Reactive nitrogen species from free nitrous acid (FNA) cause cell lysis.

Free nitrous acid (FNA, i.e. HNO2) has been demonstrated to have broad biocidal effects on a range of microorganisms, which has direct implications for wastewater management. However, the biocidal mechanisms still remain largely unknown. This study aims to test the hypothesis that FNA will induce cell lysis via cell membrane perforations, and consequently cause cell death via proteolysis, through the use of two model organisms namely Escherichia coli K12 and Pseudomonas putida KT2440. A combination of analytical techniques that included viability assays, atomic force microscopy (AFM), protein abundance assays and proteomic analysis using Quadruple-Orbitrap™ Mass spectrometry was used to evaluate the extent of cell death and possible cell lysis mechanisms. FNA treatment at 6.09 mg/L for 24 h (conditions typically applied in applications) induced 36 ± 4.2% and 91 ± 3.5% cell death/lysis of E. coli and P. putida, respectively. AFM showed that the lysis of cells was observed via perforations in the cell membrane; cells also appeared to shrink and become flat following FNA treatment. By introducing a reactive nitrogen species (RNS) scavenger to act as a treatment control, we further revealed that it was the nitrosative decomposition species of FNA, such as .NO that caused the cell lysis through the destruction of protein macromolecules found in the cell membrane (proteolysis). Subsequently, the RNS went on to cause the destruction of protein macromolecules within the cells. The death of these model organisms E. coli and P. putida following exposure to FNA treatment provides insights into the use of FNA as an antimicrobial agent in wastewater treatment.

Corrosion mitigation by nitrite spray on corroded concrete in a real sewer system.

Microbially influenced concrete corrosion (MICC) in sewers is caused by the activity of sulfide-oxidizing microorganisms (SOMs) on concrete surfaces, which greatly deteriorates the integrity of sewers. Surface treatment of corroded concrete by spraying chemicals is a low-cost and non-intrusive strategy. This study systematically evaluated the spray of nitrite solution in corrosion mitigation and re-establishment in a real sewer manhole. Two types of concrete were exposed at three heights within the sewer manhole for 21 months. Nitrite spray was applied at the 6th month for half of the coupons which had developed active corrosion. The corrosion development was monitored by measuring the surface pH, corrosion product composition, sulfide uptake rate, concrete corrosion loss, and the microbial community on the corrosion layer. Free nitrous acid (FNA, i.e. HNO2), formed by spraying a nitrite solution on acidic corrosion surfaces, was shown to inhibit the activity of SOMs. The nitrite spray reduced the corrosion loss of concrete at all heights by 40-90% for six months. The sulfide uptake rate of sprayed coupons was also reduced by about 35%, leading to 1-2 units higher surface pH, comparing to the control coupons. The microbial community analysis revealed a reduced abundance of SOMs on nitrite sprayed coupons. The long-term monitoring also showed that the corrosion mitigation effect became negligible in 15 months after the spray. The results consistently demonstrated the effectiveness of nitrite spray on the MICC mitigation and identified the re-application frequencies for full scale applications.

Characterizing the premise plumbing microbiome in both water and biofilms of a 50-year-old building.

The premise plumbing portion of drinking water distribution systems (DWDS) has several characteristics that may favor microbial growth in the form of biofilms. These microbial communities are implicated as infectious sources for the spread of opportunistic waterborne pathogens by supporting their complex ecology and transmission through DWDS outlets to susceptible individuals. However, there is limited understanding of the drinking water biofilms in real premise plumbing networks due to challenges with accessibility. Using a combination of culture-dependent and culture-independent approaches, this study comprehensively characterized the premise plumbing microbiome of a 50-year-old university building, inclusive of water and biofilm samples. Microbial diversity in the water samples were more taxonomically diverse in comparison to the mature drinking water biofilms, which were dominated with biofilm-formers and opportunistic pathogens, such as Mycobacterium spp. A model opportunistic pathogen, Legionella spp., was only detectable in water samples using quantitative PCR but could not be detected in any of the drinking water biofilms using either qPCR or culture-dependent approaches, highlighting the limitations of detection methods in these environments. This study presents preliminary findings on the microbial dynamics and complexity in premise plumbing networks, which may support public health management and the development of strategies to eliminate microbial risks to human health.

