Improved Bst DNA Polymerase Variants Derived via a Machine Learning Approach.

The DNA polymerase I from Geobacillus stearothermophilus (also known as Bst DNAP) is widely used in isothermal amplification reactions, where its strand displacement ability is prized. More robust versions of this enzyme should be enabled for diagnostic applications, especially for carrying out higher temperature reactions that might proceed more quickly. To this end, we appended a short fusion domain from the actin-binding protein villin that improved both stability and purification of the enzyme. In parallel, we have developed a machine learning algorithm that assesses the relative fit of individual amino acids to their chemical microenvironments at any position in a protein and applied this algorithm to predict sequence substitutions in Bst DNAP. The top predicted variants had greatly improved thermotolerance (heating prior to assay), and upon combination, the mutations showed additive thermostability, with denaturation temperatures up to 2.5 °C higher than the parental enzyme. The increased thermostability of the enzyme allowed faster loop-mediated isothermal amplification assays to be carried out at 73 °C, where both Bst DNAP and its improved commercial counterpart Bst 2.0 are inactivated. Overall, this is one of the first examples of the application of machine learning approaches to the thermostabilization of an enzyme.

Cytochrome OmcS Is Not Essential for Extracellular Electron Transport via Conductive Pili in Geobacter sulfurreducens Strain KN400.

The multi-heme c-type cytochrome OmcS is one of the central components used for extracellular electron transport in the Geobacter sulfurreducens strain DL-1, but its role in other microbes, including other strains of G. sulfurreducens, is currently a matter of debate. Therefore, we investigated the function of OmcS in the G. sulfurreducens strain KN400, which is even more effective in extracellular electron transfer than the DL-1 strain. We found that deleting omcS from strain KN400 did not negatively impact the rate of Fe(III) oxide reduction and that the cells expressed conductive filaments. Replacing the wild-type pilin gene with the aro-5 pilin gene eliminated the OmcS-deficient strain's ability to transport electrons to insoluble electron acceptors and diminished filament conductivity. These results are consistent with the concept that electrically conductive pili are the primary conduit for long-range electron transfer in G. sulfurreducens and closely related species. These findings, coupled with the lack of OmcS homologs in other microbes capable of extracellular electron transfer, suggest that OmcS is not a common critical component for extracellular electron transfer. IMPORTANCE OmcS has been widely studied and noted to be one of the key components for extracellular electron exchange by the Geobacter sulfurreducens strain DL-1. However, the true importance of OmcS warrants further investigation because it is well known that few bacteria, even within the Geobacteraceae family, contain OmcS homologs, and many bacteria that are capable of extracellular electron transfer lack an abundance of any type of outer surface c-type cytochrome. In addition, there is debate about the importance of OmcS filaments in the mechanism of extracellular electron transport to insoluble electron acceptors by G. sulfurreducens. It has been suggested that filaments comprised of OmcS rather than e-pili are the predominant conductive filaments expressed by G. sulfurreducens. However, the results presented here, along with multiple other sources of evidence, indicate that OmcS filaments cannot be the primary, conductive, protein nanowires expressed by G. sulfurreducens.

Early-life adversity programs long-term cytokine and microglia expression within the HPA axis in female Japanese quail.

Stress exposure during prenatal and postnatal development can have persistent and often dysfunctional effects on several physiological systems, including immune function, affecting the ability to combat infection. The neuroimmune response is inextricably linked to the action of the hypothalamic-pituitary-adrenal (HPA) axis. Cytokines released from neuroimmune cells, including microglia, activate the HPA axis, while glucocorticoids in turn regulate cytokine release from microglia. Because of the close links between these two physiological systems, coupled with potential for persistent changes to HPA axis activity following developmental stress, components of the neuroimmune system could be targets for developmental programming. However, little is known of any programming effects of developmental stress on neuroimmune function. We investigated whether developmental stress exposure via elevated prenatal corticosterone (CORT) or postnatal unpredictable food availability had long-term effects on pro- (IL-1ß) and anti-inflammatory (IL-10) cytokine and microglia-dependent gene (CSF1R) expression within HPA axis tissues in a precocial bird, the Japanese quail (Coturnix japonica). Following postnatal stress, we observed increased IL-1ß expression in the pituitary gland, reduced IL-10 expression in the amygdala and hypothalamus, and reduced CSF1R expression within the hypothalamus and pituitary gland. Postnatal stress disrupted the ratio of IL-1ß:IL-10 expression within the hippocampus and hypothalamus. Prenatal stress only increased IL-1ß expression in the pituitary gland. We found no evidence for interactive or cumulative effects across life stages on basal cytokine and glia expression in adulthood. We show that postnatal stress may have a larger impact than elevated prenatal CORT on basal immunity in HPA-axis-specific brain regions, with changes in cytokine homeostasis and microglia abundance. These results provide evidence for postnatal programming of a pro-inflammatory neuroimmune phenotype at the expense of reduced microglia, which could have implications for central nervous system health and subsequent neuroimmune responses.

