Aerobic Respiration and Its Regulation in the Metal Reducer Shewanella oneidensis.

Shewanella oneidensis MR-1 is a facultative anaerobe known for its ability to reduce metal oxides. Anaerobic respiration, especially metal reduction, has been the subject of extensive research. In contrast, S. oneidensis aerobic respiration has received less attention. S. oneidensis expresses cbb3 - and aa3 -type cytochrome c oxidases and a bd-type quinol oxidase. The aa3 -type oxidase, which in other bacteria is the major oxygen reductase under oxygen replete conditions, does not appear to contribute to aerobic respiration and growth in S. oneidensis. Our results indicated that although the aa3-type oxidase does not play a role in aerobic growth on lactate, the preferred carbon source for S. oneidensis, it is involved in growth on pyruvate or acetate. These results highlight the importance of testing multiple carbon and energy sources when attempting to identify enzyme activities and mutant phenotypes. Several regulatory proteins contribute to the regulation of aerobic growth in S. oneidensis including CRP and ArcA. The 3',5'-cAMP phosphodiesterase (CpdA) appears to play a more significant role in aerobic growth than either CRP or ArcA, yet the deficiency does not appear to be the result of reduced oxidase genes expression. Interestingly, the ∆cpdA mutant was more deficient in aerobic respiration with several carbon sources tested compared to ∆crp, which was moderately deficient only in the presence of lactate. To identify the reason for ∆cpdA aerobic growth deficiency, we isolated a suppressor mutant with transposon insertion in SO_3550. Inactivation of this gene, which encodes an anti-sigma factor, restored aerobic growth in the cpdA mutant to wild-type levels. Inactivation of SO_3550 in wild-type cells, however, did not affect aerobic growth. The S. oneidensis genome encodes two additional CRP-like proteins that we designated CrpB and CrpC. Mutants that lack crpB and crpC were deficient in aerobic growth, but this deficiency was not due to the loss of oxidase gene expression.

A Novel Bioelectronic Reporter System in Living Cells Tested with a Synthetic Biological Comparator.

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.

A Synthetic Microbial Operational Amplifier.

Synthetic biology has created oscillators, latches, logic gates, logarithmically linear circuits, and load drivers that have electronic analogs in living cells. The ubiquitous operational amplifier, which allows circuits to operate robustly and precisely has not been built with biomolecular parts. As in electronics, a biological operational-amplifier could greatly improve the predictability of circuits despite noise and variability, a problem that all cellular circuits face. Here, we show how to create a synthetic three-stage inducer-input operational amplifier with a fast CRISPR-based differential-input push-pull stage, a slow transcription-and-translation amplification stage, and a fast-enzymatic output stage. Our "Bio-OpAmp" uses only 5 proteins including dCas9. It expands the toolkit of fundamental analog circuits in synthetic biology and provides a simple circuit motif for robust and precise molecular homeostasis.

Bioelectronic measurement and feedback control of molecules in living cells.

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.

Comparison of the transport of Bacteroides fragilis and Escherichia coli within saturated sand packs.

Pathogens in groundwater accounted for ∼50% of waterborne disease outbreaks in the United States between 1971 and 2006. The fast and reliable detection of groundwater microbial contamination and the identification of the contamination sources are of critical importance to the protection of public health. Recent studies suggested that fecal anaerobe Bacteriodes spp. could be employed as an effective tool for surface water microbial source tracking (MST). The usefulness of Bacteroides spp. for groundwater MST depends strongly on its mobility within the subsurface system. This research provides laboratory results comparing transport and attachment of E. coli K12 and B. fragilis within packed quartz sands. The results indicate that at low ionic strengths both E. coli K12 and B. fragilis are readily transported through saturated sand packs. At higher ionic strengths such as may be found near concentrated sources of fecal contamination, B. fragilis displayed significantly higher mobility than E. coli K12. Analysis of the extended Derjaguin-Landau-Verweu-Overbeek (XDLVO) energy interactions for both types of bacteria showed a significant repulsive energy barrier exists between the sand surface and the bacteria, precluding attachment directly to the sand surface. However a secondary minimum energy level exists under higher ionic strength conditions. The depth of this energy low is greater for E. coli K12, which results in greater attachment of E. coli K12 than of B. fragilis. The high mobility of B. fragilis suggests that it represents a promising tool for the detection of groundwater fecal contamination as well as the identification of the microbial sources.

Lactose-inducible system for metabolic engineering of Clostridium ljungdahlii.

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.