SummaryActivated sludge used for wastewater treatment globally is composed of a high‐density microbial community of great biotechnological significance. In this study the presence and purpose of quorum sensing via N‐acylated‐l‐homoserine lactones (AHLs) in activated sludge was explored. The presence of N‐heptanoyl‐l‐homoserine lactone in organic extracts of sludge was demonstrated along with activation of a LuxR‐based AHL monitor strain deployed in sludge, indicating AHL‐mediated gene expression is active in sludge flocculates but not in the bulk aqueous phase. Bacterial isolates from activated sludge were screened for AHL production and expression of phenotypes commonly but not exclusively regulated by AHL‐mediated gene transcription. N‐acylated‐l‐homoserine lactone and exoenzyme production were frequently observed among the isolates. N‐acylated‐l‐homoserine lactone addition to sludge upregulated chitinase activity and an AHL‐ and chitinase‐producing isolate closely related to Aeromonas hydrophila was shown to respond to AHL addition with upregulation of chitinase activity. N‐acylated‐l‐homoserine lactones produced by this strain were identified and genes ahyI/R and chiA, encoding AHL production and response and chitinase activity respectively, were sequenced. These experiments provide insight into the relationship between AHL‐mediated gene expression and exoenzyme activity in activated sludge and may ultimately create opportunities to improve sludge performance.

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The metal-reducing bacterium Shewanella oneidensis MR-1 produces extensions of its outer membrane (OM) and periplasm that contain cytochromes responsible for extracellular electron transfer (EET) to external redox-active surfaces, including minerals and electrodes. While the role of multi-heme cytochromes in transporting electrons across the cell wall is well established, their distribution along S. oneidensis OM extensions is also thought to allow lateral electron transport along these filaments. These proposed bacterial nanowires, which can be several times the cell length, would thereby extend EET to more distant electron acceptors. However, it is still unclear why these extensions form, and to what extent they contribute to respiration in living cells. Here, we investigate physical contributors to their formation using in vivo fluorescence microscopy. While previous studies focused on the display of S. oneidensis outer membrane extensions (OMEs) as a response to oxygen limitation, we find that cell-to-surface contact is sufficient to trigger the production of OMEs, including some that reach >100 µm in length, irrespective of medium composition, agitation, or aeration. To visualize the extent of heme redox centers along OMEs, and help distinguish these structures from other extracellular filaments, we also performed histochemical redox-dependent staining with transmission electron microscopy on wild type and cytochrome-deficient strains. We demonstrate that redox-active components are limited to OMEs and not present on other extracellular appendages, such as pili and flagella. We also observed that the loss of 8 functional periplasmic and outer membrane cytochromes significantly decreased both the frequency and intensity of redox-dependent staining found widespread on OMEs. These results will improve our understanding of the environmental conditions that influence the formation of S. oneidensis OMEs, as well as the distribution and functionality of EET components along extracellular appendages.

Significance Multiheme cytochromes in Shewanella oneidensis MR-1 transport electrons across the cell wall, in a process called extracellular electron transfer. These electron conduits can also enable electron transport along and between cells. While the underlying mechanism is thought to involve a combination of electron hopping and lateral diffusion of cytochromes along membranes, these diffusive dynamics have never been observed in vivo. Here, we observe the mobility of quantum dot-labeled cytochromes on living cell surfaces and membrane nanowires, quantify their diffusion with single-particle tracking techniques, and simulate the contribution of these dynamics to electron transport. This work reveals the impact of redox molecule dynamics on bacterial electron transport, with implications for understanding and harnessing this process in the environment and bioelectronics.

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