Chlorine disinfection facilitates natural transformation through ROS-mediated oxidative stress.

The bacterial infection that involves antimicrobial resistance is a rising global threat to public health. Chlorine-based water disinfection processes can inactivate antibiotic resistant bacteria. However, at the same time, these processes may cause the release of antibiotic resistance genes into the water as free DNA, and consequently increase the risk to disseminate antibiotic resistance via natural transformation. Presently, little is known about the contribution of residual chlorine affecting the transformation of extracellular antibiotic resistance genes (ARGs). This study investigates whether chloramine and free chlorine promote the transformation of ARGs and how this may occur. We reveal that both chloramine and free chlorine, at practically relevant concentrations, significantly stimulated the transformation of plasmid-encoded ARGs by the recipient Acinetobacter baylyi ADP1, by up to a 10-fold increase. The underlying mechanisms underpinning the increased transformations were revealed. Disinfectant exposure induced a series of cell responses, including increased levels of reactive oxygen species (ROS), bacterial membrane damage, ROS-mediated DNA damage, and increased stress response. These effects thus culminated in the enhanced transformation of ARGs. This promoted transformation was observed when exposing disinfectant-pretreated A. baylyi to free plasmid. In contrast, after pretreating free plasmid with disinfectants, the transformation of ARGs decreased due to the damage of plasmid integrity. These findings provide important insight on the roles of disinfectants affecting the horizontal transfer of ARGs, which could be crucial in the management of antibiotic resistance in our water systems.

Non-antibiotic pharmaceuticals promote the transmission of multidrug resistance plasmids through intra- and intergenera conjugation.

Antibiotic resistance is a global threat to public health. The use of antibiotics at sub-inhibitory concentrations has been recognized as an important factor in disseminating antibiotic resistance via horizontal gene transfer. Although non-antibiotic, human-targeted pharmaceuticals are widely used by society (95% of the pharmaceuticals market), the potential contribution to the spread of antibiotic resistance is not clear. Here, we report that commonly consumed, non-antibiotic pharmaceuticals, including nonsteroidal anti-inflammatories (ibuprofen, naproxen, diclofenac), a lipid-lowering drug (gemfibrozil), and a ß-blocker (propranolol), at clinically and environmentally relevant concentrations, significantly accelerated the dissemination of antibiotic resistance via plasmid-borne bacterial conjugation. Various indicators were used to study the bacterial response to these drugs, including monitoring reactive oxygen species (ROS) and cell membrane permeability by flow cytometry, cell arrangement, and whole-genome RNA and protein sequencing. Enhanced conjugation correlated well with increased production of ROS and cell membrane permeability. Additionally, these non-antibiotic pharmaceuticals induced responses similar to those detected when bacteria are exposed to antibiotics, such as inducing the SOS response and enhancing efflux pumps. The findings advance understanding of the transfer of antibiotic resistance genes, emphasizing the concern that non-antibiotic, human-targeted pharmaceuticals enhance the spread of antibiotic resistance among bacterial populations.

Nonnutritive sweeteners can promote the dissemination of antibiotic resistance through conjugative gene transfer.

