Monochloramine Induces Release of DNA and RNA from Bacterial Cells: Quantification, Sequencing Analyses, and Implications.

Monochloramine (MCA) is a widely used secondary disinfectant to suppress microbial growth in drinking water distribution systems. In monochloraminated drinking water, a significant amount of extracellular DNA (eDNA) has been reported, which has many implications ranging from obscuring DNA-based drinking water microbiome analyses to posing potential health concerns. To address this, it is imperative for us to know the origin of the eDNA in drinking water. Using Pseudomonas aeruginosa as a model organism, we report for the first time that MCA induces the release of nucleic acids from both biofilms and planktonic cells. Upon exposure to 2 mg/L MCA, massive release of DNA from suspended cells in both MilliQ water and 0.9% NaCl was directly visualized using live cell imaging in a CellASIC ONIX2 microfluidic system. Exposing established biofilms to MCA also resulted in DNA release from the biofilms, which was confirmed by increased detection of eDNA in the effluent. Intriguingly, massive release of RNA was also observed, and the extracellular RNA (eRNA) was also found to persist in water for days. Sequencing analyses of the eDNA revealed that it could be used to assemble the whole genome of the model organism, while in the water, certain fragments of the genome were more persistent than others. RNA sequencing showed that the eRNA contains non-coding RNA and mRNA, implying its role as a possible signaling molecule in environmental systems and a snapshot of the past metabolic state of the bacterial cells.

Increased particle size of goethite enhances the antibacterial effect on human pathogen Escherichia coli O157:H7: A Raman spectroscopic study.

The persistence of Escherichia coli O157:H7 in soil is one of the most common causes of the food-borne outbreaks. Nano-sized iron oxide minerals in soil, especially goethite, have been found to reduce bacterial viability, which helps to control the spread of human pathogens. However, little is known about the antibacterial effects of iron oxides with different particle sizes. Our result revealed that the micro-sized goethite exhibited a more effective antibacterial activity against E. coli O157:H7 than the nano-sized goethite. The underlying antibacterial mechanisms were further investigated via single-cell Raman microspectroscopy. The exposure to nano-sized goethite increased the levels of ribonucleoside-related substances, phenylalanine and adenosine 5'-triphosphate, while decreased those of glycogen, protein and lipopolysaccharide & outer membrane porins (LPS & OMPs). Meanwhile, micro-sized goethite triggered less variation in ribonucleoside-related substances and induced more reduction in LPS & OMPs. Therefore, the antibacterial effects of nano-sized goethite were mediated by both ROS-dependent RNA damage and cell membrane destruction, whereas micro-sized goethite induced severer membrane damage and less ROS-dependent oxidative stress. This work demonstrates the role of particle sizes in antibacterial effects of iron oxides and provides implications for the pathogen control in soil.

Shewanella biofilm development and engineering for environmental and bioenergy applications.

The genus Shewanella comprises about 70 species of Gram-negative, facultative anaerobic bacteria inhabiting various environments, which have shown great potential in various biotechnological applications ranging from environmental bioremediation, metal(loid) recovery and material synthesis to bioenergy generation. Most environmental and energy applications of Shewanella involve the biofilm mode of growth on surfaces of solid minerals or electrodes. In this article, we first provide an overview of Shewanella biofilm biology with the focus on biofilm dynamics, biofilm matrix, and key signalling systems involved in Shewanella biofilm development. Then we review strategies recently exploited to engineer Shewanella biofilms to improve biofilm-mediated bioprocesses.

Direct interspecies electron transfer (DIET) can be suppressed under ammonia-stressed condition - Reevaluate the role of conductive materials.

