Biofilm shows spatially stratified metabolic responses to contaminant exposure

Biofilms are core to a range of biological processes, including the bioremediation of environmental contaminants. Within a biofilm population, cells with diverse genotypes and phenotypes coexist, suggesting that distinct metabolic pathways may be expressed based on the local environmental conditions in a biofilm. However, metabolic responses to local environmental conditions in a metabolically active biofilm interacting with environmental contaminants have never been quantitatively elucidated. In this study, we monitored the spatiotemporal metabolic responses of metabolically active Shewanella oneidensis MR‐1 biofilms to U(VI) (uranyl, UO₂ ²⁺) and Cr(VI) (chromate, CrO₄ ^2?) using non‐invasive nuclear magnetic resonance imaging (MRI) and spectroscopy (MRS) approaches to obtain insights into adaptation in biofilms during biofilm‐contaminant interactions. While overall biomass distribution was not significantly altered upon exposure to U(VI) or Cr(VI), MRI and spatial mapping of the diffusion revealed localized changes in the water diffusion coefficients in the biofilms, suggesting significant contaminant‐induced changes in structural or hydrodynamic properties during bioremediation. Finally, we quantitatively demonstrated that the metabolic responses of biofilms to contaminant exposure are spatially stratified, implying that adaptation in biofilms is custom‐developed based on local microenvironments.     

Use of an Electrochemical Split Cell Technique to Evaluate the Influence of Shewanella oneidensis Activities on Corrosion of Carbon Steel

Microbially induced corrosion (MIC) is a complex problem that affects various industries. Several techniques have been developed to monitor corrosion and elucidate corrosion mechanisms, including microbiological processes that induce metal deterioration. We used zero resistance ammetry (ZRA) in a split chamber configuration to evaluate the effects of the facultatively anaerobic Fe(III) reducing bacterium Shewanella oneidensis MR-1 on the corrosion of UNS {"type":"entrez-nucleotide","attrs":{"text":"G10180","term_id":"942029"}}G10180 carbon steel. We show that activities of S. oneidensis inhibit corrosion of steel with which that organism has direct contact. However, when a carbon steel coupon in contact with S. oneidensis was electrically connected to a second coupon that was free of biofilm (in separate chambers of the split chamber assembly), ZRA-based measurements indicated that current moved from the S. oneidensis-containing chamber to the cell-free chamber. This electron transfer enhanced the O₂ reduction reaction on the coupon deployed in the cell free chamber, and consequently, enhanced oxidation and corrosion of that electrode. Our results illustrate a novel mechanism for MIC in cases where metal surfaces are heterogeneously covered by biofilms.     

Microbial reduction of Cr(VI) in the presence of chromate conversion coating constituents

The reduction of Cr(VI) by the metal-reducing bacterium Shewanella oneidensis MR-1 was evaluated, to determine the potential for exploiting Cr(VI) bioreduction as a means of treating chromate conversion coating (CCC) waste streams. Inclusion of Cr(VI) at concentrations ≥1 mM inhibited aerobic growth of S. oneidensis, but that organism was able to reduce Cr(VI) at a concentration of up to 1 mM under anaerobic, nongrowth conditions. S. oneidensis reduced Cr(VI) in the presence of common CCC constituents, with the exception of ferricyanide, when these CCC constituents were included at concentrations typical of CCC waste streams. Ferricyanide inhibited neither aerobic growth nor metabolism under aerobic, nitrate- or iron-reducing conditions, suggesting that the ferricyanide-depended inhibition of Cr(VI) reduction is not due to broad metabolic inhibition, but is specific to Cr(VI) reduction. Results indicate that under some conditions, the activities of metal-reducing bacteria, such as S. oneidensis, could be exploited for the removal of Cr(VI) from CCC waste streams under appropriate conditions.     

Iron Triggers λSo Prophage Induction and Release of Extracellular DNA in Shewanella oneidensis MR-1 Biofilms

Prophages are ubiquitous elements within bacterial chromosomes and affect host physiology and ecology in multiple ways. We have previously demonstrated that phage-induced lysis is required for extracellular DNA (eDNA) release and normal biofilm formation in Shewanella oneidensis MR-1. Here, we investigated the regulatory mechanisms of prophage λSo spatiotemporal induction in biofilms. To this end, we used a functional fluorescence fusion to monitor λSo activation in various mutant backgrounds and in response to different physiological conditions. λSo induction occurred mainly in a subpopulation of filamentous cells in a strictly RecA-dependent manner, implicating oxidative stress-induced DNA damage as the major trigger. Accordingly, mutants affected in the oxidative stress response (ΔoxyR) or iron homeostasis (Δfur) displayed drastically increased levels of phage induction and abnormal biofilm formation, while planktonic cells were not or only marginally affected. To further investigate the role of oxidative stress, we performed a mutant screen and identified two independent amino acid substitutions in OxyR (T104N and L197P) that suppress induction of λSo by hydrogen peroxide (H₂O₂). However, λSo induction was not suppressed in biofilms formed by both mutants, suggesting a minor role of intracellular H₂O₂ in this process. In contrast, addition of iron to biofilms strongly enhanced λSo induction and eDNA release, while both processes were significantly suppressed at low iron levels, strongly indicating that iron is the limiting factor. We conclude that uptake of iron during biofilm formation triggers λSo-mediated lysis of a subpopulation of cells, likely by an increase in iron-mediated DNA damage sensed by RecA.     

