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ABSTRACT A type II VapB14 antitoxin regulates biofilm dispersal in the archaeal thermoacidophile Sulfolobus acidocaldarius through traditional toxin neutralization but also through noncanonical transcriptional regulation. Type II VapC toxins are ribonucleases that are neutralized by their proteinaceous cognate type II VapB antitoxin. VapB antitoxins have a flexible tail at their C terminus that covers the toxin’s active site, neutralizing its activity. VapB antitoxins also have a DNA-binding domain at their N terminus that allows them to autorepress not only their own promoters but also distal targets. VapB14 antitoxin gene deletion in S. acidocaldarius stunted biofilm and planktonic growth and increased motility structures (archaella). Conversely, planktonic cells were devoid of archaella in the Δ vapC14 cognate toxin mutant. VapB14 is highly conserved at both the nucleotide and amino acid levels across the Sulfolobales, extremely unusual for type II antitoxins, which are typically acquired through horizontal gene transfer. Furthermore, homologs of VapB14 are found across the Crenarchaeota , in some Euryarchaeota , and even bacteria. S. acidocaldarius vapB14 and its homolog in the thermoacidophile Metallosphaera sedula (Msed_0871) were both upregulated in biofilm cells, supporting the role of the antitoxin in biofilm regulation. In several Sulfolobales species, including M. sedula, homologs of vapB14 and vapC14 are not colocalized. Strikingly, Sulfuracidifex tepidarius has an unpaired VapB14 homolog and lacks a cognate VapC14, illustrating the toxin-independent conservation of the VapB14 antitoxin. The findings here suggest that a stand-alone VapB-type antitoxin was the product of selective evolutionary pressure to influence biofilm formation in these archaea, a vital microbial community behavior. IMPORTANCE Biofilms allow microbes to resist a multitude of stresses and stay proximate to vital nutrients. The mechanisms of entering and leaving a biofilm are highly regulated to ensure microbial survival, but are not yet well described in archaea. Here, a VapBC type II toxin-antitoxin system in the thermoacidophilic archaeon Sulfolobus acidocaldarius was shown to control biofilm dispersal through a multifaceted regulation of the archaeal motility structure, the archaellum. The VapC14 toxin degrades an RNA that causes an increase in archaella and swimming. The VapB14 antitoxin decreases archaella and biofilm dispersal by binding the VapC14 toxin and neutralizing its activity, while also repressing the archaellum genes. VapB14-like antitoxins are highly conserved across the Sulfolobales and respond similarly to biofilm growth. In fact, VapB14-like antitoxins are also found in other archaea, and even in bacteria, indicating an evolutionary pressure to maintain this protein and its role in biofilm formation. 

ABSTRACT Quorum sensing (QS) enables coordinated, population-wide behavior. QS-active bacteria “communicate” their number density using autoinducers which they synthesize, collect, and interpret. Tangentially, chemotactic bacteria migrate, seeking out nutrients and other molecules. It has long been hypothesized that bacterial behaviors, such as chemotaxis, were the primordial progenitors of complex behaviors of higher-order organisms. Recently, QS was linked to chemotaxis, yet the notion that these behaviors can together contribute to higher-order behaviors has not been shown. Here, we mathematically link flocking behavior, commonly observed in fish and birds, to bacterial chemotaxis and QS by constructing a phenomenological model of population-scale QS-mediated phenomena. Specifically, we recast a previously developed mathematical model of flocking and found that simulated bacterial behaviors aligned well with well-known QS behaviors. This relatively simple system of ordinary differential equations affords analytical analysis of asymptotic behavior and describes cell position and velocity, QS-mediated protein expression, and the surrounding concentrations of an autoinducer. Further, heuristic explorations of the model revealed that the emergence of “migratory” subpopulations occurs only when chemotaxis is directly linked to QS. That is, behaviors were simulated when chemotaxis was coupled to QS and when not. When coupled, the bacterial flocking model predicts the formation of two distinct groups of cells migrating at different speeds in their journey toward an attractant. This is qualitatively similar to phenomena spotted in our Escherichia coli chemotaxis experiments as well as in analogous work observed over 50 years ago. IMPORTANCE Our modeling efforts show how cell density can affect chemotaxis; they help to explain the roots of subgroup formation in bacterial populations. Our work also reinforces the notion that bacterial mechanisms are at times exhibited in higher-order organisms. 

The Clostridium genus contains a diverse range of Gram-positive, sporulating, obligate anaerobes that have been of historical biotechnological interest due to their acetone–butanol–ethanol (ABE) solvent production. Within the last few decades, interest has grown in the Clostridium spp. capable of consuming a wider variety of feedstocks, which include gaseous and renewable biomass sources. Additionally, attenuated pathogens have been of interest as potential therapeutics. The fruition of the genus's great promise has been limited by the slow progress in genetic engineering and synthetic biology methods and tools, relative to workhorse organisms such as Escherichia coli . Recent advances in these areas, not least of which include CRISPR-based tools, renew the promise of metabolic engineering for a broad range of feedstock consumption and production of chemicals. In this chapter, we describe the current state of engineering in the Clostridium genus by describing efforts and continued challenges in directed evolution, the use of systems biology for greater understanding, methods for performing genomic editing, and the expanding library of genetic parts. These new capabilities and tools have expanded the number of species that are able to be engineered for biotechnological purposes, increased the throughput of genetic studies, and expanded the range of products made from Clostridium.