Thermal hydrolysis pretreatment (THP) and anaerobic digestion (AD) integrated (THP-AD) process is a promising process for sludge management. However, the high ammonia production during the THP-AD process severely affects system's stability and performance. Conductive materials are widely reported to stimulate AD, thus they are potentially helpful in alleviating ammonia inhibition. This study investigated the effects of three widely studied conductive materials, i.e. zero-valent iron (ZVI), magnetite nanoparticles (Mag.) and powder activated carbon (PAC), on THP-AD process. Results showed that all the tested materials could effectively stimulate methanogenesis process under non-ammonia inhibition conditions. However, upon ammonia stress, these materials behaved distinctively with the best methanogenic performance in ZVI group followed by Mag. Group, and even worsened inhibition occurred in PAC group. The mechanisms behind were investigated from two levels-the reaction kinetics of each anaerobic digestion step and the responses of intracellular metabolism. It is revealed that ZVI effectively promoted all AD reactions, especially the energy unfavorable propanoate and butanoate metabolism and overall methanogenesis. In addition, ZVI likely acted as intracellular electron shuttles, and the conjunction point of ZVI to electron transfer system was identified as EtfAB: quinone oxidoreductase. On the contrary, the declined methanogenic performance in PAC group was attributed to selectively stimulated the growth of acetoclastic methanogen - Methanosaeta, which is sensitive to ammonia toxicity. The proteomic information further revealed that ammonia stress was unfavorable to the formation of direct interspecies electron transfer between syntrophic anaerobes. Overall, the present study provides fundamental knowledge about the role of different conductive materials in AD systems from intracellular proteomic level.

Responses of Exogenous Bacteria to Soluble Extracellular Polymeric Substances in Wastewater: A Mechanistic Study and Implications on Bioaugmentation.

Compared with the chemically defined synthetic wastewater (SynWW), real wastewater has been reported to exhibit distinct effects on microbial community development. Whether and how soluble microbial products in real wastewater contribute to different effects of synthetic and real wastewater on the fate of exogenous bacteria remains elusive. In this study, using a model wastewater bacterium Comamonas testosteroni, we first examined the influences of microfiltration filter-sterilized real wastewater (MF-WW) and SynWW on the retention of C. testosteroni in established wastewater flocs during bioaugmentation. In bioreactors fed with MF-WW, augmentation of C. testosteroni to wastewater flocs resulted in a substantially higher abundance of the augmented bacterial cells than those fed with SynWW. To identify the soluble microbial products in MF-WW contributing to the observed differences between bioaugmentation reactors fed with MF-WW and SynWW, we examined the effect of MF-WW and SynWW on the growth, floc formation, and biofilm development of C. testosteroni. When C. testosteroni grew in MF-WW, visible flocs formed within 2 h, which is in contrast to cell growth in SynWW where floc formation was not observed. We further demonstrated that the observed differences were mainly attributed to the high molecular weight fraction of the soluble extracellular polymeric substances (EPS) in MF-WW, in particular, proteins and extracellular DNA. The DLVO analysis suggested that, in the presence of soluble EPS, the bacterial cell surface exhibits an increased hydrophobicity and a diminished energy barrier, leading to irreversible attachment of planktonic cells and floc formation. The RNA-seq based transcriptional analysis revealed that, in the presence of soluble EPS, genes involved in nonessential metabolisms were downregulated while genes coding for Cco (cbb3-type) and Cox (aa3-type) oxidases with different oxygen affinities were upregulated, facilitating bacterial survival in flocs. Taken together, this study reveals the mechanisms underlying the contribution of soluble EPS in real wastewater to the recruitment of exogenous bacteria by microbial aggregates and provides implications to bioaugmentation.

Comparison of nitrous oxide emission between a partial and full nitrification enriched ammonia-oxidising culture.

Nitrification systems are known to be a source of nitrous oxide (N2O) emission, however, the contribution from partial and full nitrification systems remains controversial. In this study, N2O emission from a partial and full nitrification culture was investigated. In all tests, nitrite, dissolved oxygen concentration and pH levels were controlled within a similar range limiting ammonium concentration to be the only variable. The results reveal with the same amount of ammonium removed, the full nitrification culture produced far greater N2O than the partial nitrification culture for both pulse (25-36 times) and continuous feeding modes (2-110 times). The relative gene expression data indicate that under pulse feeding there is a decreasing trend of nirK and norB genes for the partial and full nitrification culture respectively while under continuous feeding, increasing norB trends were observed for both. This possibly indicated the hydroxylamine pathway was favoured for the partial nitrification culture while the hybrid N-nitrosation pathway maybe the major contributor for the full nitrification culture. These findings improve our understanding on N2O production pathways and enable researchers to propose better mitigation strategies.

Bacterial Metabolism During Biofilm Growth Investigated by 13C Tracing.