Antimicrobial resistance (AMR) poses a worldwide threat to human health and biosecurity. The spread of antibiotic resistance genes (ARGs) via conjugative plasmid transfer is a major contributor to the evolution of this resistance. Although permitted as safe food additives, compounds such as saccharine, sucralose, aspartame, and acesulfame potassium that are commonly used as nonnutritive sweeteners have recently been associated with shifts in the gut microbiota similar to those caused by antibiotics. As antibiotics can promote the spread of antibiotic resistance genes (ARGs), we hypothesize that these nonnutritive sweeteners could have a similar effect. Here, we demonstrate for the first time that saccharine, sucralose, aspartame, and acesulfame potassium could promote plasmid-mediated conjugative transfer in three established conjugation models between the same and different phylogenetic strains. The real-time dynamic conjugation process was visualized at the single-cell level. Bacteria exposed to the tested compounds exhibited increased reactive oxygen species (ROS) production, the SOS response, and gene transfer. In addition, cell membrane permeability increased in both parental bacteria under exposure to the tested compounds. The expression of genes involved in ROS detoxification, the SOS response, and cell membrane permeability was significantly upregulated under sweetener treatment. In conclusion, exposure to nonnutritive sweeteners enhances conjugation in bacteria. Our findings provide insight into AMR spread and indicate the potential risk associated with the presence of nonnutritive sweeteners.

Structural changes in model compounds of sludge extracellular polymeric substances caused by exposure to free nitrous acid.

Previous studies demonstrate that free nitrous acid (FNA i.e. HNO2) detaches sewer biofilms, breaks down flocs of waste activated sludge (WAS) and enhances biogas production from WAS. This suggests possible interactions of FNA with organic extracellular polymeric substances (EPS) that bind the cells into biofilms or sludge flocs. This study evaluates the chemical interactions and reaction mechanisms between FNA and molecules representative of key EPS in biofilm and sludge flocs. Molecules chosen to represent components found in the extracellular polymeric matrix were treated with FNA at 6.09 mgN/L (NO2- = 250 mgN/L, pH = 5.0 ± 0.2, T = 22 °C) for 24 hours (conditions typically used in applications) so as to consider the hypothesized chemical interactions and the consequent reaction pathways. A number of analytical techniques were employed to measure the molecular changes in the EPS molecules including; proton (1H) nuclear magnetic resonance spectroscopy (NMR), electrospray ionisation mass spectrometry (ESI-MS) and gel permeation chromatography (GPC). The results demonstrated that FNA broke down a range of large EPS molecules including carbohydrates, protein and lipids to smaller molecules. Two mechanistic pathways have been proposed including electrophilic substitution, whereby the nitrosium ion (NO+) was the reactive electrophile, and oxidative radical reactions, through which the nitrogen radicals (.NO2, .NO) and reactive nitrogen intermediates (RNIs) (e.g. N2O3 and N2O4) formed from the decomposition of FNA became part of the reaction products. Larger, more complex organic molecules such as humic acid, required higher concentrations of FNA (6.09 mgN/L or greater) to cause molecular breakdown, whereas smaller molecules, such as calcium alginate, was broken down at lower concentrations (3.04 mgN/L). The study contributes to the understanding of the fundamental mechanisms behind the application of FNA for biofilm control and flocular sludge disintegration.

Structural Changes in Cell-Wall and Cell-Membrane Organic Materials Following Exposure to Free Nitrous Acid.

Previous studies demonstrate that free nitrous acid (FNA, i.e., HNO2) is biocidal for a range of microorganisms. The biocidal mechanisms of FNA are largely unknown. In this work, it is hypothesized that FNA will break bonds in molecules found in the cell envelope, thus causing cell lysis. Selected molecules representing components found in the cell envelope were treated with FNA at 6.09 mg N/L (NO2- = 250 mg N/L, pH 5.0) for 24 h (conditions typically used in applications) to evaluate the hypothesized chemical interactions. Molecular changes were observed using analytical techniques including proton (1H) nuclear magnetic resonance spectroscopy (NMR) and electrospray ionization mass spectrometry (ESI-MS). It was found that FNA broke down a range of cell envelope molecules. The spectral data demonstrated that the FNA reactions proceeded via two general pathways. One consisted of electrophilic substitution, whereby the nitrosonium ion (NO+) was the reactive electrophile. The other was via oxidative reactions involving nitrogen radicals (e.g., •NO2 and •NO) formed from the decomposition of FNA. We further revealed that it was HNO2 that caused the breakdown, rather than the exclusive action of the acid (H+) or nitrite (NO2-) counterparts. The fragmentation of these representative cell envelope molecules provides insight into the biocidal effects of FNA on microorganisms.