In situ effective diffusion coefficient profiles in live biofilms using pulsed‐field gradient nuclear magnetic resonance

Diffusive mass transfer in biofilms is characterized by the effective diffusion coefficient. It is well documented that the effective diffusion coefficient can vary by location in a biofilm. The current literature is dominated by effective diffusion coefficient measurements for distinct cell clusters and stratified biofilms showing this spatial variation. Regardless of whether distinct cell clusters or surface‐averaging methods are used, position‐dependent measurements of the effective diffusion coefficient are currently: (1) invasive to the biofilm, (2) performed under unnatural conditions, (3) lethal to cells, and/or (4) spatially restricted to only certain regions of the biofilm. Invasive measurements can lead to inaccurate results and prohibit further (time‐dependent) measurements which are important for the mathematical modeling of biofilms. In this study our goals were to: (1) measure the effective diffusion coefficient for water in live biofilms, (2) monitor how the effective diffusion coefficient changes over time under growth conditions, and (3) correlate the effective diffusion coefficient with depth in the biofilm. We measured in situ two‐dimensional effective diffusion coefficient maps within Shewanella oneidensis MR‐1 biofilms using pulsed‐field gradient nuclear magnetic resonance methods, and used them to calculate surface‐averaged relative effective diffusion coefficient (D_rs) profiles. We found that (1) D_rs decreased from the top of the biofilm to the bottom, (2) D_rs profiles differed for biofilms of different ages, (3) D_rs profiles changed over time and generally decreased with time, (4) all the biofilms showed very similar D_rs profiles near the top of the biofilm, and (5) the D_rs profile near the bottom of the biofilm was different for each biofilm. Practically, our results demonstrate that advanced biofilm models should use a variable effective diffusivity which changes with time and location in the biofilm. Biotechnol. Bioeng. 2010;106: 928–937. © 2010 Wiley Periodicals, Inc.     

In Situ Analysis of a Silver Nanoparticle-Precipitating Shewanella Biofilm by Surface Enhanced Confocal Raman Microscopy

Shewanella oneidensis MR-1 is an electroactive bacterium, capable of reducing extracellular insoluble electron acceptors, making it important for both nutrient cycling in nature and microbial electrochemical technologies, such as microbial fuel cells and microbial electrosynthesis. When allowed to anaerobically colonize an Ag/AgCl solid interface, S. oneidensis has precipitated silver nanoparticles (AgNp), thus providing the means for a surface enhanced confocal Raman microscopy (SECRaM) investigation of its biofilm. The result is the in-situ chemical mapping of the biofilm as it developed over time, where the distribution of cytochromes, reduced and oxidized flavins, polysaccharides and phosphate in the undisturbed biofilm is monitored. Utilizing AgNp bio-produced by the bacteria colonizing the Ag/AgCl interface, we could perform SECRaM while avoiding the use of a patterned or roughened support or the introduction of noble metal salts and reducing agents. This new method will allow a spatially and temporally resolved chemical investigation not only of Shewanella biofilms at an insoluble electron acceptor, but also of other noble metal nanoparticle-precipitating bacteria in laboratory cultures or in complex microbial communities in their natural habitats.     

Bacteriophage-Mediated Dispersal of Campylobacter jejuni Biofilms

Bacteria in their natural environments frequently exist as mixed surface-associated communities, protected by extracellular material, termed biofilms. Biofilms formed by the human pathogen Campylobacter jejuni may arise in the gastrointestinal tract of animals but also in water pipes and other industrial situations, leading to their possible transmission into the human food chain either directly or via farm animals. Bacteriophages are natural predators of bacteria that usually kill their prey by cell lysis and have potential application for the biocontrol and dispersal of target bacteria in biofilms. The effects of virulent Campylobacter specific-bacteriophages CP8 and CP30 on C. jejuni biofilms formed on glass by strains NCTC 11168 and PT14 at 37°C under microaerobic conditions were investigated. Independent bacteriophage treatments (n ≥ 3) led to 1 to 3 log₁₀ CFU/cm² reductions in the viable count 24 h postinfection compared with control levels. In contrast, bacteriophages applied under these conditions effected a reduction of less than 1 log₁₀ CFU/ml in planktonic cells. Resistance to bacteriophage in bacteria surviving bacteriophage treatment of C. jejuni NCTC 11168 biofilms was 84% and 90% for CP8 and CP30, respectively, whereas bacteriophage resistance was not found in similarly recovered C. jejuni PT14 cells. Dispersal of the biofilm matrix by bacteriophage was demonstrated by crystal violet staining and transmission electron microscopy. Bacteriophage may play an important role in the control of attachment and biofilm formation by Campylobacter in situations where biofilms occur in nature, and they have the potential for application in industrial situations leading to improvements in food safety.     