Glucocorticoid programming of neuroimmune function.

Throughout life physiological systems strive to maintain homeostasis and these systems are susceptible to exposure to maternal or environmental perturbations, particularly during embryonic development. In some cases, these perturbations may influence genetic and physiological processes that permanently alter the functioning of these physiological systems; a process known as developmental programming. In recent years, the neuroimmune system has garnered attention for its fundamental interactions with key hormonal systems, such as the hypothalamic pituitary adrenal (HPA) axis. The ultimate product of this axis, the glucocorticoid hormones, play a key role in modulating immune responses within the periphery and the CNS as part of the physiological stress response. It is well-established that elevated glucocorticoids induced by developmental stress exert profound short and long-term physiological effects, yet there is relatively little information of how these effects are manifested within the neuroimmune system. Pre and post-natal periods are prime candidates for manipulation in order to uncover the physiological mechanisms that underlie glucocorticoid programming of neuroimmune responses. Understanding the potential programming role of glucocorticoids may be key in uncovering vulnerable windows of CNS susceptibility to stressful experiences during embryonic development and improve our use of glucocorticoids as therapeutics in the treatment of neurodegenerative diseases.

Arsenic Detoxification by Geobacter Species.

Insight into the mechanisms for arsenic detoxification by Geobacter species is expected to improve the understanding of global cycling of arsenic in iron-rich subsurface sedimentary environments. Analysis of 14 different Geobacter genomes showed that all of these species have genes coding for an arsenic detoxification system (ars operon), and several have genes required for arsenic respiration (arr operon) and methylation (arsM). Genes encoding four arsenic repressor-like proteins were detected in the genome of G. sulfurreducens; however, only one (ArsR1) regulated transcription of the ars operon. Elimination of arsR1 from the G. sulfurreducens chromosome resulted in enhanced transcription of genes coding for the arsenic efflux pump (Acr3) and arsenate reductase (ArsC). When the gene coding for Acr3 was deleted, cells were not able to grow in the presence of either the oxidized or reduced form of arsenic, while arsC deletion mutants could grow in the presence of arsenite but not arsenate. These studies shed light on how Geobacter influences arsenic mobility in anoxic sediments and may help us develop methods to remediate arsenic contamination in the subsurface. IMPORTANCE: This study examines arsenic transformation mechanisms utilized by Geobacter, a genus of iron-reducing bacteria that are predominant in many anoxic iron-rich subsurface environments. Geobacter species play a major role in microbially mediated arsenic release from metal hydroxides in the subsurface. This release raises arsenic concentrations in drinking water to levels that are high enough to cause major health problems. Therefore, information obtained from studies of Geobacter should shed light on arsenic cycling in iron-rich subsurface sedimentary environments, which may help reduce arsenic-associated illnesses. These studies should also help in the development of biosensors that can be used to detect arsenic contaminants in anoxic subsurface environments. We examined 14 different Geobacter genomes and found that all of these species possess genes coding for an arsenic detoxification system (ars operon), and some also have genes required for arsenic respiration (arr operon) and arsenic methylation (arsM).

Metatranscriptomic Evidence for Direct Interspecies Electron Transfer between Geobacter and Methanothrix Species in Methanogenic Rice Paddy Soils.