This study investigated the metabolism of Pseudomonas aeruginosa PAO1 during its biofilm development via microscopy imaging, gene expression analysis, and 13C-labeling. First, dynamic labeling was employed to investigate glucose utilization rate in fresh biofilms (thickness 40∼60 micrometer). The labeling turnover time of glucose-6-P indicated biofilm metabolism was substantially slower than planktonic cells. Second, PAO1 was cultured in continuous tubular biofilm reactors or shake flasks. Then 13C-metabolic flux analysis of PAO1 was performed based on the isotopomer patterns of proteinogenic amino acids. The results showed that PAO1 biofilm cells during growth conserved the flux features as their planktonic mode. (1) Glucose could be degraded by two cyclic routes (the TCA cycle and the Entner-Doudoroff-Embden-Meyerhof-Parnas loop) that facilitated NAD(P)H supplies. (2) Anaplerotic pathways (including pyruvate shunt) increased flux plasticity. (3) Biofilm growth phenotype did not require significant intracellular flux rewiring (variations between biofilm and planktonic flux network, normalized by glucose uptake rate as 100%, were less than 20%). (4) Transcription analysis indicated that key catabolic genes in fresh biofilm cells had expression levels comparable to planktonic cells. Finally, PAO1, Shewanella oneidensis (as the comparing group), and their c-di-GMP transconjugants (with different biofilm formation capabilities) were 13C-labeled under biofilm reactors or planktonic conditions. Analysis of amino acid labeling variances from different cultures indicated Shewanella flux network was more flexibly changed than PAO1 during its biofilm formation.

Engineering a light-responsive, quorum quenching biofilm to mitigate biofouling on water purification membranes.

Quorum quenching (QQ) has been reported to be a promising approach for membrane biofouling control. Entrapment of QQ bacteria in porous matrices is required to retain them in continuously operated membrane processes and to prevent uncontrollable biofilm formation by the QQ bacteria on membrane surfaces. It would be more desirable if the formation and dispersal of biofilms by QQ bacteria could be controlled so that the QQ bacterial cells are self-immobilized, but the QQ biofilm itself still does not compromise membrane performance. In this study, we engineered a QQ bacterial biofilm whose growth and dispersal can be modulated by light through a dichromatic, optogenetic c-di-GMP gene circuit in which the bacterial cells sense near-infrared (NIR) light and blue light to adjust its biofilm formation by regulating the c-di-GMP level. We also demonstrated the potential application of the engineered light-responsive QQ biofilm in mitigating biofouling of water purification forward osmosis membranes. The c-di-GMP-targeted optogenetic approach for controllable biofilm development we have demonstrated here should prove widely applicable for designing other controllable biofilm-enabled applications such as biofilm-based biocatalysis.

A near-infrared light responsive c-di-GMP module-based AND logic gate in Shewanella oneidensis.

A novel, biofilm-based AND logic gate was constructed in Shewanella oneidensis through a near-infrared (NIR) light responsive c-di-GMP module. The logic gate was demonstrated in microbial fuel cells with isopropyl ß-d-thiogalactoside (IPTG) and NIR light as the inputs and electrical signals as the output.

Interspecific diversity reduces and functionally substitutes for intraspecific variation in biofilm communities.

Diversity has a key role in the dynamics and resilience of communities and both interspecific (species) and intraspecific (genotypic) diversity can have important effects on community structure and function. However, a critical and unresolved question for understanding the ecology of a community is to what extent these two levels of diversity are functionally substitutable? Here we show, for a mixed-species biofilm community composed of Pseudomonas aeruginosa, P. protegens and Klebsiella pneumoniae, that increased interspecific diversity reduces and functionally substitutes for intraspecific diversity in mediating tolerance to stress. Biofilm populations generated high percentages of genotypic variants, which were largely absent in biofilm communities. Biofilms with either high intra- or interspecific diversity were more tolerant to SDS stress than biofilms with no or low diversity. Unexpectedly, genotypic variants decreased the tolerance of biofilm communities when experimentally introduced into the communities. For example, substituting P. protegens wild type with its genotypic variant within biofilm communities decreased SDS tolerance by twofold, apparently due to perturbation of interspecific interactions. A decrease in variant frequency was also observed when biofilm populations were exposed to cell-free effluents from another species, suggesting that extracellular factors have a role in selection against the appearance of intraspecific variants. This work demonstrates the functional substitution of inter- and intraspecific diversity for an emergent property of biofilms. It also provides a potential explanation for a long-standing paradox in microbiology, in which morphotypic variants are common in laboratory grown biofilm populations, but are rare in diverse, environmental biofilm communities.

Involvement in Denitrification is Beneficial to the Biofilm Lifestyle of Comamonas testosteroni: A Mechanistic Study and Its Environmental Implications.