Non-antibiotic pharmaceuticals enhance the transmission of exogenous antibiotic resistance genes through bacterial transformation.

Antibiotic resistance is a serious global threat for public health. Considering the high abundance of cell-free DNA encoding antibiotic resistance genes (ARGs) in both clinical and environmental settings, natural transformation is an important horizontal gene transfer pathway to transmit antibiotic resistance. It is acknowledged that antibiotics are key drivers for disseminating antibiotic resistance, yet the contributions of non-antibiotic pharmaceuticals on transformation of ARGs are overlooked. In this study, we report that some commonly consumed non-antibiotic pharmaceuticals, at clinically and environmentally relevant concentrations, significantly facilitated the spread of antibiotic resistance through the uptake of exogenous ARGs. This included nonsteroidal anti-inflammatories, ibuprofen, naproxen, diclofenac, the lipid-lowering drug, gemfibrozil, and the ß-blocker propranolol. Based on the results of flow cytometry, whole-genome RNA sequencing and proteomic analysis, the enhanced transformation of ARGs was affiliated with promoted bacterial competence, enhanced stress levels, over-produced reactive oxygen species and increased cell membrane permeability. In addition, a mathematical model was proposed and calibrated to predict the dynamics of transformation during exposure to non-antibiotic pharmaceuticals. Given the high consumption of non-antibiotic pharmaceuticals, these findings reveal new concerns regarding antibiotic resistance dissemination exacerbated by non-antibiotic pharmaceuticals.

Efficient inactivation of antibiotic resistant bacteria and antibiotic resistance genes by photo-Fenton process under visible LED light and neutral pH.

Antibiotic resistance has been recognized as a major threat to public health worldwide. Inactivation of antibiotic resistant bacteria (ARB) and degradation of antibiotic resistance genes (ARGs) are critical to prevent the spread of antibiotic resistance in the environment. Conventional disinfection processes are effective to inactivate water-borne pathogens, yet they are unable to completely eliminate the antibiotic resistance risk. This study explored the potential of the photo-Fenton process to inactivate ARB, and to degrade both extracellular and intracellular ARGs (e-ARGs and i-ARGs, respectively). Using Escherichia coli DH5α with two plasmid-encoded ARGs (tetA and blaTEM-1) as a model ARB, a 6.17 log ARB removal was achieved within 30 min of applying photo-Fenton under visible LED and neutral pH conditions. In addition, no ARB regrowth occurred after 48-h, demonstrating that this process is very effective to induce permanent disinfection on ARB. The photo-Fenton process was validated under various water matrices, including ultrapure water (UPW), simulated wastewater (SWW) and phosphate buffer (PBS). The higher inactivation efficiency was observed in SWW as compared to other matrices. The photo-Fenton process also caused a 6.75 to 8.56-log reduction in eARGs based on quantitative real-time PCR of both short- and long amplicons. Atomic force microscopy (AFM) further confirmed that the extracellular DNA was sheared into short DNA fragments, thus eliminating the risk of the transmission of antibiotic resistance. As compared with e-ARGs, a higher dosage of Fenton reagent was required to damage i-ARGs. In addition, the tetA gene was more easily degraded than the blaTEM-1 gene. Collectively, our results demonstrate the photo-Fenton process is a promising technology for disinfecting water to prevent the spread of antibiotic resistance.

Adaptive Evolution of Geobacter sulfurreducens in Coculture with Pseudomonas aeruginosa.

