scholarly journals Scarless Genome Editing and Stable Inducible Expression Vectors for Geobacter sulfurreducens

2015 ◽  
Vol 81 (20) ◽  
pp. 7178-7186 ◽  
Author(s):  
Chi Ho Chan ◽  
Caleb E. Levar ◽  
Lori Zacharoff ◽  
Jonathan P. Badalamenti ◽  
Daniel R. Bond
Keyword(s):  
Gene Clusters ◽  
Genetic System ◽  
Geobacter Sulfurreducens ◽  
Expression Vectors ◽  
Extracellular Electron Transfer ◽  
Gene Encoding ◽  
Biofilm Growth ◽  
Content Type ◽  
Biofilm Development ◽  
Citrate Reduction

ABSTRACTMetal reduction by members of theGeobacteraceaeis encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent ofGeobacterbiofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacteriumGeobacter sulfurreducens. A motile strain ofG. sulfurreducenswas constructed by precise removal of a transposon interrupting thefgrMflagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochromeimcH. We also show that RK2-based plasmids were maintained inG. sulfurreducensfor over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutiveGeobacterpromoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented ΔimcHmutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerousG. sulfurreducensstrain constructs simultaneously and shows the sensitivity ofimcHexpression by the vanillate system. These tools will enable more sophisticated genetic studies inG. sulfurreducenswithout polar insertion effects or need for multiple antibiotics.

scholarly journals Bacteriophage-Mediated Control of a Two-Species Biofilm Formed by Microorganisms Causing Catheter-Associated Urinary Tract Infections in anIn VitroUrinary Catheter Model

2014 ◽  
Vol 59 (2) ◽  
pp. 1127-1137 ◽  
Author(s):  
Susan M. Lehman ◽  
Rodney M. Donlan
Keyword(s):  
Mixed Species ◽  
Biofilm Inhibition ◽  
Biofilm Growth ◽  
Content Type ◽  
Patient Morbidity ◽  
Phage Cocktail ◽  
Artificial Urine ◽  
Tract Infections ◽  
Biofilm Development

ABSTRACTMicroorganisms from a patient or their environment may colonize indwelling urinary catheters, forming biofilm communities on catheter surfaces and increasing patient morbidity and mortality. This study investigated the effect of pretreating hydrogel-coated silicone catheters with mixtures ofPseudomonas aeruginosaandProteus mirabilisbacteriophages on the development of single- and two-species biofilms in a multiday continuous-flowin vitromodel using artificial urine. Novel phages were purified from sewage, characterized, and screened for their abilities to reduce biofilm development by clinical isolates of their respective hosts. Our screening data showed that artificial urine medium (AUM) is a valid substitute for human urine for the purpose of evaluating uropathogen biofilm control by these bacteriophages. Defined phage cocktails targetingP. aeruginosaandP. mirabiliswere designed based on the biofilm inhibition screens. Hydrogel-coated catheters were pretreated with one or both cocktails and challenged with approximately 1 × 103CFU/ml of the corresponding pathogen(s). The biofilm growth on the catheter surfaces in AUM was monitored over 72 to 96 h. Phage pretreatment reducedP. aeruginosabiofilm counts by 4 log10CFU/cm2(P≤ 0.01) andP. mirabilisbiofilm counts by >2 log10CFU/cm2(P≤ 0.01) over 48 h. The presence ofP. mirabiliswas always associated with an increase in lumen pH from 7.5 to 9.5 and with eventual blockage of the reactor lines. The results of this study suggest that pretreatment of a hydrogel urinary catheter with a phage cocktail can significantly reduce mixed-species biofilm formation by clinically relevant bacteria.

scholarly journals Identification of Different Putative Outer Membrane Electron Conduits Necessary for Fe(III) Citrate, Fe(III) Oxide, Mn(IV) Oxide, or Electrode Reduction byGeobacter sulfurreducens

2018 ◽  
Vol 200 (19) ◽  
Author(s):  
Fernanda Jiménez Otero ◽  
Chi Ho Chan ◽  
Daniel R. Bond
Keyword(s):  
Electron Transfer ◽  
Outer Membrane ◽  
Electron Acceptor ◽  
Gene Clusters ◽  
Geobacter Sulfurreducens ◽  
Transcriptional Analysis ◽  
Metal Reduction ◽  
Wild Type ◽  
Redox Proteins ◽  
Content Type

