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Microbial iron reduction (MIR) is an important and ubiquitous natural process in the biogeochemical cycling of iron and carbon in anaerobic sedimentary and subsurface environments. The objectives of this study were (1) to determine if the MIR process can enhance the inactivation of Escherichia coli cells under anaerobic conditions and (2) to identify potential inactivation mechanisms. Laboratory microcosm experiments showed that the presence of MIR activity significantly enhanced E. coli inactivation, and the inactivation rate under the MIR condition was significantly larger than those under other anaerobic redox conditions. Under anoxic condition, higher Fe2+concentrations exhibited a linear function to larger E. coli inactivation rates, indicating that the production of Fe2+by MIR was one of the important roles in E. coli inactivation. When E. coli cells were amended as the sole electron source to the MIR process, increased Fe2+ production was observed, which corresponded to decreasing TOC concentration. Together, the results suggest that MIR enhanced E. coli inactivation through the production of Fe2+ as metabolic waste, and the inactivation benefited the MIR process as the inactivated cells were used as an electron source, which represents a potential new mechanism for bacterial inter-species competition. This knowledge could further improve our understanding of the fate of fecal bacteria in natural environments where the MIR process is prevalent, and may also be explored for enhanced removal of bacterial pathogens in engineering processes.

International audience; This study examined the combined effect of hydraulic retention time (HRT), organic loading rate (OLR) and heat pretreatment of manure (70 degrees C, 1 h) on the fate of E. coli, enterococci, C. perfringens, C. difficile, and on chemical parameters (volatile fatty acids and ammonia) that may inactivate pathogens. Semi-continuous mesophilic anaerobic reactors were fed with pig manure and horse feed. The operating conditions were 2, 3, 4 COD.L-1.d(-1) (OLR), 24, 35, 46 days (HRT) and use or not of a thermal pretreatment. The levels of the chemical parameters did not reach concentrations capable of inactivating the four bacteria. Anaerobic digestion led to a Log io removal > 3 (E. coli), 0.9-2.1 (enterococci), 0.1-0.6 (C. perfringens) and 0-1 (C. difficile). Increasing HRT only reduced the concentration of E. coli in the digestate. Increasing OLR reduced the Log(10) removal of enterococci and C. difficile. The heat pretreatment led to non-detection of E. coli in the digestate, reduced the concentration of C. perfringens by 0.8-1.3 Log(10) and increased the concentration of C. difficile by 0.04-0.7 Log(10). Enterococci, not detected in the heated manure, were present in the digestate. The distribution of genes encoding virulence factors of C. difficile (tcdA and tcdB) and C. perfringens (cpa, cpb2 and cpb) was not impacted by anaerobic digestion or by the heat pretreatment. Enterococci, C. perfringens, C. difficile were present in the digestate at relatively stable concentrations regardless of the operating conditions, indicating that even with heat pretreatment, the biosafety of digestate cannot be guaranteed in mesophilic conditions.

Temperate bacteriophages are viruses that can incorporate their genomes into their bacterial hosts, existing there as prophages that refrain from killing the host cell until induced. Prophages are largely quiescent, but they can alter host phenotype through factors encoded in their genomes (often virulence factors) or by disrupting host genes as a result of integration. Here we describe another mechanism by which a prophage can modulate host phenotype. We show that a temperate phage that integrates in Escherichia coli reprograms host regulation of an anaerobic respiratory system, thereby inhibiting a bet hedging strategy. The phage exerts this effect by upregulating a host-encoded signal transduction protein through transcription initiated from a phage-encoded promoter. We further show that this phenomenon occurs not only in a laboratory strain of E. coli, but also in a natural isolate that contains a prophage at this site., eLife digest Animals and plants can all fall prey to viruses – and so can bacteria. The viruses that infect bacteria are called bacteriophages (or phages for short), and they are found everywhere bacteria live and probably outnumber bacteria by at least ten to one. While some phages quickly kill every bacterial cell they infect, others enter a dormant state by inserting their DNA into the DNA of their host cell. Here they lie in wait for a signal that reactivates them, triggering the production of more phages and the death of the host cell. While the phage lies dormant its DNA may harm the host by interfering with nearby bacterial genes, or it may actually provide new genes that benefit the host. In most cases the effects of dormant phages are unknown. A bacterium known as Escherichia coli is commonly found in the intestines of humans and other mammals. It can use a nutrient called trimethylamine oxide (TMAO) to help it survive rapid decreases in oxygen levels that can occur in its environment. When a phage called HK022 infects E. coli, the phage enters a dormant state by inserting its DNA between two genes that are critical for E. coli to use TMAO. However, it is not clear what effect, if any, HK022 has on E. coli’s behavior. To address this question, Carey et al. used genetic approaches to study E. coli cells carrying dormant HK022 phages. The experiments showed that the bacteria lost the ability to use TMAO to survive rapid decreases in oxygen because the dormant phages switched on one of the neighboring E. coli genes. Unexpectedly, the phage achieved this by neatly replacing the gene’s own promoter – the stretch of DNA that contains information about when the gene should be switched on, and how strongly – with a substitute promoter carried in the phage’s DNA. This substitute promoter is stronger than the normal version – meaning that the gene is more active than it should be. Phages are key players in every natural population of microbes and are therefore entwined in the health of humans and the environment. The findings of Carey et al. show a new mechanism through which phages modify their hosts. In the future it may be possible to develop this mechanism into a tool to manipulate bacteria in complex environments like infection sites, for example by introducing phages that block the mechanisms that allow bacteria to tolerate antibiotics.

