Transcriptional analysis was performed on Clostridium acetobutylicum with the goal of identifying sugar-specific mechanisms for the transcriptional regulation of transport and metabolism genes. DNA microarrays were used to determine transcript levels from total RNA isolated from cells grown on media containing eleven different carbohydrates, including two pentoses (xylose, arabinose), four hexoses (glucose, mannose, galactose, fructose), four disaccharides (sucrose, lactose, maltose, cellobiose) and one polysaccharide (starch). Sugar-specific induction of many transport and metabolism genes indicates that these processes are regulated at the transcriptional level and are subject to carbon catabolite repression. The results show that C. acetobutylicum utilizes symporters and ATP-binding cassette (ABC) transporters for the uptake of pentose sugars, while disaccharides and hexoses are primarily taken up by phosphotransferase system (PTS) transporters and a gluconate : H + (GntP) transporter. The transcription of some transporter genes was induced by specific sugars, while others were induced by a subset of the sugars tested. Sugar-specific transport roles are suggested, based on expression comparisons, for various transporters of the PTS, the ABC superfamily and members of the major facilitator superfamily (MFS), including the GntP symporter family and the glycoside-pentoside-hexuronide (GPH)-cation symporter family. Additionally, updates to the C. acetobutylicum genome annotation are proposed, including the identification of genes likely to encode proteins involved in the metabolism of arabinose and xylose via the pentose phosphate pathway.

Many reports have elucidated the mechanisms and consequences of bacterial quorum sensing (QS), a molecular communication system by which bacterial cells enumerate their cell density and organize collective behavior. In few cases, however, the numbers of bacteria exhibiting this collective behavior have been reported, either as a number concentration or a fraction of the whole. Not all cells in the population, for example, take on the collective phenotype. Thus, the specific attribution of the postulated benefit can remain obscure. This is partly due to our inability to independently assemble a defined quorum, for natural and most artificial systems the quorum itself is a consequence of the biological context (niche and signaling mechanisms). Here, we describe the intentional assembly of quantized quorums. These are made possible by independently engineering the autoinducer signal transduction cascade of Escherichia coli (E. coli) and the sensitivity of detector cells so that upon encountering a particular autoinducer level, a discretized sub-population of cells emerges with the desired phenotype. In our case, the emergent cells all express an equivalent amount of marker protein, DsRed, as an indicator of a specific QS-mediated activity. The process is robust, as detector cells are engineered to target both large and small quorums. The process takes about 6 h, irrespective of quorum level. We demonstrate sensitive detection of autoinducer-2 (AI-2) as an application stemming from quantized quorums. We then demonstrate sub-population partitioning in that AI-2-secreting cells can 'call' groups neighboring cells that 'travel' and establish a QS-mediated phenotype upon reaching the new locale.

Escherichia coli were engineered to enable programmed motility, sensing and phenotypic response to the density of epidermal growth factor receptor expressed on the surface of cancer cells.

In this report, a novel zymogram assay and coupled phosphoketolase assay were employed to demonstrate that Clostridium acetobutylicum gene CAC1343 encodes a bi-functional xylulose-5-P/fructose-6-P phosphoketolase (XFP). The specific activity of purified recombinant XFP was 6.9 U/mg on xylulose-5-P and 21 U/mg on fructose-6-P, while the specific activity of XFP in concentrated C. acetobutylicum whole-cell extract was 0.094 and 0.52 U/mg, respectively. Analysis of crude cell extracts indicated that XFP activity was present in cells grown on arabinose but not glucose and quantitative PCR was used to show that CAC1343 mRNA expression was induced 185-fold during growth on arabinose when compared to growth on glucose. HPLC analysis of metabolites revealed that during growth on xylose and glucose more butyrate than acetate was formed with final acetate:butyrate ratios of 0.72 and 0.83, respectively. Growth on arabinose caused a metabolic shift to more oxidized products with a final acetate:butyrate ratio of 1.95. The shift towards more oxidized products is consistent with the presence of an XFP, suggesting that arabinose is metabolized via a phosphoketolase pathway while xylose is probably metabolized via the pentose phosphate pathway.

Advances in nanotechnology have provided unprecedented physical means to sample molecular space. Living cells provide additional capability in that they identify molecules within complex environments and actuate function. We have merged cells with nanotechnology for an integrated molecular processing network. Here we show that an engineered cell consortium autonomously generates feedback to chemical cues. Moreover, abiotic components are readily assembled onto cells, enabling amplified and ‘binned' responses. Specifically, engineered cell populations are triggered by a quorum sensing (QS) signal molecule, autoinducer-2, to express surface-displayed fusions consisting of a fluorescent marker and an affinity peptide. The latter provides means for attaching magnetic nanoparticles to fluorescently activated subpopulations for coalescence into colour-indexed output. The resultant nano-guided cell network assesses QS activity and conveys molecular information as a ‘bio-litmus' in a manner read by simple optical means.