ABSTRACTAt least five gene clusters in theGeobacter sulfurreducensgenome encode putative “electron conduits” implicated in electron transfer across the outer membrane, each containing a periplasmic multihemec-type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single-gene-cluster deletions and all possible multiple-deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III) and Mn(IV) oxides, and graphite electrodes poised at +0.24 V and −0.1 V versus the standard hydrogen electrode (SHE). Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously describedomcBCcluster caused defects, but deletion of additional components in an ΔomcBCbackground, such asextEFG, were needed to produce defects greater than 50% compared to findings with the wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the ΔextABCDmutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III) oxide, Mn(IV) oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than the wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth conditions showed all of theseextclusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing onlyextABCDdetected no significant changes in expression of genes encoding known redox proteins or pilus components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth ofG. sulfurreducens, depending on the available extracellular electron acceptor.IMPORTANCEGram-negative metal-reducing bacteria utilize electron conduits, chains of redox proteins spanning the outer membrane, to transfer electrons to the extracellular surface. Only one pathway for electron transfer across the outer membrane ofGeobacter sulfurreducenshas been linked to Fe(III) reduction. However,G. sulfurreducensis able to respire a wide array of extracellular substrates. Here we present the first combinatorial genetic analysis of five different electron conduits via creation of new markerless deletion strains and complementation vectors. Multiple conduit gene clusters appear to have overlapping roles, including two that have never been linked to metal reduction. Another recently described cluster (ExtABCD) was the only electron conduit essential during electrode reduction, a substrate of special importance to biotechnological applications of this organism.

scholarly journals Outer Cell Surface Components Essential for Fe(III) Oxide Reduction by Geobacter metallireducens

2012 ◽  
Vol 79 (3) ◽  
pp. 901-907 ◽  
Author(s):  
Jessica A. Smith ◽  
Derek R. Lovley ◽  
Pier-Luc Tremblay
Keyword(s):  
Outer Surface ◽  
Surface Protein ◽  
Transcript Abundance ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Outer Cell ◽  
Gene Transcript ◽  
Oxide Reduction ◽  
Geobacter Metallireducens ◽  
Content Type

ABSTRACTGeobacterspecies are important Fe(III) reducers in a diversity of soils and sediments. Mechanisms for Fe(III) oxide reduction have been studied in detail inGeobacter sulfurreducens, but a number of the most thoroughly studied outer surface components ofG. sulfurreducens, particularlyc-type cytochromes, are not well conserved amongGeobacterspecies. In order to identify cellular components potentially important for Fe(III) oxide reduction inGeobacter metallireducens, gene transcript abundance was compared in cells grown on Fe(III) oxide or soluble Fe(III) citrate with whole-genome microarrays. Outer-surface cytochromes were also identified. Deletion of genes forc-type cytochromes that had higher transcript abundance during growth on Fe(III) oxides and/or were detected in the outer-surface protein fraction identified sixc-type cytochrome genes, that when deleted removed the capacity for Fe(III) oxide reduction. Several of thec-type cytochromes which were essential for Fe(III) oxide reduction inG. metallireducenshave homologs inG. sulfurreducensthat are not important for Fe(III) oxide reduction. Other genes essential for Fe(III) oxide reduction included a gene predicted to encode an NHL (Ncl-1–HT2A–Lin-41) repeat-containing protein and a gene potentially involved in pili glycosylation. Genes associated with flagellum-based motility, chemotaxis, and pili had higher transcript abundance during growth on Fe(III) oxide, consistent with the previously proposed importance of these components in Fe(III) oxide reduction. These results demonstrate that there are similarities in extracellular electron transfer betweenG. metallireducensandG. sulfurreducensbut the outer-surfacec-type cytochromes involved in Fe(III) oxide reduction are different.

scholarly journals Elucidating the Genetic Basis of Crystalline Biofilm Formation in Proteus mirabilis

2014 ◽  
Vol 82 (4) ◽  
pp. 1616-1626 ◽  
Author(s):  
N. Holling ◽  
D. Lednor ◽  
S. Tsang ◽  
A. Bissell ◽  
L. Campbell ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Proteus Mirabilis ◽  
Biofilm Formation ◽  
Efflux Pump ◽  
Model Systems ◽  
Multidrug Efflux Pump ◽  
Gene Encoding ◽  
Content Type ◽  
Biofilm Development