The possibility that Methanothrix (formerly Methanosaeta) and Geobacter species cooperate via direct interspecies electron transfer (DIET) in terrestrial methanogenic environments was investigated in rice paddy soils. Genes with high sequence similarity to the gene for the PilA pilin monomer of the electrically conductive pili (e-pili) of Geobacter sulfurreducens accounted for over half of the PilA gene sequences in metagenomic libraries and 42% of the mRNA transcripts in RNA sequencing (RNA-seq) libraries. This abundance of e-pilin genes and transcripts is significant because e-pili can serve as conduits for DIET. Most of the e-pilin genes and transcripts were affiliated with Geobacter species, but sequences most closely related to putative e-pilin genes from genera such as Desulfobacterium, Deferribacter, Geoalkalibacter, and Desulfobacula, were also detected. Approximately 17% of all metagenomic and metatranscriptomic bacterial sequences clustered with Geobacter species, and the finding that Geobacter spp. were actively transcribing growth-related genes indicated that they were metabolically active in the soils. Genes coding for e-pilin were among the most highly transcribed Geobacter genes. In addition, homologs of genes encoding OmcS, a c-type cytochrome associated with the e-pili of G. sulfurreducens and required for DIET, were also highly expressed in the soils. Methanothrix species in the soils highly expressed genes for enzymes involved in the reduction of carbon dioxide to methane. DIET is the only electron donor known to support CO2 reduction in Methanothrix Thus, these results are consistent with a model in which Geobacter species were providing electrons to Methanothrix species for methane production through electrical connections of e-pili.IMPORTANCEMethanothrix species are some of the most important microbial contributors to global methane production, but surprisingly little is known about their physiology and ecology. The possibility that DIET is a source of electrons for Methanothrix in methanogenic rice paddy soils is important because it demonstrates that the contribution that Methanothrix makes to methane production in terrestrial environments may extend beyond the conversion of acetate to methane. Furthermore, defined coculture studies have suggested that when Methanothrix species receive some of their energy from DIET, they grow faster than when acetate is their sole energy source. Thus, Methanothrix growth and metabolism in methanogenic soils may be faster and more robust than generally considered. The results also suggest that the reason that Geobacter species are repeatedly found to be among the most metabolically active microorganisms in methanogenic soils is that they grow syntrophically in cooperation with Methanothrix spp., and possibly other methanogens, via DIET.

A genetic assay for gene essentiality in Clostridium.

Essential genes of pathogens are potential therapeutic targets, but are difficult to verify. Here, gene essentiality was determined by targeted knockout following engineered gene duplication. Null mutants of candidate essential genes of Clostridium difficile were viable only in the presence of a stable second copy of the gene.

"Walking with Only Non-Invasive Ventilation in Stable Hypercapnic COPD Patients: Sufficient or Not?".

We are grateful to Ulasli and Esquinas for their comments to our paper.. They argued that arterial blood gas analyses were not performed without oxygen prior to the 6-minute walk test with noninvasive ventilation (6MWT-NPPV). This point has already been discussed in our original work by indicating the limitations of our study. The reason for using oxygen prior to exercise testing was to guarantee comparable starting conditions.

Minute ventilation during spontaneous breathing, high-intensity noninvasive positive pressure ventilation and intelligent volume assured pressure support in hypercapnic COPD.

BACKGROUND: High-intensity noninvasive positive pressure ventilation (HI-NPPV) is an effective treatment option in patients with stable hypercapnic chronic obstructive pulmonary disease (COPD). However, the effect of HI-NPPV compared with spontaneous breathing (SB) on minute ventilation (MV) in patients receiving long-term treatment remains to be determined. This study compared MV during HI-NPPV and SB. In addition, the ability of intelligent volume assured pressure support (iVAPS) to increase MV to the same extent as HI-NPPV was determined. METHODS: Daytime pneumotachographic measurements were performed during SB, HI-NPPV and iVAPS. RESULTS: Twenty-seven stable hypercapnic COPD patients (mean FEV1 34 ± 15% predicted) who had been treated with HI-NPPV for a median of 22 months (interquartile range 8.5-84 months) were enrolled. Mean MV was 9.5 ± 1.7 L/min during SB and 12.1 ± 2.8 L/min during HI-NPPV, an increase of 2.5 L/min (95% CI [1.5-3.6] p < 0.001), or 26%. MV during iVAPS was 11.7 ± 3.6 L/min, an increase of 1.8 L/min (95%CI [0.7-3.0], p = 0.003) compared with SB. There was no difference in MV between HI-NPPV and iVAPS (p = 0.25). CONCLUSION: Long-term HI-NPPV increased MV by an average of 26% compared with SB in stable hypercapnic COPD patients. A similar increase in MV was observed during use of iVAPS.