Interactions between microorganisms in mixed communities are highly complex, being either syntrophic, neutral, predatory, or competitive. Evolutionary changes can occur in the interaction dynamics between community members as they adapt to coexistence. Here, we report that the syntrophic interaction between Geobacter sulfurreducens and Pseudomonas aeruginosa coculture change in their dynamics over evolutionary time. Specifically, Geobacter sp. dominance increases with adaptation within the cocultures, as determined through quantitative PCR and fluorescence in situ hybridization. This suggests a transition from syntrophy to competition and demonstrates the rapid adaptive capacity of Geobacter spp. to dominate in cocultures with P. aeruginosa Early in coculture establishment, two single-nucleotide variants in the G. sulfurreducensfabI and tetR genes emerged that were strongly selected for throughout coculture evolution with P. aeruginosa phenazine wild-type and phenazine-deficient mutants. Sequential window acquisition of all theoretical spectra-mass spectrometry (SWATH-MS) proteomics revealed that the tetR variant cooccurred with the upregulation of an adenylate cyclase transporter, CyaE, and a resistance-nodulation-division (RND) efflux pump notably known for antibiotic efflux. To determine whether antibiotic production was driving the increased expression of the multidrug efflux pump, we tested Pseudomonas-derived phenazine-1-carboxylic acid (PHZ-1-CA) for its potential to inhibit Geobacter growth and drive selection of the tetR and fabI genetic variants. Despite its inhibitory properties, PHZ-1-CA did not drive variant selection, indicating that other antibiotics may drive overexpression of the efflux pump and CyaE or that a novel role exists for these proteins in the context of this interaction.IMPORTANCEGeobacter and Pseudomonas spp. cohabit many of the same environments, where Geobacter spp. often dominate. Both bacteria are capable of extracellular electron transfer (EET) and play important roles in biogeochemical cycling. Although they recently in 2017 were demonstrated to undergo direct interspecies electron transfer (DIET) with one another, the genetic evolution of this syntrophic interaction has not been examined. Here, we use whole-genome sequencing of the cocultures before and after adaptive evolution to determine whether genetic selection is occurring. We also probe their interaction on a temporal level and determine whether their interaction dynamics change over the course of adaptive evolution. This study brings to light the multifaceted nature of interactions between just two microorganisms within a controlled environment and will aid in improving metabolic models of microbial communities comprising these two bacteria.

Increased Resistance of Nitrite-Admixed Concrete to Microbially Induced Corrosion in Real Sewers.

Microbially induced concrete corrosion is a major deterioration process in sewers, causing a huge economic burden, and improved mitigating technologies are required. This study reports a novel and promising effective solution to attenuate the corrosion in sewers using calcium nitrite-admixed concrete. This strategy aims to suppress the development and activity of corrosion-inducing microorganisms with the antimicrobial free nitrous acid, which is generated in situ from calcium nitrite that is added to the concrete. Concrete coupons with calcium nitrite as an admixture were exposed in a sewer manhole, together with control coupons that had no nitrite admixture, for 18 months. The corrosion process was monitored by measuring the surface pH, corrosion product composition, concrete corrosion loss, and the microbial community on the corrosion layer. During the exposure, the corrosion loss of the admixed concrete coupons was 30% lower than that of the control coupons. The sulfide uptake rate of the admixed concrete was also 30% lower, leading to a higher surface pH (0.5-0.6 unit), in comparison to that of the control coupons. A negative correlation between the calcium nitrite admixture in concrete and the abundance of sulfide-oxidizing microorganisms was determined by DNA sequencing. The results obtained in this field study demonstrated that this novel use of calcium nitrite as an admixture in concrete is a promising strategy to mitigate the microbially induced corrosion in sewers.

Improving wastewater management using free nitrous acid (FNA).