ABSTRACTProteus mirabilisforms extensive crystalline biofilms on urethral catheters that occlude urine flow and frequently complicate the management of long-term-catheterized patients. Here, using random transposon mutagenesis in conjunction within vitromodels of the catheterized urinary tract, we elucidate the mechanisms underpinning the formation of crystalline biofilms byP. mirabilis. Mutants identified as defective in blockage of urethral catheters had disruptions in genes involved in nitrogen metabolism and efflux systems but were unaffected in general growth, survival in bladder model systems, or the ability to elevate urinary pH. Imaging of biofilms directly on catheter surfaces, along with quantification of levels of encrustation and biomass, confirmed that the mutants were attenuated specifically in the ability to form crystalline biofilms compared with that of the wild type. However, the biofilm-deficient phenotype of these mutants was not due to deficiencies in attachment to catheter biomaterials, and defects in later stages of biofilm development were indicated. For one blocking-deficient mutant, the disrupted gene (encoding a putative multidrug efflux pump) was also found to be associated with susceptibility to fosfomycin, and loss of this system or general inhibition of efflux pumps increased sensitivity to this antibiotic. Furthermore, homologues of this system were found to be widely distributed among other common pathogens of the catheterized urinary tract. Overall, our findings provide fundamental new insight into crystalline biofilm formation byP. mirabilis, including the link between biofilm formation and antibiotic resistance in this organism, and indicate a potential role for efflux pump inhibitors in the treatment or prevention ofP. mirabiliscrystalline biofilms.

scholarly journals Metabolic Remodeling during Biofilm Development ofBacillus subtilis

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Tippapha Pisithkul ◽  
Jeremy W. Schroeder ◽  
Edna A. Trujillo ◽  
Ponlkrit Yeesin ◽  
David M. Stevenson ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Regulatory Networks ◽  
Developmental Process ◽  
Central Carbon Metabolism ◽  
Bacterial Biofilm ◽  
Biofilm Growth ◽  
Time Resolved ◽  
Content Type ◽  
Metabolic Remodeling ◽  
Biofilm Development

ABSTRACTBiofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such asBacillus subtilishas been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development inB. subtilisby combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring duringB. subtilisbiofilm development that will serve as a useful road map for future studies on biofilm physiology.IMPORTANCEBacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such asBacillus subtilis, very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development inB. subtilisby combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism.

scholarly journals Real-Time Microscopic Observation of Candida Biofilm Development and Effects Due to Micafungin and Fluconazole

2013 ◽  
Vol 57 (5) ◽  
pp. 2226-2230 ◽  
Author(s):  
Yukihiro Kaneko ◽  
Susumu Miyagawa ◽  
On Takeda ◽  
Masateru Hakariya ◽  
Satoru Matsumoto ◽  
...  
Keyword(s):  
Real Time ◽  
Time Lapse ◽  
Time Data ◽  
Biofilm Growth ◽  
Content Type ◽  
Biofilm Thickness ◽  
Ergosterol Synthesis ◽  
Glucan Synthesis ◽  
Biofilm Development ◽  
Observation Period

ABSTRACTTo understand the process ofCandidabiofilm development and the effects of antifungal agents on biofilms, we analyzed real-time data comprising time-lapse images taken at times separated by brief intervals. The growth rate was calculated by measuring the change of biofilm thickness every hour. For the antifungal study, 5-h-old biofilms ofCandida albicanswere treated with either micafungin (MCFG) or fluconazole (FLCZ). MCFG began to suppress biofilm growth a few minutes after the initiation of the treatment, and this effect was maintained over the course of the observation period. In contrast, the suppressive effects of FLCZ on biofilm growth took longer to manifest: biofilms grew in the first 5 h after treatment, and then their growth was suppressed over the next 10 h, finally producing results similar to those observed with MCFG. MCFG was also involved in the disruption of cells in the biofilms, releasing string-like structures (undefined extracellular component) from the burst hyphae. Thus, MCFG inhibited the detachment of yeast cell clusters from the tips of hyphae. In contrast, FLCZ did not disrupt biofilm cells. MCFG also showed fast antifungal activity againstCandida parapsilosisbiofilms. In conclusion, our results show that inhibition of glucan synthesis due to MCFG contributed not only to fungicidal activity but also to the immediate suppression of biofilm growth, while FLCZ suppressed growth by inhibiting ergosterol synthesis. Therefore, those characteristic differences should be considered when treating clinical biofilm infections.

scholarly journals Adaptive Evolution of Geobacter sulfurreducens in Coculture with Pseudomonas aeruginosa

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Lucie Semenec ◽  
Ismael A. Vergara ◽  
Andrew E. Laloo ◽  
Steve Petrovski ◽  
Philip L. Bond ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Electron Transfer ◽  
Adaptive Evolution ◽  
Efflux Pump ◽  
Antibiotic Production ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Content Type ◽  
Interaction Dynamics ◽  
Syntrophic Interaction

ABSTRACT 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. sulfurreducens fabI 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. IMPORTANCE Geobacter 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.

scholarly journals Extracellular Electron Transfer: Respiratory or Nutrient Homeostasis?