Life on the edge: characterising the edges of mutually non-dominating sets.

Multi-objective optimisation yields an estimated Pareto front of mutually non- dominating solutions, but with more than three objectives, understanding the relationships between solutions is challenging. Natural solutions to use as landmarks are those lying near to the edges of the mutually non-dominating set. We propose four definitions of edge points for many-objective mutually non-dominating sets and examine the relations between them. The first defines edge points to be those that extend the range of the attainment surface. This is shown to be equivalent to finding points which are not dominated on projection onto subsets of the objectives. If the objectives are to be minimised, a further definition considers points which are not dominated under maximisation when projected onto objective subsets. A final definition looks for edges via alternative projections of the set. We examine the relations between these definitions and their efficacy in many dimensions for synthetic concave- and convex-shaped sets, and on solutions to a prototypical many-objective optimisation problem, showing how they can reveal information about the structure of the estimated Pareto front. We show that the "controlling dominance area of solutions" modification of the dominance relation can be effectively used to locate edges and interior points of high-dimensional mutually non-dominating sets.

Deletion of CD44 promotes adipogenesis by regulating PPARγ and cell cycle-related pathways.

CD44, a cell surface adhesion receptor and stem cell biomarker, is recently implicated in chronic metabolic diseases. Ablation of CD44 ameliorates adipose tissue inflammation and insulin resistance in obesity. Here, we investigated cell type-specific CD44 expression in human and mouse adipose tissue and further studied how CD44 in preadipocytes regulates adipocyte function. Using Crispr Cas9-mdediated gene deletion and lentivirus-mediated gene re-expression, we discovered that deletion of CD44 promotes adipocyte differentiation and adipogenesis, whereas re-expression of CD44 abolishes this effect and decreases insulin responsiveness and adiponectin secretion in 3T3-L1 cells. Mechanistically, CD44 does so via suppressing Pparg expression. Using quantitative proteomics analysis, we further discovered that cell cycle-regulated pathways were mostly decreased by deletion of CD44. Indeed, re-expression of CD44 moderately restored expression of proteins involved in all phases of the cell cycle. These data were further supported by increased preadipocyte proliferation rates in CD44-deficient cells and re-expression of CD44 diminished this effect. Our data suggest that CD44 plays a crucial role in regulating adipogenesis and adipocyte function possibly through regulating PPARγ and cell cycle-related pathways. This study provides evidence for the first time that CD44 expressed in preadipocytes plays key roles in regulating adipocyte function outside immune cells where CD44 is primarily expressed. Therefore, targeting CD44 in (pre)adipocytes may provide therapeutic potential to treat obesity-associated metabolic complications.

Respiratory muscle function in interstitial lung disease.

Interstitial lung diseases limit daily activities, impair quality of life and result in (exertional) dyspnoea. This has mainly been attributed to a decline in lung function and impaired gas exchange. However, the contribution of respiratory muscle dysfunction to these limitations remains to be conclusively investigated. Interstitial lung disease patients and matched controls performed body plethysmography, a standardised 6-min walk test, volitional tests (respiratory drive (P0.1), global maximal inspiratory mouth occlusion pressure (PImax), sniff nasal pressure (SnPna) and inspiratory muscle load) and nonvolitional tests on respiratory muscle function and strength (twitch mouth and transdiaphragmatic pressure during bilateral magnetic phrenic nerve stimulation (TwPmo and TwPdi)). 25 patients and 24 controls were included in the study. PImax and SnPna remained unaltered (both p>0.05), whereas P0.1 and the load on the inspiratory muscles were higher (both p<0.05) in interstitial lung disease patients compared with controls. TwPmo and TwPdi were lower in interstitial lung disease patients (mean±sd TwPmo 0.86±0.4 versus 1.32±0.4, p<0.001; TwPdi 1.34±0.6 versus 1.88±0.5, p=0.022). Diaphragmatic force generation seems to be impaired in this cohort of interstitial lung disease patients while global respiratory muscle strength remains preserved. Central respiratory drive and the load imposed on the inspiratory muscles are increased. Whether impaired respiratory muscle function impacts morbidity and mortality in interstitial lung disease patients needs to be investigated in future studies.

The effect of continuous positive airway pressure on stair-climbing performance in severe COPD patients.