Free nitrous acid (FNA), the protonated form of nitrite, has historically been an unwanted substance in wastewater systems due to its inhibition on a wide range of microorganisms. However, in recent years, advanced understanding of FNA inhibitory and biocidal effects on microorganisms has led to the development of a series of FNA-based applications that improve wastewater management practices. FNA has been used in sewer systems to control sewer corrosion and odor; in wastewater treatment to achieve carbon and energy efficient nitrogen removal; in sludge management to improve the sludge reduction and energy recovery; in membrane systems to address membrane fouling; and in wastewater algae systems to facilitate algae harvesting. This paper aims to comprehensively and critically review the current status of FNA-based applications in improving wastewater management. The underlying mechanisms of FNA inhibitory and biocidal effects are also reviewed and discussed. Knowledge gaps and current limitations of the FNA-based applications are identified; and perspectives on the development of FNA-based applications are discussed. We conclude that the FNA-based technologies have great potential for enhancing the performance of wastewater systems; however, further development and demonstration at larger scales are still required for their wider applications.

New insights of the bacterial response to exposure of differently sized silver nanomaterials.

The release of silver nanomaterials (AgNMs) from extensive use poses potential risks to human health and ecological environments. Although previous studies have reported the negative effects of AgNMs on various microorganisms, little is known about the response of bacteria under the exposure of AgNMs at the cellular level. Here, we report the multiple responses of Pseudomonas aeruginosa PAO1 (PAO1) under the exposure of two types of AgNMs, including spherical silver nanoparticles (AgNPs) and fibrous silver nanorods (AgNRs), by physiological experiments, microscopy, synchrotron-based X-ray Absorption Spectroscopy (XAS), flow cytometry and genome-wide RNA sequencing. Our results demonstrated that the exposure to both types of AgNMs could inhibit the growth of PAO1, accompanied by the overproduction of oxidative stress and inducing cell membrane damage. Transmission electron microscopy revealed the roughened cell membrane under both AgNMs treatment. In addition, both AgNMs repressed the expression of quorum sensing and metal efflux-related genes in PAO1, but stimulated denitrification, glycerol and amino acid metabolisms, SOS response and pyocin overproduction of PAO1. Compared to AgNRs, AgNPs exposure showed a much lower threshold concentration to trigger the inhibitory effect and induced greater transcriptional responses of PAO1. This study suggested that AgNMs could cause multiple effects on the proliferation, metabolism, virulence and pathogenesis of PAO1, which might further affect the corresponding environmental microbial communities. Overall, our findings offer insights into the interactions between AgNMs and bacteria at the molecular level.

A comparative proteomic analysis of Desulfovibrio vulgaris Hildenborough in response to the antimicrobial agent free nitrous acid.

Sulfate reducing bacteria (SRB) can contribute to facilitating serious concrete corrosion through the production of hydrogen sulfide in sewers. Recently, free nitrous acid (FNA) was discovered as a promising antimicrobial agent to inhibit SRB activities thereby limiting hydrogen sulfide production in sewers. However, knowledge of the bacterial response to increasing levels of the antimicrobial agent is unknown. Here we report the proteomic response of Desulfovibrio vulgaris Hildenborough and reveal that the antimicrobial effect of FNA is multi-targeted and dependent on the FNA levels. This was achieved using a sequential window acquisition of all theoretical mass spectrometry analysis to determine protein abundance variations in D. vulgaris during exposure to different FNA concentrations. When exposed to 1.0 µg N/L FNA, nitrite reduction (nitrite reductase) related proteins and nitrosative stress related proteins, including the hybrid cluster protein, showed distinct increased abundances. When exposed to 4.0 and 8.0 µg N/L FNA, increased abundance was detected for proteins putatively involved in nitrite reduction. Abundance of proteins involved in the sulfate reduction pathway (from adenylylphophosulfate to sulfite) and lactate oxidation pathway (from pyruvate to acetate) were initially inhibited in response to FNA at 8 h incubation, and then recovered at 12 h incubation. Lowered ribosomal protein abundance in D. vulgaris was detected, however, total cellular protein levels were mostly constant in the presence or absence of FNA. In addition, this study indicates that proteins coded by genes DVU2543, DVU0772, and DVU3212 potentially participate in resisting oxidative stress with FNA exposure. These findings share new insights for understanding the dynamic responses of D. vulgaris to FNA and could be useful to guide and improve the practical applications of FNA-based technologies for control of sewer corrosion.

