Targeted Cell Labeling and Sorting of Prokaryotes for Cultivation and Omics Approaches.

To date, the vast majority of prokaryotic organisms escapes detailed characterization because they cannot be isolated in axenic cultures. These organisms are referred to as microbial dark matter. Targeted labelling and sorting of these microorganisms pave the way for single-cell, enrichment, or cultivation approaches. In this review, we describe an array of different methods ranging from labeling-free to specific labelling techniques. In addition, different cell sorting methods and their combinations with targeting strategies are summarized and downstream applications like sequencing and cultivation are reviewed. Recent advances, challenges, and limitations of the particular methods are discussed with respect to cell viability, genome integrity as well as throughput, in order to help researchers select the most suitable methods for their specific research questions.

Extracellular riboflavin induces anaerobic biofilm formation in Shewanella oneidensis.

BACKGROUND: Some microorganisms can respire with extracellular electron acceptors using an extended electron transport chain to the cell surface. This process can be applied in bioelectrochemical systems in which the organisms produce an electrical current by respiring with an anode as electron acceptor. These organisms apply flavin molecules as cofactors to facilitate one-electron transfer catalyzed by the terminal reductases and in some cases as endogenous electron shuttles. RESULTS: In the model organism Shewanella oneidensis, riboflavin production and excretion trigger a specific biofilm formation response that is initiated at a specific threshold concentration, similar to canonical quorum-sensing molecules. Riboflavin-mediated messaging is based on the overexpression of the gene encoding the putrescine decarboxylase speC which leads to posttranscriptional overproduction of proteins involved in biofilm formation. Using a model of growth-dependent riboflavin production under batch and biofilm growth conditions, the number of cells necessary to produce the threshold concentration per time was deduced. Furthermore, our results indicate that specific retention of riboflavin in the biofilm matrix leads to localized concentrations, which by far exceed the necessary threshold value. CONCLUSION: This study describes a new quorum-sensing mechanism in S. oneidensis. Biofilm formation of S. oneidensis is induced by low concentrations of riboflavin resulting in an upregulation of the ornithine-decarboxylase speC. The results can be applied for the development of strains catalyzing increased current densities in bioelectrochemical systems.

Improvement of the electron transfer rate in Shewanella oneidensis MR-1 using a tailored periplasmic protein composition.

Periplasmic c-type cytochromes are essential for the electron transport between the cytoplasmic membrane bound menquinol oxidase CymA and the terminal ferric iron reductase MtrABC in the outer membrane of Shewanella oneidensis cells. Either STC or FccA are necessary for periplasmic electron transfer. We followed the hypothesis that the elimination of potential competing reactions in the periplasm and the simultaneous overexpression of STC (cctA) could lead to an accelerated electron transfer to the cell surface. The genes nrfA, ccpA, napB and napA were replaced by cctA. This led to a 1.7-fold increased ferric iron reduction rate and a 23% higher current generation in a bioelectrochemical system. Moreover, the quadruple mutant had a higher periplasmic flavin content. Further deletion of fccA and its replacement by cctA resulted in a strain with ferric iron reduction rates similar to the wild type and a lower concentration of periplasmic flavin compared to the quadruple mutant. A transcriptomic analysis revealed that the quadruple mutant had a 3.7-fold higher cctA expression which could not be further increased by the replacement of fccA. This work indicates that a synthetic adaptation of Shewanella towards extracellular respiration holds potential for increased respiratory rates and consequently higher current densities.

Extracellular Electron Transfer and Biosensors.

This chapter summarizes in the beginning our current understanding of extracellular electron transport processes in organisms belonging to the genera Shewanella and Geobacter. Organisms belonging to these genera developed strategies to transport respiratory electrons to the cell surface that are defined by modules of which some seem to be rather unique for one or the other genus while others are similar. We use this overview regarding our current knowledge of extracellular electron transfer to explain the physiological interaction of microorganisms in direct interspecies electron transfer, a process in which one organism basically comprises the electron acceptor for another microbe and that depends also on extended electron transport chains. This analysis of mechanisms for the transport of respiratory electrons to insoluble electron acceptors ends with an overview of questions that remain so far unanswered. Moreover, we use the description of the biochemistry of extracellular electron transport to explain the fundamentals of biosensors based on this process and give an overview regarding their status of development and applicability. Graphical Abstract.

Chromate Resistance Mechanisms in Leucobacter chromiiresistens.