Stair climbing is associated with dynamic pulmonary hyperinflation and the development of severe dyspnea in patients with chronic obstructive pulmonary disease (COPD). This study aimed to assess whether (i) continuous positive airway pressure (CPAP) applied during stair climbing prevents dynamic hyperinflation and thereby reduces exercise-induced dyspnea in oxygen-dependent COPD-patients, and (ii) the CPAP-device and oxygen tank can be carried in a hip belt. In a randomised cross-over design, oxygen-dependent COPD patients performed two stair-climbing tests (44 steps): with supplemental oxygen only, then with the addition of CPAP (7 mbar). The oxygen tank and CPAP-device were carried in a hip belt during both trials. Eighteen COPD patients were included in the study. Although all patients could tolerate stair climbing with oxygen alone, 4 patients were unable to perform stair climbing while using CPAP. Fourteen COPD patients (mean FEV1 36 ± 14% pred.) completed the trial and were analyzed. The mean flow rate of supplemental oxygen was 3 ± 2 l/min during stair climbing. Lung hyperinflation, deoxygenation, hypoventilation, blood lactate production, dyspnea and the time needed to manage stair climbing were not improved by the application of CPAP (all p > 0.05). However, in comparison to climbing with oxygen alone, limb discomfort was reduced when oxygen was supplemented with CPAP (p = 0.008). In conclusion, very severe COPD patients are able to carry supporting devices such as oxygen tanks or CPAP-devices in a hip belt during stair climbing. However, the application of CPAP in addition to supplemental oxygen during stair climbing prevents neither exercise-induced dynamic hyperinflation, nor dyspnea.

Canine Pyothorax: Comparison of Culture and Susceptibility Results to the BSAVA PROTECT ME Poster and Other Published Recommended Antimicrobial Use Guidelines.

The most common bacterial isolates in dogs with pyothorax include mixed anaerobes, Enterobacteriaceae (especially Escherichia coli), Pasteurella spp., Streptococcus spp., and Staphylococcus spp. A fluoroquinolone with amoxicillin (±clavulanate) or a fluoroquinolone with clindamycin are the most commonly recommended empirical antimicrobials whilst pending bacterial culture of the pleural effusion. The aim of this study is to review and compare the pleural effusion culture and antimicrobial susceptibility results to the PROTECT ME poster and other published antimicrobial use guidelines. The medical records of 53 dogs diagnosed with pyothorax between 2014 and 2020 at two veterinary referral centres were reviewed. Information, including culture and susceptibility results, was assessed. Antimicrobial susceptibility panels varied; susceptibility to a particular antibiotic was calculated as a percentage of isolates tested against the same antibiotic. A total of 30 of 53 dogs (57.7%) had a positive pleural fluid culture. The most common isolates were Pasteurella species (23.3%), Escherichia coli (23.3%), and mixed anaerobes (20%). From the aerobic isolates, 73-83% were susceptible to a fluoroquinolone, 14/19 (74%) to amoxicillin, and 20/22 (91%) to potentiated amoxicillin. Resistance to clindamycin was documented in 9/13 (69%) aerobic isolates, with all Gram-negative bacteria (9/9) being resistant. The combination of potentiated amoxicillin with marbofloxacin would have been appropriate in most of the dogs (75-92.9%). This study shows a high rate of resistance to clindamycin, which is not a suitable option for monotherapy and may be less effective in combination therapy compared to potentiated amoxicillin.

Engineering Geobacter pili to produce metal:organic filaments.

The organized self-assembly of conductive biological structures holds promise for creating new bioelectronic devices. In particular, Geobacter sulfurreducens type IVa pili have proven to be a versatile material for fabricating protein nanowire-based devices. To scale the production of conductive pili, we designed a strain of Shewanella oneidensis that heterologously expressed abundant, conductive Geobacter pili when grown aerobically in liquid culture. S. oneidensis expressing a cysteine-modified pilin, designed to enhance the capability to bind to gold, generated conductive pili that self-assembled into biohybrid filaments in the presence of gold nanoparticles. Elemental composition analysis confirmed the filament-metal interactions within the structures, which were several orders of magnitude larger than previously described metal:organic filaments. The results demonstrate that the S. oneidensis chassis significantly advances the possibilities for facile conductive protein nanowire design and fabrication.

