The development of tools for genetic manipulation of Clostridium ljungdahlii has increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox for C. ljungdahlii , the possibility of adapting a lactose-inducible system for gene expression, previously reported for Clostridium perfringens , was investigated. The plasmid pAH2, originally developed for C. perfringens with a gusA reporter gene, functioned as an effective lactose-inducible system in C. ljungdahlii . Lactose induction of C. ljungdahlii containing pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression of adhE1 30-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression of adhE1 in a strain in which adhE1 and the adhE1 homolog adhE2 had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression of adhE2 similarly failed to restore ethanol production, suggesting that adhE1 is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA , cyaB , and cyaC , predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

As the fields of biotechnology and synthetic biology expand, cheap and sensitive tools are needed to measure increasingly complicated genetic circuits. In order to bypass some drawbacks of optical fluorescent reporting systems, we have designed and created a co-culture microbial fuel cell (MFC) system for electronic reporting. This system leverages the syntrophic growth of Escheriachia. coli (E. coli) and an electrogenic bacterium Shewanella oneidensis MR-1 (S. oneidensis). The fermentative products of E. coli provide a carbon and electron source for S. oneidensis MR-1, which then reports on such activity electrically at the anode of the MFC. To further test the capability of electrical reporting of complicated synthetic circuits, a novel synthetic biological comparator was designed and tested with both fluorescent and electrical reporting systems. The results suggest that the electrical reporting system is a good alternative to commonly used optical fluorescent reporter systems since it is a non-toxic reporting system with a much wider dynamic range.

We describe an electrochemical measurement technique that enables bioelectronic measurements of reporter proteins in living cells as an alternative to traditional optical fluorescence. Using electronically programmable microfluidics, the measurement is in turn used to control the concentration of an inducer input that regulates production of the protein from a genetic promoter. The resulting bioelectronic and microfluidic negative-feedback loop then serves to regulate the concentration of the protein in the cell. We show measurements wherein a user-programmable set-point precisely alters the protein concentration in the cell with feedback-loop parameters affecting the dynamics of the closed-loop response in a predictable fashion. Our work does not require expensive optical fluorescence measurement techniques that are prone to toxicity in chronic settings, sophisticated time-lapse microscopy, or bulky/expensive chemo-stat instrumentation for dynamic measurement and control of biomolecules in cells. Therefore, it may be useful in creating a: cheap, portable, chronic, dynamic, and precise all-electronic alternative for measurement and control of molecules in living cells.