Chromate is one of the major anthropogenic contaminants on Earth. Leucobacter chromiiresistens is a highly chromate-resistant strain, tolerating chromate concentrations in LB medium of up to 400 mM. In response to chromate stress, L. chromiiresistens forms biofilms, which are held together via extracellular DNA. Inhibition of biofilm formation leads to drastically decreased chromate tolerance. Moreover, chromate is reduced intracellularly to the less-toxic Cr(III). The oxidation status and localization of chromium in cell aggregates were analyzed by energy-dispersive X-ray spectroscopy coupled to scanning transmission electron microscopy and X-ray absorption spectroscopy measurements. Most of the heavy metal is localized as Cr(III) at the cytoplasmic membrane. As a new cellular response to chromate stress, we observed an increased production of the carotenoid lutein. Carotenoid production could increase membrane stability and reduce the concentration of reactive oxygen species. Bioinformatic analysis of the L. chromiiresistens genome revealed several gene clusters that could enable heavy-metal resistance. The extreme chromate tolerance and the unique set of resistance factors suggest the use of L. chromiiresistens as a new model organism to study microbial chromate resistance.IMPORTANCE Chromate is a highly toxic oxyanion. Extensive industrial use and inadequate waste management has caused the toxic pollution of several field sites. Understanding the chromate resistance mechanisms that enable organisms to thrive under these conditions is fundamental to develop (micro)biological strategies and applications aiming at bioremediation of contaminated soils or waters. Potential detoxifying microorganisms are often not sufficient in their resistance characteristics to effectively perform, e.g., chromate reduction or biosorption. In this study, we describe the manifold strategies of L. chromiiresistens to establish an extremely high level of chromate resistance. The multitude of mechanisms conferring it make this organism suitable for consideration as a new model organism to study chromate resistance.

Development of a production chain from vegetable biowaste to platform chemicals.

BACKGROUND: A future bioeconomy relies on the development of technologies to convert waste into valuable compounds. We present here an attempt to design a biotechnological cascade for the conversion of vegetable waste into acetoin and electrical energy. RESULTS: A vegetable waste dark fermentation effluent containing mainly acetate, butyrate and propionate was oxidized in a bioelectrochemical system. The achieved average current at a constant anode potential of 0 mV against standard hydrogen electrode was 177.5 ± 52.5 µA/cm2. During this step, acetate and butyrate were removed from the effluent while propionate was the major remaining component of the total organic carbon content comprising on average 75.6%. The key players with regard to carbon oxidation and electrode reduction were revealed using amplicon sequencing and metatranscriptomic analysis. Using nanofiltration, it was possible to concentrate the propionate in the effluent. The effluent was revealed to be a suitable medium for biotechnological production strains. As a proof of principle, the propionate in the effluent of the bioelectrochemical system was converted into the platform chemical acetoin with a carbon recovery of 86%. CONCLUSIONS: To the best of our knowledge this is the first report on a full biotechnological production chain leading from vegetable waste to the production of a single valuable platform chemical that integrates carbon elimination steps leading to the production of the valuable side product electrical energy.

Unbalanced fermentation of glycerol in Escherichia coli via heterologous production of an electron transport chain and electrode interaction in microbial electrochemical cells.

Microbial electrochemical cells are an emerging technology for achieving unbalanced fermentations. However, organisms that can serve as potential biocatalysts for this application are limited by their narrow substrate spectrum. This study describes the reprogramming of Escherichia coli for the efficient use of anodes as electron acceptors. Electron transfer into the periplasm was accelerated by 183% via heterologous expression of the c-type cytochromes CymA, MtrA and STC from Shewanella oneidensis. STC was identified as a target for heterologous expression via a two-stage screening approach. First, mass spectroscopic analysis revealed natively expressed cytochromes in S. oneidensis. Thereafter, the corresponding genes were cloned and expressed in E. coli to quantify periplasmic electron transfer activity using methylene blue. This redox dye was further used to expand electron transfer to carbon electrode surfaces. The results demonstrate that E. coli can be reprogrammed from glycerol fermentation to respiration upon production of the new electron transport chain.

A dynamic periplasmic electron transfer network enables respiratory flexibility beyond a thermodynamic regulatory regime.