Negotiating targets with patients: choice of target in relation to occupational state.

OBJECTIVES: Following the recent National Institute for Health and Clinical Excellence guidance on the management of RA, we were interested to see if we could negotiate targets for treatment with patients in routine clinics, how they would express this and whether staying at work would be a target. METHODS: One hundred RA patients were recruited. They were consecutive within clinics, but not all clinics were used. They were asked their understanding of the DAS score and a target for treatment negotiated. Any impact of the RA on their paid employment was then explored. RESULTS: Four participants were unable to specify a target for their RA. Negotiated targets were expressed as restricted activities and either as maintaining an activity (70) if the disease was stable, or regaining an activity (26) if the treatment was being increased. Targets were walking a distance for 50% of patients; leisure activities for 18%; domestic activities for 17%; work for 14% and personal care for 2%. For the 21 participants currently working, maintaining work was the target for 12, with 1 wishing to regain lost hours. No patient currently not working expressed returning to work as a target. There were some differences in targets between men and women. CONCLUSIONS: Patients are able to negotiate a target for their treatment, expressed as maintaining or regaining a physical activity. Work ceases to be a target once it is lost. Therefore, preventing loss of occupation is likely to be more effective than trying to regain it.

Radiation-induced neuroinflammation: a potential protective role for poly(ADP-ribose) polymerase inhibitors?

Radiotherapy (RT) plays a fundamental role in the treatment of glioblastoma (GBM). GBM are notoriously invasive and harbor a subpopulation of cells with stem-like features which exhibit upregulation of the DNA damage response (DDR) and are radioresistant. High radiation doses are therefore delivered to large brain volumes and are known to extend survival but also cause delayed toxicity with 50%-90% of patients developing neurocognitive dysfunction. Emerging evidence identifies neuroinflammation as a critical mediator of the adverse effects of RT on cognitive function. In addition to its well-established role in promoting repair of radiation-induced DNA damage, activation of poly(ADP-ribose) polymerase (PARP) can exacerbate neuroinflammation by promoting secretion of inflammatory mediators. Therefore, PARP represents an intriguing mechanistic link between radiation-induced activation of the DDR and subsequent neuroinflammation. PARP inhibitors (PARPi) have emerged as promising new agents for GBM when given in combination with RT, with multiple preclinical studies demonstrating radiosensitizing effects and at least 3 compounds being evaluated in clinical trials. We propose that concomitant use of PARPi could reduce radiation-induced neuroinflammation and reduce the severity of radiation-induced cognitive dysfunction while at the same time improving tumor control by enhancing radiosensitivity.

Splenectomy in the management of primary immune-mediated hemolytic anemia and primary immune-mediated thrombocytopenia in dogs.

BACKGROUND: Current reports about the use of splenectomy for the management of immune-mediated hemolytic anemia (IMHA) or immune-mediated thrombocytopenia (ITP) or both in dogs are limited. OBJECTIVES: To retrospectively describe the use of splenectomy as part of the management for IMHA, ITP, and concurrent IMHA and severe thrombocytopenia (CIST) in dogs. It was hypothesized that splenectomy would be beneficial in allowing for reduction of dose of immunosuppressive drugs or discontinuation in 1 or more of these groups. ANIMALS: Seventeen client-owned dogs (7 with IMHA, 7 with ITP, and 3 with CIST) were identified across 7 UK-based referral hospitals from a study period of 2005 to 2016. METHODS: Data were collected retrospectively via questionnaires and included information about diagnosis, management and treatment response before and after splenectomy. Based on clinical outcome, treatment with splenectomy as part of the management protocol was classified as either successful or unsuccessful. RESULTS: Six of 7 dogs with ITP were managed successfully with splenectomy as part of their management protocol (3 complete and 3 partial responses), although 1 subsequently developed suspected IMHA. Of the 7 dogs with IMHA, splenectomy was part of a successful management protocol in 4 dogs (2 complete and 2 partial responses). In the CIST group, 1 case (1/3) responded completely to management with splenectomy as part of the management protocol. CONCLUSIONS AND CLINICAL IMPORTANCE: Splenectomy was considered successful and well tolerated in most cases of isolated ITP. Whether there is a benefit of splenectomy in cases of IMHA and CIST could not be determined in the current study.