Cellular and biochemical response to Cr(VI) in Stenotrophomonas sp.

Chromium (Cr)-resistant bacteria isolated from a soil with 6 g kg⁻¹ of Cr were identified based on 16S rRNA gene sequence analysis as a Stenotrophomonas , and designated as JD1. Growth of JD1 was accompanied by transformation of Cr(VI) to Cr(III) in liquid medium initially containing 300 mg L⁻¹ Cr(VI), the maximum concentration allowing growth. JD1 produced the highest levels of a Cr(VI)-binding exopolysaccharide when grown in medium with 100 mg L⁻¹ Cr(VI). The relative exopolysaccharide monosaccharide composition was analysed by HPLC, which showed that rhamnose+galactose was the major component, and that its relative level increased when cells were grown with Cr(VI). JD1 grew as a biofilm on various inert surfaces. Biofilm macromolecular composition analysis indicated that the relative levels of exopolysaccharide and protein were more abundant in biofilms grown in 100 mg L⁻¹ Cr(VI), whereas relative uronic acid levels remained constant. Biofilm cells exposed to Cr(VI) were elongated, grouped in clusters and exopolysaccharide obtained from the biofilm extracellular matrix had an enhanced capacity to bind Cr(VI). Exopolysaccharide production and composition, and biofilm growth are discussed as a mechanism of protection that allows survival during Cr(VI) stress.     

Influence of Hydrodynamics and Cell Signaling on the Structure and Behavior of Pseudomonas aeruginosa Biofilms

Biofilms were grown from wild-type (WT) Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (P < 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies.     

Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.Subject terms: Microbiology, Diseases     

Enhanced Exopolymer Production and Chromium Stabilization in Pseudomonas putida Unsaturated Biofilms

Chromium-contaminated soils threaten surface and groundwater quality at many industrial sites. In vadose zones, indigenous bacteria can reduce Cr(VI) to Cr(III), but the subsequent fate of Cr(III) and the roles of bacterial biofilms are relatively unknown. To investigate, we cultured Pseudomonas putida, a model organism for vadose zone bioremediation, as unsaturated biofilms on membranes overlaying iron-deficient solid media either containing molecular dichromate from potassium dichromate (Cr-only treatment) or with deposits of solid, dichromate-coated hematite (Fe+Cr treatment) to simulate vadose zone conditions. Controls included iron-deficient solid medium and an Fe-only treatment using solid hematite deposits. Under iron-deficient conditions, chromium exposure resulted in lower cell yield and lower amounts of cellular protein and carbohydrate, but providing iron in the form of hematite overcame these toxic effects of Cr. For the Cr and Fe+Cr treatments, Cr(VI) was completely reduced to Cr(III) that accumulated on biofilm cells and extracellular polymeric substances (EPSs). Chromium exposure resulted in elevated extracellular carbohydrates, protein, DNA, and EPS sugars that were relatively enriched in N-acetyl-glucosamine, rhamnose, glucose, and mannose. The proportions of EPS protein and carbohydrate relative to intracellular pools suggested Cr toxicity-mediated cell lysis as the origin. However, DNA accumulated extracellularly in amounts far greater than expected from cell lysis, and Cr was liberated when extracted EPS was treated with DNase. These results demonstrate that Cr accumulation in unsaturated biofilms occurs with enzymatic reduction of Cr(VI), cellular lysis, cellular association, and extracellular DNA binding of Cr(III), which altogether can facilitate localized biotic stabilization of Cr in contaminated vadose zones.     

Magnesium-induced biofilm development in Arthrobacter sp. SUK 1201 and removal of hexavalent chromium

This study reports the influence of Mg ions on the development and architecture of biofilms by a chromium resistant and reducing bacterium Arthrobacter sp. SUK 1201 and their utilization in the removal of toxic hexavalent chromium. Among the different metal ions tested, Mg(II) greatly influenced the biofilm growth in peptone yeast extract glucose medium. Both Scanning and Confocal Laser Scanning Microscopy revealed that biofilms formed under the induction of Mg(II) had characteristic higher cell densities. The cells remain embedded in thick porous layers of extracellular polymeric substances as evident from the fluorescein isothiocyanate labeled lectin concanavalin A and 4, 6- diamino-2-phenylindole staining. COMSTAT analysis also indicated maximum thickness and roughness coefficient of the biofilm grown in presence of Mg(II). Biofilms of Arthrobacter sp. SUK 1201 developed under such Mg (II) influenced condition showed complete removal of 0.5 mM Cr(VI) in mineral salts medium. The biofilm of this isolate grown in presence of Mg(II) was also able to remove 60µM Cr(VI) from mine seepage water suggesting its possible implication in effective bioremediation of chromium polluted environments.     