Microorganisms show an astonishing versatility in energy metabolism. They can use a variety of different catabolic electron acceptors, but they use them according to a thermodynamic hierarchy, which is determined by the redox potential of the available electron acceptors. This hierarchy is reflected by a regulatory machinery that leads to the production of respiratory chains in dependence of the availability of the corresponding electron acceptors. In this study, we showed that the γ-proteobacterium Shewanella oneidensis produces several functional electron transfer chains simultaneously. Furthermore, these chains are interconnected, most likely with the aid of c-type cytochromes. The cytochrome pool of a single S. oneidensis cell consists of ca. 700 000 hemes, which are reduced in the absence on an electron acceptor, but can be reoxidized in the presence of a variety of electron acceptors, irrespective of prior growth conditions. The small tetraheme cytochrome (STC) and the soluble heme and flavin containing fumarate reductase FccA have overlapping activity and appear to be important for this electron transfer network. Double deletion mutants showed either delayed growth or no growth with ferric iron, nitrate, dimethyl sulfoxide or fumarate as electron acceptor. We propose that an electron transfer machinery that is produced irrespective of a thermodynamic hierarchy not only enables the organism to quickly release catabolic electrons to a variety of environmental electron acceptors, but also offers a fitness benefit in redox-stratified environments.

Draft genome sequence of Leucobacter chromiiresistens, an extremely chromium-tolerant strain.

Here we present the draft genome of Leucobacter chromiiresistens. This is the first genome sequence of an organism belonging to the genus Leucobacter. L. chromiiresistens was sequenced due to its capability to tolerate up to 300 mM Cr(VI) in the medium, which is so far a unique feature for microorganisms.

Investigation of the electron transport chain to and the catalytic activity of the diheme cytochrome c peroxidase CcpA of Shewanella oneidensis.

Bacterial diheme c-type cytochrome peroxidases (BCCPs) catalyze the periplasmic reduction of hydrogen peroxide to water. The gammaproteobacterium Shewanella oneidensis produces the peroxidase CcpA under a number of anaerobic conditions, including dissimilatory iron-reducing conditions. We wanted to understand the function of this protein in the organism and its putative connection to the electron transport chain to ferric iron. CcpA was isolated and tested for peroxidase activity, and its structural conformation was analyzed by X-ray crystallography. CcpA exhibited in vitro peroxidase activity and had a structure typical of diheme peroxidases. It was produced in almost equal amounts under anaerobic and microaerophilic conditions. With 50 mM ferric citrate and 50 µM oxygen in the growth medium, CcpA expression results in a strong selective advantage for the cell, which was detected in competitive growth experiments with wild-type and ΔccpA mutant cells that lack the entire ccpA gene due to a markerless deletion. We were unable to reduce CcpA directly with CymA, MtrA, or FccA, which are known key players in the chain of electron transport to ferric iron and fumarate but identified the small monoheme ScyA as a mediator of electron transport between CymA and BCCP. To our knowledge, this is the first detailed description of a complete chain of electron transport to a periplasmic c-type cytochrome peroxidase. This study furthermore reports the possibility of establishing a specific electron transport chain using c-type cytochromes.

Leucobacter chromiiresistens sp. nov., a chromate-resistant strain.

A Gram-positive, irregular rod-shaped, non-motile, yellow-pigmented bacterium, strain JG 31(T), was isolated in the course of identifying chromium-resistant soil bacteria. 16S rRNA gene sequence analysis of the isolated bacterium indicated its phylogenetic position within the genus Leucobacter. Binary 16S rRNA gene sequence alignments of the isolated bacterium with the 11 species of the genus recognized at the time of writing revealed sequence similarities of more than 97 % with Leucobacter alluvii (GenBank accession no: AM072820; 99.4 %), Leucobacter iarius (AM040493; 98.2 %), Leucobacter aridicollis (AJ781047; 97.8 %), Leucobacter komagatae (AB007419; 97.4 %), Leucobacter chironomi (EU346911; 97.1 %) and Leucobacter luti (AM072819; 97.1 %). In contrast, DNA-DNA hybridization experiments showed similarity values below 28 % for DNA samples from the most closely related type strains of L. alluvii, L. aridicollis and L. iarius. Protein analysis by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and automated RiboPrinting using the restriction enzyme PvuII differentiated strain JG 31(T) from all type strains of the genus Leucobacter. The dominant fatty acids of the novel isolate were anteiso-C(15 : 0), anteiso-C(17 : 0) and iso-C(16 : 0), while the quinone system consisted of menaquinones MK-11, MK-10, MK-9 and MK-8. In a B-type cross-linked peptidoglycan, the cell-wall amino acids were alanine, glycine, threonine, glutamic acid and 2,4-diaminobutyric acid. Strain JG 31(T) was able to grow in a medium containing up to 300 mM K(2)CrO(4) and showed cellular aggregation in response to chromate stress. From biochemical and genomic analyses, the new strain is considered to represent a novel species of the genus Leucobacter, for which the name Leucobacter chromiiresistens sp. nov. is proposed. The type strain is strain JG 31(T) ( = DSM 22788(T)  = CCOS 200(T)).