The Gram-positive bacterium Bacillus subtilis can initiate the process of sporulation under conditions of nutrient limitation. Here, we review some of the last five years of work in this area, with a particular focus on the decision to initiate sporulation, DNA translocation, cell-cell communication, protein localization and spore morphogenesis. The progress we describe has implications not just for the study of sporulation but also for other biological systems where homologs of sporulation-specific proteins are involved in vegetative growth.

Vibrio cholerae, the causative agent of the human disease cholera, uses cell-to-cell communication to control pathogenicity and biofilm formation. This process, known as quorum sensing, relies on the secretion and detection of signalling molecules called autoinducers. At low cell density V. cholerae activates the expression of virulence factors and forms biofilms. At high cell density the accumulation of two quorum-sensing autoinducers represses these traits. These two autoinducers, cholerae autoinducer-1 (CAI-1) and autoinducer-2 (AI-2), function synergistically to control gene regulation, although CAI-1 is the stronger of the two signals. V. cholerae AI-2 is the furanosyl borate diester (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate. Here we describe the purification of CAI-1 and identify the molecule as (S)-3-hydroxytridecan-4-one, a new type of bacterial autoinducer. We provide a synthetic route to both the R and S isomers of CAI-1 as well as simple homologues, and we evaluate their relative activities. Synthetic (S)-3-hydroxytridecan-4-one functions as effectively as natural CAI-1 in repressing production of the canonical virulence factor TCP (toxin co-regulated pilus). These findings suggest that CAI-1 could be used as a therapy to prevent cholera infection and, furthermore, that strategies to manipulate bacterial quorum sensing hold promise in the clinical arena.

Vibrio cholerae, the bacterium that causes the disease cholera, controls virulence factor production and biofilm development in response to two extracellular quorum-sensing molecules, called autoinducers. The strongest autoinducer, called CAI-1 (for cholera autoinducer-1), was previously identified as (S)-3-hydroxytridecan-4-one. Biosynthesis of CAI-1 requires the enzyme CqsA. Here, we determine the CqsA reaction mechanism, identify the CqsA substrates as (S)-2-aminobutyrate and decanoyl coenzyme A, and demonstrate that the product of the reaction is 3-aminotridecan-4-one, dubbed amino-CAI-1. CqsA produces amino-CAI-1 by a pyridoxal phosphate (PLP)-dependent acyl-CoA transferase reaction. Amino-CAI-1 is converted to CAI-1 in a subsequent step via a CqsA-independent mechanism. Consistent with this, we find cells release ≥100 times more CAI-1 than amino-CAI-1. Nonetheless, V. cholerae responds to amino-CAI-1 as well as CAI-1, whereas other CAI-1 variants do not elicit a quorum-sensing response. Thus, both CAI-1 and amino-CAI-1 have potential as lead molecules in the development of an anti-cholera treatment.

Agricultural productivity relies on synthetic nitrogen fertilizers, yet half of that reactive nitrogen is lost to the environment. There is an urgent need for alternative nitrogen solutions to reduce the water pollution, ozone depletion, atmospheric particulate formation, and global greenhouse gas emissions associated with synthetic nitrogen fertilizer use. One such solution is biological nitrogen fixation (BNF), a component of the complex natural nitrogen cycle. BNF application to commercial agriculture is currently limited by fertilizer use and plant type. This paper describes the identification, development, and deployment of the first microbial product optimized using synthetic biology tools to enable BNF for corn (Zea mays) in fertilized fields, demonstrating the successful, safe commercialization of root-associated diazotrophs and realizing the potential of BNF to replace and reduce synthetic nitrogen fertilizer use in production agriculture. Derived from a wild nitrogen-fixing microbe isolated from agricultural soils, Klebsiella variicola 137-1036 (“Kv137-1036”) retains the capacity of the parent strain to colonize corn roots while increasing nitrogen fixation activity 122-fold in nitrogen-rich environments. This technical milestone was then commercialized in less than half of the time of a traditional biological product, with robust biosafety evaluations and product formulations contributing to consumer confidence and ease of use. Tested in multi-year, multi-site field trial experiments throughout the U.S. Corn Belt, fields grown with Kv137-1036 exhibited both higher yields (0.35 ± 0.092 t/ha ± SE or 5.2 ± 1.4 bushels/acre ± SE) and reduced within-field yield variance by 25% in 2018 and 8% in 2019 compared to fields fertilized with synthetic nitrogen fertilizers alone. These results demonstrate the capacity of a broad-acre BNF product to fix nitrogen for corn in field conditions with reliable agronomic benefits.

Imidazolium ionic liquids (IILs) have a range of biotechnological applications, including as pretreatment solvents that extract cellulose from plant biomass for microbial fermentation into sustainable bioenergy. However, residual levels of IILs, such as 1-ethyl-3-methylimidazolium chloride ([C2C1im]Cl), are toxic to biofuel-producing microbes, including the yeast Saccharomyces cerevisiae. S. cerevisiae strains isolated from diverse ecological niches differ in genomic sequence and in phenotypes potentially beneficial for industrial applications, including tolerance to inhibitory compounds present in hydrolyzed plant feedstocks. We evaluated >100 genome-sequenced S. cerevisiae strains for tolerance to [C2C1im]Cl and identified one strain with exceptional tolerance. By screening a library of genomic DNA fragments from the [C2C1im]Cl-tolerant strain for improved IIL tolerance, we identified SGE1, which encodes a plasma membrane multidrug efflux pump, and a previously uncharacterized gene that we named ionic liquid tolerance 1 (ILT1), which encodes a predicted membrane protein. Analyses of SGE1 sequences from our panel of S. cerevisiae strains together with growth phenotypes implicated two single nucleotide polymorphisms (SNPs) that associated with IIL tolerance and sensitivity. We confirmed these phenotypic effects by transferring the SGE1 SNPs into a [C2C1im]Cl-sensitive yeast strain using CRISPR/Cas9 genome editing. Further studies indicated that these SNPs affect Sge1 protein stability and cell surface localization, influencing the amount of toxic IILs that cells can pump out of the cytoplasm. Our results highlight the general potential for discovering useful biotechnological functions from untapped natural sequence variation and provide functional insight into emergent SGE1 alleles with reduced capacities to protect against IIL toxicity.