Technical guide for genetic advancement of underdeveloped and intractable Clostridium

Abstract

In recent years, the genus Clostridium has risen to the forefront of both medical biotechnology and industrial biotechnology owing to its potential in applications as diverse as anticancer therapy and production of commodity chemicals and biofuels. The prevalence of hyper-virulent strains of C. difficile within medical institutions has also led to a global epidemic that demands a more thorough understanding of clostridial genetics, physiology, and pathogenicity. Unfortunately, Clostridium suffers from a lack of sophisticated genetic tools and techniques which has hindered the biotechnological exploitation of this important bacterial genus. This review provides a comprehensive summary of biotechnological progress made in clostridial genetic tool development, while also aiming to serve as a technical guide for the advancement of underdeveloped clostridial strains, including recalcitrant species, novel environmental samples, and non-type strains. Relevant strain engineering techniques, from genome sequencing and establishment of a gene transfer methodology through to deployment of advanced genome editing procedures, are discussed in detail to provide a blueprint for future clostridial strain construction endeavors. It is expected that a more thorough and rounded-out genetic toolkit available for use in the clostridia will bring about the construction of superior bioprocessing strains and a more complete understanding of clostridial genetics, physiology, and pathogenicity.

Introduction

Clostridium is one of the largest genera of prokaryotes and is comprised of approximately 200 distinct species of bacteria (Wiegel et al., 2006). Prior to the advent of 16S rRNA sequencing, subscription to the genus was traditionally granted based on the fulfillment of three simple criteria, namely the capacity to form endospores, an obligately anaerobic metabolism, and an inability to perform sulfate reduction (Gottschalk et al., 1981). In practice, the clostridia are more generally recognized by distinct rod-shaped cells, the presence of Gram-positive cell walls, and DNA of low guanine + cytosine content (GC content; typically 26–32%), although these characteristics are not strict taxonomic markers. As a result of such simple classification requirements, Clostridium is notorious for its extreme phylogenetic heterogeneity and possesses more genetic diversity than almost all other microbial genera (Wiegel et al., 2006). In fact, Clostridium contains numerous uncharacteristic phenotypes, including species that are cocci-shaped, asporogenous, aerotolerant, intermediate in GC content, and Gram-negative or Gram-variable (Finegold et al., 2002). As such, 16S rRNA cataloging and next generation genome sequencing have led to several massive proposed rearrangements of Clostridium (Gupta and Gao, 2009, Ludwig et al., 2009, Rainey et al., 2009, Yutin and Galperin, 2013). Only 73 species were found to possess sufficient relatedness to the type species, C. butyricum, leading to the proposal of a new core genus, termed Clostridium sensu stricto (Lawson et al., 1993, Wiegel et al., 2006). The heterogeneity of the genus allows Clostridium as a whole to thrive within diverse habitats, as the clostridia are truly ubiquitous in nature. Owing to their ability to form endospores that are resistant to oxygen, heat, desiccation, acid, and alcohol, dormant spores of Clostridium are commonly found within soil, aquatic sediments, intestinal tracts of mammals, and unpasteurized or spoiled foods (Jones and Woods, 1986). From this rich diversity many species of Clostridium have arisen in recent years in both the medical and industrial sectors of biotechnology.

Clostridium can be divided into pathogenic and apathogenic species. Some of the most notorious and potent human toxins are produced by the pathogenic clostridia, which encompasses C. botulinum, C. difficile, C. perfringens, C. septicum, and C. tetani (Shone and Hambleton, 1989). C. botulinum and C. tetani produce two of the most potent neurotoxins and are the causative agents of the well-known diseases botulism and tetanus, respectively. The botulism toxin is the most toxic agent currently known to humans, as type H toxin was recently found to have a lethal dose of 2–13 ng for an average-sized human (Barash and Arnon, 2014). The advent of proper food handling and pasteurization practices in the case of botulism, and access to effective vaccination programs for the prevention of tetanus, have drastically diminished the prevalence of these fatal diseases within the developed world. Still, an estimated 58,000 fatal cases of newborn tetanus were reported globally in 2010 and, as of 2012, 31 countries have yet to effectively eliminate maternal and neonatal tetanus (World Health Organization; http://www.who.int/). C. difficile, on the other hand, produces two highly virulent exotoxins and is the leading cause of antibiotic-associated diarrhea and nosocomial infection (McFarland et al., 1989). Dormant spores of C. difficile often survive routine hospital sanitation practices, including exposure to alcohol-based hand rubs, and can be ingested through the fecal–oral route, allowing proliferation in the small intestine of patients with compromised gut flora, typically resulting from antibiotic treatment. Infection by C. difficile causes a range of intestinal maladies, including severe diarrhea, pseudomembranous colitis, septicemia, and death (Shone and Hambleton, 1989). The occurrence of healthcare-associated C. difficile infection has increased drastically over the past decade and has risen to epidemic status (Chalmers et al., 2010, Kelly and LaMont, 2008). Other pathogenic clostridia, particularly C. perfringens and C. septicum, are associated with food poisoning, gas gangrene, and meningitis, among other ailments (Shone and Hambleton, 1989). It is estimated that C. perfringens accounts for more than 10% of the 9.4 million annual cases of foodborne illness in the United States (Scallan et al., 2011).

