTOR; P < 0.05). Moreover, the apoptosis rate of the D3 and D4 groups was significantly increased (P < 0.05). Taken together, these results indicated that the optimal level of CAP-replacing fish meal should be <50% that has no negative effect on the growth performance and liver health of juvenile largemouth bass. In addition, excessive CAP inclusion may damage liver health by activating autophagy and apoptosis signaling pathways.

Spacer2PAM: A computational framework for identification of functional PAM sequences for endogenous CRISPR systems

RNA-guided nucleases from clustered regularly interspaced short palindromic repeats (CRISPR) systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR systems in many bacteria and archaea are particularly attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR effectors. However, each CRISPR system recognizes a unique set of PAM sequences, which requires extensive screening of randomized DNA libraries. This challenge makes it difficult to develop endogenous CRISPR systems, especially in organisms that are slow-growing or have transformation idiosyncrasies. To address this limitation, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to identify potential PAM sequences for any CRISPR system given its corresponding CRISPR array as input. Spacer2PAM can be used in Quick mode to generate a single PAM prediction that is likely to be functional or in Comprehensive mode to inform targeted, unpooled PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-predicted PAM library for the type I-B CRISPR system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR systems for industrial biotechnology and synthetic biology.