Direct Observation of Electrically Conductive Pili Emanating from Geobacter sulfurreducens.

Geobacter sulfurreducens is a model microbe for elucidating the mechanisms for extracellular electron transfer in several biogeochemical cycles, bioelectrochemical applications, and microbial metal corrosion. Multiple lines of evidence previously suggested that electrically conductive pili (e-pili) are an essential conduit for long-range extracellular electron transport in G. sulfurreducens. However, it has recently been reported that G. sulfurreducens does not express e-pili and that filaments comprised of multi-heme c-type cytochromes are responsible for long-range electron transport. This possibility was directly investigated by examining cells, rather than filament preparations, with atomic force microscopy. Approximately 90% of the filaments emanating from wild-type cells had a diameter (3 nm) and conductance consistent with previous reports of e-pili harvested from G. sulfurreducens or heterologously expressed in Escherichia coli from the G. sulfurreducens pilin gene. The remaining 10% of filaments had a morphology consistent with filaments comprised of the c-type cytochrome OmcS. A strain expressing a modified pilin gene designed to yield poorly conductive pili expressed 90% filaments with a 3-nm diameter, but greatly reduced conductance, further indicating that the 3-nm diameter conductive filaments in the wild-type strain were e-pili. A strain in which genes for five of the most abundant outer-surface c-type cytochromes, including OmcS, were deleted yielded only 3-nm-diameter filaments with the same conductance as in the wild type. These results demonstrate that e-pili are the most abundant conductive filaments expressed by G. sulfurreducens, consistent with previous functional studies demonstrating the need for e-pili for long-range extracellular electron transfer. IMPORTANCE Electroactive microbes have significant environmental impacts, as well as applications in bioenergy and bioremediation. The composition, function, and even existence of electrically conductive pili (e-pili) has been one of the most contentious areas of investigation in electromicrobiology, in part because e-pili offer a mechanism for long-range electron transport that does not involve the metal cofactors common in much of biological electron transport. This study demonstrates that e-pili are abundant filaments emanating from Geobacter sulfurreducens, which serves as a model for long-range extracellular electron transfer in direct interspecies electron transfer, dissimilatory metal reduction, microbe-electrode exchange, and corrosion caused by direct electron uptake from Fe(0). The methods described in this study provide a simple strategy for evaluating the distribution of conductive filaments throughout the microbial world with an approach that avoids artifactual production and/or enrichment of filaments that may not be physiologically relevant.

Generation of High Current Densities in Geobacter sulfurreducens Lacking the Putative Gene for the PilB Pilus Assembly Motor.

Geobacter sulfurreducens is commonly employed as a model for the study of extracellular electron transport mechanisms in the Geobacter species. Deletion of pilB, which is known to encode the pilus assembly motor protein for type IV pili in other bacteria, has been proposed as an effective strategy for evaluating the role of electrically conductive pili (e-pili) in G. sulfurreducens extracellular electron transfer. In those studies, the inhibition of e-pili expression associated with pilB deletion was not demonstrated directly but was inferred from the observation that pilB deletion mutants produced lower current densities than wild-type cells. Here, we report that deleting pilB did not diminish current production. Conducting probe atomic force microscopy revealed filaments with the same diameter and similar current-voltage response as e-pili harvested from wild-type G. sulfurreducens or when e-pili are expressed heterologously from the G. sulfurreducens pilin gene in Escherichia coli. Immunogold labeling demonstrated that a G. sulfurreducens strain expressing a pilin monomer with a His tag continued to express His tag-labeled filaments when pilB was deleted. These results suggest that a reinterpretation of the results of previous studies on G. sulfurreducens pilB deletion strains may be necessary. IMPORTANCE Geobacter sulfurreducens is a model microbe for the study of biogeochemically and technologically significant processes, such as the reduction of Fe(III) oxides in soils and sediments, bioelectrochemical applications that produce electric current from waste organic matter or drive useful processes with the consumption of renewable electricity, direct interspecies electron transfer in anaerobic digestors and methanogenic soils and sediments, and metal corrosion. Elucidating the phenotypes associated with gene deletions is an important strategy for determining the mechanisms for extracellular electron transfer in G. sulfurreducens. The results reported here demonstrate that we cannot replicate the key phenotype reported for a gene deletion that has been central to the development of models for long-range electron transport in G. sulfurreducens.