In addition to the pathogenic clostridia, a number of medically-important species have garnered significant research focus in recent years. Of special interest is the anticancer property exhibited by certain apathogenic species (Van Mellaert et al., 2006). As deoxygenated tissue is generally only found in tumors, spores of Clostridium can be injected and targeted to anoxic regions of tumors with impeccable specificity, where they proliferate and promote cytotoxicity and oncolysis (tumor destruction). This idea was first demonstrated in 1955 when only cancerous mice contracted tetanus upon injection of C. tetani spores into both healthy and tumor-bearing specimens (Malmgren and Flanigan, 1955). Species of Clostridium found to possess significant anticancer properties include C. acetobutylicum, C. butyricum, C. novyi, C. sporogenes, and C. tyrobutyricum (Dang et al., 2001, Thiele et al., 1964, Van Mellaert et al., 2006). Strains exhibiting natural cytotoxic properties can also be modified genetically to express an anticancer agent, such as cytotoxic proteins, cytokines, and antigens or antibodies (Forbes, 2010). Expression of the corresponding anticancer genes can then be controlled through the use of an inducible genetic promoter, such as one induced by radiation (Nuyts et al., 2001a, Nuyts et al., 2001b). As such, Clostridium anticancer therapy remains a highly active area of research, as reports detailing clostridial therapies lag only behind work with Salmonella (Forbes, 2010).

Within the apathogenic clostridia, several species have garnered immense interest in the field of industrial biotechnology as a result of their diversity of substrate utilization and unique metabolic capabilities (Tracy et al., 2012). While many clostridia produce standard fermentation products, including organic acids and carbon dioxide and hydrogen gases, a number of species produce varying amounts of alcohols and solvents, such as acetone, ethanol, 1,3-propanediol, isopropanol, and butanol, which have industrial potential as bulk solvents and prospective biofuels (Gottschalk et al., 1981). In fact, the exploitation of clostridia for large-scale production of commodity chemicals represents one of the first worldwide industrial bioprocesses (Jones and Woods, 1986). Prior to the dominance of the current petrochemical industry, large-scale production of acetone, and later butanol, as solvents (AB fermentation) was carried out by species of Clostridium. Fluctuating costs of molasses and maize feedstocks, coupled to the establishment of more economical petrochemical processes during the 1950s, however, led to the eventual downfall of fermentative AB production. A potential revival of the industrial AB fermentation for the production of butanol, this time as a promising biofuel, is currently underway in response to the mounting environmental and political issues surrounding the production and consumption of petroleum-based fuels (Awang et al., 1988). Unfortunately, many process shortcomings have yet to be resolved, including high feedstock costs, poor solvent yields, and product toxicity (Zheng et al., 2009). The revival of a competitive AB fermentation process depends upon the resolution of these fundamental issues. However, it is the capacity to genetically manipulate the solventogenic clostridia that determines the future success of clostridial production of bulk solvents and biofuels (Papoutsakis, 2008).

The overall state of genetic engineering within the Clostridium is sparse given the immense medical and industrial biotechnological potentials surrounding many species of Clostridium. Of the approximately 200 traditionally-classified species, only a small subset of the clostridia has been probed with genome sequencing and manipulated using gene transfer methods (Table 1). Further, numerous landmark advancements have been made since the advent of recombinant DNA technology (Papoutsakis, 2008), yet even the most genetically advanced clostridia lag far behind other microbes, especially the model organisms Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis, in terms of access to genetic tools and technologies. In fact, only a small collection of clostridia have experienced any significant degree of genetic advancement. Accordingly, the objective of this review is two-fold. First, we aim to provide a comprehensive review of the genetic tools and methodologies currently available for the manipulation of clostridial species, with an emphasis on significant advancements made in recent years. Second, we present a series of technical guidelines for researchers aiming to genetically manipulate a Clostridium species or strain for which few or no previous genetic methodologies have been described. Comprehensive genetic work within the genus, including genome sequencing, the development of gene transfer procedures, host–vector systems, gene overexpression, knockout, and knockdown tools, and advanced genome editing technologies, is required for the advancement of lesser known species, non-type strains, and environmental isolates of Clostridium. In-depth experimental aspects of relevant genetic tools and techniques are discussed. A schematic template depicting the workflow involved in the development of clostridial host strains, from establishing plasmid transformation and genome sequencing through to advanced genome editing technologies, is shown in Fig. 1. Readers are also directed toward recent reviews pertaining to various aspects of clostridial genetics, metabolism, and pathogenicity discussed herein (Gheshlaghi et al., 2009, Lee et al., 2008, Lutke-Eversloh and Bahl, 2011, Minton, 2003, Papoutsakis, 2008, Tracy et al., 2012, Van Mellaert et al., 2006). Our hope is that this review facilitates the expansion and development of the genetic engineering tools available to the clostridia, so that the full potential of this important genus can be realized.