Comamonas is one of the most abundant microorganisms in biofilm communities driving wastewater treatment. Little has been known about the role of this group of organisms and their biofilm mode of life. In this study, using Comamonas testosteroni as a model organism, we demonstrated the involvement of Comamonas biofilms in denitrification under bulk aerobic conditions and elucidated the influence of nitrate respiration on its biofilm lifestyle. Our results showed that C. testosteroni could use nitrate as the sole electron acceptor for anaerobic growth. Under bulk aerobic condition, biofilms of C. testosteroni were capable of reducing nitrate, and intriguingly, nitrate reduction significantly enhanced viability of the biofilm-cells and reduced cell detachment from the biofilms. Nitrate respiration was further shown to play an essential role in maintaining high cell viability in the biofilms. RNA-seq analysis, quantitative polymerase chain reaction, and liquid chromatography-mass spectrometry revealed a higher level of bis(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) in cells respiring on nitrate than those grown aerobically (1.3 × 10(-4) fmol/cell vs 7.9 × 10(-6) fmol/cell; P < 0.01). C-di-GMP is one universal signaling molecule that regulates the biofilm mode of life, and a higher c-di-GMP concentration reduces cell detachment from biofilms. Taking these factors together, this study reveals that nitrate reduction occurs in mature biofilms of C. testosteroni under bulk aerobic conditions, and the respiratory reduction of nitrate is beneficial to the biofilm lifestyle by providing more metabolic energy to maintain high viability and a higher level of c-di-GMP to reduce cell detachment.

Surface display of roGFP for monitoring redox status of extracellular microenvironments in Shewanella oneidensis biofilms.

Biofilms are the most ubiquitous and resilient form of microbial life on earth. One most important feature of a biofilm is the presence of a self-produced matrix, which creates highly heterogeneous and dynamic microenvironments within biofilms. Redox status in biofilm microenvironments plays a critical role in biofilm development and function. However, there is a lack of non-intrusive tools to quantify extracellular redox status of microenvironments within a biofilm matrix. In this study, using Shewanella oneidensis as a model organism, we demonstrated a novel approach to monitor extracellular redox status in biofilm microenvironments. Specifically, we displayed a redox sensitive fluorescence protein roGFP onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed roGFP as a sensor to quantify the extracellular redox status in the matrix of S. oneidensis biofilms. The fusion of roGFP into BpfA has no negative impacts on cell growth and biofilm formation. Upon exposure to oxidizing agents such as H2 O2 , Ag(+) , and SeO3 (2-) , S. oneidensis BpfA-roGFP cells exhibited a characteristic fluorescence of roGFP. Proteinase treatment assay and super-resolution structured illumination microscopy confirmed the surface localization of BpfA-roGFP. We further used the surface displayed roGFP monitored the extracellular redox status in the matrix at different depths of a biofilm exposed to H2 O2 . This study provides a novel approach to non-invasively monitor extracellular redox status in microenvironments within biofilms, which can be used to understand redox responses of biofilms to environmental perturbations.

Biofilm development and enhanced stress resistance of a model, mixed-species community biofilm.

Most studies of biofilm biology have taken a reductionist approach, where single-species biofilms have been extensively investigated. However, biofilms in nature mostly comprise multiple species, where interspecies interactions can shape the development, structure and function of these communities differently from biofilm populations. Hence, a reproducible mixed-species biofilm comprising Pseudomonas aeruginosa, Pseudomonas protegens and Klebsiella pneumoniae was adapted to study how interspecies interactions affect biofilm development, structure and stress responses. Each species was fluorescently tagged to determine its abundance and spatial localization within the biofilm. The mixed-species biofilm exhibited distinct structures that were not observed in comparable single-species biofilms. In addition, development of the mixed-species biofilm was delayed 1-2 days compared with the single-species biofilms. Composition and spatial organization of the mixed-species biofilm also changed along the flow cell channel, where nutrient conditions and growth rate of each species could have a part in community assembly. Intriguingly, the mixed-species biofilm was more resistant to the antimicrobials sodium dodecyl sulfate and tobramycin than the single-species biofilms. Crucially, such community level resilience was found to be a protection offered by the resistant species to the whole community rather than selection for the resistant species. In contrast, community-level resilience was not observed for mixed-species planktonic cultures. These findings suggest that community-level interactions, such as sharing of public goods, are unique to the structured biofilm community, where the members are closely associated with each other.