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Lars J. C. Jeuken ◽  
Kiel Hards ◽  
Yoshio Nakatani
Keyword(s):  
Electron Transfer ◽  
Iron Reduction ◽  
Ferric Iron ◽  
Shewanella Oneidensis ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Nutrient Homeostasis

ABSTRACT Exoelectrogens are able to transfer electrons extracellularly, enabling them to respire on insoluble terminal electron acceptors. Extensively studied exoelectrogens, such as Geobacter sulfurreducens and Shewanella oneidensis, are Gram negative. More recently, it has been reported that Gram-positive bacteria, such as Listeria monocytogenes and Enterococcus faecalis, also exhibit the ability to transfer electrons extracellularly, although it is still unclear whether this has a function in respiration or in redox control of the environment, for instance, by reducing ferric iron for iron uptake. In this issue of Journal of Bacteriology, Hederstedt and colleagues report on experiments that directly compare extracellular electron transfer (EET) pathways for ferric iron reduction and respiration and find a clear difference (L. Hederstedt, L. Gorton, and G. Pankratova, J Bacteriol 202:e00725-19, 2020, https://doi.org/10.1128/JB.00725-19), providing further insights and new questions into the function and metabolic pathways of EET in Gram-positive bacteria.

scholarly journals Role ofepaQ, a Previously UncharacterizedEnterococcus faecalisGene, in Biofilm Development and Antimicrobial Resistance

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Michelle L. Korir ◽  
Jennifer L. Dale ◽  
Gary M. Dunny
Keyword(s):  
Antibiotic Resistance ◽  
Biofilm Formation ◽  
Cell Surface Hydrophobicity ◽  
Opportunistic Pathogen ◽  
Surface Hydrophobicity ◽  
Biofilm Growth ◽  
Content Type ◽  
Biofilm Architecture ◽  
High Level ◽  
Biofilm Development

ABSTRACTEnterococcus faecalisis a commensal of the human gastrointestinal tract; it is also an opportunistic pathogen and one of the leading causes of hospital-acquired infections.E. faecalisproduces biofilms that are highly resistant to antibiotics, and it has been previously reported that certain genes of theepaoperon contribute to biofilm-associated antibiotic resistance. Despite several studies examining theepaoperon, many gene products of this operon remain annotated as hypothetical proteins. Here, we further explore theepaoperon; we identifiedepaQ, currently annotated as encoding a hypothetical membrane protein, as being important for biofilm formation in the presence of the antibiotic daptomycin. Mutants with disruptions ofepaQwere more susceptible to daptomycin relative to the wild type, suggesting its importance in biofilm-associated antibiotic resistance. Furthermore, the ΔepaQmutant exhibited an altered biofilm architectural arrangement and formed small aggregates in liquid cultures. Our cumulative data show thatepamutations result in altered polysaccharide content, increased cell surface hydrophobicity, and decreased membrane potential. Surprisingly, severalepamutations significantly increased resistance to the antibiotic ceftriaxone, indicating that the way in which theepaoperon impacts antibiotic resistance is antibiotic dependent. These results further define the key role ofepain antibiotic resistance in biofilms and in biofilm architecture.IMPORTANCEE. faecalisis a common cause of nosocomial infection, has a high level of antibiotic resistance, and forms robust biofilms. Biofilm formation is associated with increased antibiotic resistance. Therefore, a thorough understanding of biofilm-associated antibiotic resistance is important for combating resistance. Several genes from theepaoperon have previously been implicated in biofilm-associated antibiotic resistance, pathogenesis, and competitive fitness in the GI tract, but most genes in this locus remain uncharacterized. Here, we examineepaQ,which has not been characterized functionally. We show that the ΔepaQmutant exhibits reduced biofilm formation in the presence of daptomycin, altered biofilm architecture, and increased resistance to ceftriaxone, further expanding our understanding of the contribution of this operon to intrinsic enterococcal antibiotic resistance and biofilm growth.

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