The phenotype of Escherichia coli is governed by global transcriptional regulators under variable environmental conditions. Fnr, ArcA, IhfA-B, Crp, and Fis are amongst the major global transcription regulators that change their activity across the range of aeration, hence forming the core transcriptional network responsible for survival under changing aeration conditions. Effect of deletion of these global transcription factors on the kinetics of cell growth and mixed acid production under anaerobic fermentation conditions has not been characterized. To quantify the kinetic parameters in the absence of global transcription factors, experiments were performed using single deletion mutants of the above-mentioned global transcription regulators. The absence of global transcription regulators resulted in a relatively higher glucose uptake rate than that required for the observed growth rate. This further resulted in a higher yield of mixed acids per unit biomass in mutants as compared to the parent strain (E. coli BW25113). Thus, the increased channeling of carbon towards mixed acid secretion resulted in a lower growth rate in the mutants.

Six antimicrobial-producing seaweed-derived Bacillus strains were evaluated in vitro as animal probiotics, in comparison to two Bacillus from an EU-authorized animal probiotic product. Antimicrobial activity was demonstrated on solid media against porcine Salmonella and E. coli. The marine isolates were most active against the latter, had better activity than the commercial probiotics and Bacillus pumilus WIT 588 also reduced E. coli counts in broth. All of the marine Bacillus tolerated physiological concentrations of bile, with some as tolerant as one of the probiotics. Spore counts for all isolates remained almost constant during incubation in simulated gastric and ileum juices. All of the marine Bacillus grew anaerobically and the spores of all except one isolate germinated under anaerobic conditions. All were sensitive to a panel of antibiotics and none harbored Bacillus enterotoxin genes but all, except B. pumilus WIT 588, showed some degree of β-hemolysis. However, trypan blue dye exclusion and xCELLigence assays demonstrated a lack of toxicity in comparison to two pathogens; in fact, the commercial probiotics appeared more cytotoxic than the majority of the marine Bacillus. Overall, some of the marine-derived Bacillus, in particular B. pumilus WIT 588, demonstrate potential for use as livestock probiotics.

For adaptation between anaerobic, micro-aerobic and aerobic conditions Escherichia coli's metabolism and in particular its electron transport chain (ETC) is highly regulated. Although it is known that the global transcriptional regulators FNR and ArcA are involved in oxygen response it is unclear how they interplay in the regulation of ETC enzymes under micro-aerobic chemostat conditions. Also, there are diverse results which and how quinones (oxidised/reduced, ubiquinone/other quinones) are controlling the ArcBA two-component system. In the following a mathematical model of the E. coli ETC linked to basic modules for substrate uptake, fermentation product excretion and biomass formation is introduced. The kinetic modelling focusses on regulatory principles of the ETC for varying oxygen conditions in glucose-limited continuous cultures. The model is based on the balance of electron donation (glucose) and acceptance (oxygen or other acceptors). Also, it is able to account for different chemostat conditions due to changed substrate concentrations and dilution rates. The parameter identification process is divided into an estimation and a validation step based on previously published and new experimental data. The model shows that experimentally observed, qualitatively different behaviour of the ubiquinone redox state and the ArcA activity profile in the micro-aerobic range for different experimental conditions can emerge from a single network structure. The network structure features a strong feed-forward effect from the FNR regulatory system to the ArcBA regulatory system via a common control of the dehydrogenases of the ETC. The model supports the hypothesis that ubiquinone but not ubiquinol plays a key role in determining the activity of ArcBA in a glucose-limited chemostat at micro-aerobic conditions.

Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative anaerobe Escherichia coli has elaborate sensing and signal transduction mechanisms for redox control in response to the availability of O2 and other electron acceptors. The ArcBA two-component system consists of ArcB, a membrane-associated sensor kinase, and ArcA, the cognate response regulator. The tripartite hybrid kinase ArcB possesses a transmembrane, a PAS, a primary transmitter (H1), a receiver (D1), and a phosphotransfer (H2) domain. Metabolic fluxes were compared under anoxic conditions in a wild-type E. coli strain, its ΔarcB derivative, and two partial arcB deletion mutants in which ArcB lacked either the H1 domain or the PAS-H1-D1 domains. These analyses revealed that elimination of different segments in ArcB determines a distinctive distribution of D-glucose catabolic fluxes, different from that observed in the ΔarcB background. Metabolite profiles, enzyme activity levels, and gene expression patterns were also investigated in these strains. Relevant alterations were observed at the P-enol-pyruvate/pyruvate and acetyl coenzyme A metabolic nodes, and the formation of reduced fermentation metabolites, such as succinate, D-lactate, and ethanol, was favored in the mutant strains to different extents compared to the wild-type strain. These phenotypic traits were associated with altered levels of the enzymatic activities operating at these nodes, as well as with elevated NADH/NAD+ ratios. Thus, targeted modification of global regulators to obtain different metabolic flux distributions under anoxic conditions is emerging as an attractive tool for metabolic engineering purposes. © 2012, American Society for Microbiology. Fil:Ruiz, J.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:de Almeida, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Méndez, B.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Background Anaerobic rather than aerobic fermentation is preferred for conversion of biomass derived sugars to high value redox-neutral and reduced commodities. This will likely result in a higher yield of substrate to product conversion and decrease production cost since substrate often accounts for a significant portion of the overall cost. To this goal, metabolic pathway engineering has been used to optimize substrate carbon flow to target products. This approach works well for the production of redox neutral products such as lactic acid from redox neutral sugars using the reducing power NADH (nicotinamide adenine dinucleotide, reduced) generated from glycolysis (2 NADH per glucose equivalent). Nevertheless, greater than two NADH per glucose catabolized is needed for the production of reduced products (such as xylitol) from redox neutral sugars by anaerobic fermentation. Results The Escherichia coli strain AI05 (ΔfrdBC ΔldhA ΔackA Δ(focA-pflB) ΔadhE ΔptsG ΔpdhR::pflBp6-(aceEF-lpd)), previously engineered for reduction of xylose to xylitol using reducing power (NADH equivalent) of glucose catabolism, was further engineered by 1) deleting xylAB operon (encoding for xylose isomerase and xylulokinase) to prevent xylose from entering the pentose phosphate pathway; 2) anaerobically expressing the sdhCDAB-sucABCD operon (encoding for succinate dehydrogenase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase) to enable an anaerobically functional tricarboxcylic acid cycle with a theoretical 10 NAD(P)H equivalent per glucose catabolized. These reducing equivalents can be oxidized by synthetic respiration via xylose reduction, producing xylitol. The resulting strain, AI21 (pAI02), achieved a 96 % xylose to xylitol conversion, with a yield of 6 xylitol per glucose catabolized (molar yield of xylitol per glucose consumed (YRPG) = 6). This represents a 33 % improvement in xylose to xylitol conversion, and a 63 % increase in xylitol yield per glucose catabolized over that achieved by AI05 (pAI02). Conclusions Increasing reducing power (NADH equivalent) output per glucose catabolized was achieved by anaerobic expression of both the pdh operon (pyruvate dehydrogenase) and the sdhCDAB-sucABCD operon, resulting in a strain capable of generating 10 NADH equivalent per glucose under anaerobic condition. The new E. coli strain AI21 (pAI02) achieved an actual 96 % conversion of xylose to xylitol (via synthetic respiration), and 6 xylitol (from xylose) per glucose catabolized (YRPG = 6, the highest known value). This strategy can be used to engineer microbial strains for the production of other reduced products from redox neutral sugars using glucose as a source of reducing power.