Correction to: Enhanced Growth of Pilin-Deficient Geobacter sulfurreducens Mutants in Carbon Poor and Electron Donor Limiting Conditions.

The published version of this article contained an old version of Fig. 2.

Periodic deprivation of gaseous hydrogen sulfide affects the activity of the concrete corrosion layer in sewers.

Sulfide induced concrete corrosion significantly reduces the service life of the sewer systems. Gaseous hydrogen sulfide (H2S) levels are a key factor affecting the corrosion rate and these fluctuate due to the diurnal flow pattern of sewers. Currently, there is little known about how such fluctuations, in particular the periodic deprivation of H2S, may affect the corrosion activity. This study investigated the impact of the deprivation of H2S on the sulfide uptake rate (SUR) of concrete coupons incubated in laboratory corrosion chambers. After systematic evaluation of the gaseous H2S concentration profiles of two sewer systems, two types of profiles, i.e. short- (1 h) and long- (12 h) term deprivation of H2S, were applied to the concrete coupons. In comparison to the baseline SUR, exposing the concrete coupon to 0 ppm of H2S for 1 h consistently caused a temporary increase of the SUR (i.e. 3.2%-12.5%) following re-supply of H2S at baseline levels. With the continuous re-supply of H2S, there was gradual and steady decrease of SUR to the level close to the baseline SUR. However, for the case after deprivation of H2S for 12 h, the SUR was 5.1% lower than baseline SUR and gradually increased to a level similar to the baseline SUR during the 20-30 min of continuous re-supply of H2S. In addition, the simultaneous deprivation of H2S and O2 for 1 h had negligible impact on the SUR. Further analysis suggests that the historically accumulated intermediates of sulfide oxidation could act as electron donors for sulfide oxidizing bacteria (SOB). The replenishment of the intermediates upon the re-supply of H2S could play a key role in the increase of SUR after short-term deprivation of H2S. However, the activity of SOB could be diminished after long-term deprivation of H2S, although the sulfur intermediates still could be available. Estimating the sulfide uptake by concrete using the SUR of the average H2S concentration could lead to overestimation of the sulfide uptake. There could be more significant overestimation for the case with longer deprivation of H2S.

Enhanced Growth of Pilin-Deficient Geobacter sulfurreducens Mutants in Carbon Poor and Electron Donor Limiting Conditions.

Geobacter sulfurreducens pili enable extracellular electron transfer and play a role in secretion of c-type cytochromes such as OmcZ. PilA-deficient mutants of G. sulfurreducens have previously been shown to accumulate cytochromes within their membranes. This cytochrome retaining phenotype allowed for enhanced growth of PilA-deficient mutants in electron donor and carbon-limited conditions where formate and fumarate are provided as the sole electron donor and acceptor with no supplementary carbon source. Conversely, wild-type G. sulfurreducens, which has normal secretion of cytochromes, has comparative limited growth in these conditions. This growth is further impeded for OmcZ-deficient and OmcS-deficient mutants. A PilB-deficient mutant which prevents pilin production but allows for secretion of OmcZ had moderate growth in these conditions, indicating a role for cytochrome localization to enabling survival in the electron donor and carbon-limited conditions. To determine which pathways enhanced growth using formate, Sequential Window Acquisition of all Theoretical Mass Spectra mass spectrometry (SWATH-MS) proteomics of formate adapted PilA-deficient mutants and acetate grown wild type was performed. PilA-deficient mutants had an overall decrease in tricarboxylic acid (TCA) cycle enzymes and significant upregulation of electron transport chain associated proteins including many c-type cytochromes and [NiFe]-hydrogenases. Whole genome sequencing of the mutants shows strong convergent evolution and emergence of genetic subpopulations during adaptation to growth on formate. The results described here suggest a role for membrane constrained c-type cytochromes to the enhancement of survival and growth in electron donor and carbon-limited conditions.