Carbon nanotubes promote Cr(VI) reduction by alginate-immobilized Shewanella oneidensis MR-1

Bioreduction of hexavalent chromium (Cr(VI)) into trivalent one (Cr(III)) based on microbial immobilization techniques has been recognized as a promising way to remove Cr contaminants from wastewater. However, such a bioreduction process is inefficient due to limited electron transfer through the immobilization matrix. In this study, a modified immobilization process was proposed by impregnating carbon nanotubes (CNTs) into Ca-alginate beads, which were then used to immobilize Shewanella oneidensis MR-1 for enhanced Cr(VI) reduction. Compared with the free cells and the beads without CNTs, the AL/CNT/cell beads showed up to 4 times higher reduction rates, mainly attributed to an enhanced electron transfer by the CNTs. In addition, the dose of CNTs greatly improved the stability of beads, suggesting a high feasibility of the AL/CNT/cell beads for repeated use. The optimized CNT concentration, temperature and pH for Cr(VI) reduction by the AL/CNT/cell beads were 0.5%, 30 °C and 6.0–7.0, respectively.     

Spatiometabolic Stratification of Shewanella oneidensis Biofilms

Biofilms, or surface-attached microbial communities, are both ubiquitous and resilient in the environment. Although much is known about how biofilms form, develop, and detach, very little is understood about how these events are related to metabolism and its dynamics. It is commonly thought that large subpopulations of cells within biofilms are not actively producing proteins or generating energy and are therefore dead. An alternative hypothesis is that within the growth-inactive domains of biofilms, significant populations of living cells persist and retain the capacity to dynamically regulate their metabolism. To test this, we employed unstable fluorescent reporters to measure growth activity and protein synthesis in vivo over the course of biofilm development and created a quantitative routine to compare domains of activity in independently grown biofilms. Here we report that Shewanella oneidensis biofilm structures reproducibly stratify with respect to growth activity and metabolism as a function of size. Within domains of growth-inactive cells, genes typically upregulated under anaerobic conditions are expressed well after growth has ceased. These findings reveal that, far from being dead, the majority of cells in mature S. oneidensis biofilms have actively turned-on metabolic programs appropriate to their local microenvironment and developmental stage.     

Prostaglandin E2 from Candida albicans Stimulates the Growth of Staphylococcus aureus in Mixed Biofilms

Background

Previous studies showed that Staphylococcus aureus and Candida albicans interact synergistically in dual species biofilms resulting in enhanced mortality in animal models.

Methodology/Principal Findings

The aim of the current study was to test possible candidate molecules which might mediate this synergistic interaction in an in vitro model of mixed biofilms, such as farnesol, tyrosol and prostaglandin (PG) E₂. In mono-microbial and dual biofilms of C.albicans wild type strains PGE₂ levels between 25 and 250 pg/mL were measured. Similar concentrations of purified PGE₂ significantly enhanced S.aureus biofilm formation in a mode comparable to that observed in dual species biofilms. Supernatants of the null mutant deficient in PGE₂ production did not stimulate the proliferation of S.aureus and the addition of the cyclooxygenase inhibitor indomethacin blocked the S.aureus biofilm formation in a dose-dependent manner. Additionally, S. aureus biofilm formation was boosted by low and inhibited by high farnesol concentrations. Supernatants of the farnesol-deficient C. albicans ATCC10231 strain significantly enhanced the biofilm formation of S. aureus but at a lower level than the farnesol producer SC5314. However, C. albicans ATCC10231 also produced PGE₂ but amounts were significantly lower compared to SC5314.

Conclusion/Significance

In conclision, we identified C. albicans PGE₂ as a key molecule stimulating the growth and biofilm formation of S. aureus in dual S. aureus/C. albicans biofilms, although C. albicans derived farnesol, but not tyrosol, may also contribute to this effect but to a lesser extent.     

Temporal root responses in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> L. to chromate reveal structural and regulatory mechanisms involving the SOLITARY ROOT/IAA14 repressor for maintenance of identity meristem genes

The Arabidopsis root system is modified in response to stress generated by high concentrations of nonessential ions such as chromate [Cr(VI)]. In this work, the distribution of auxin and its transporters PIN1 and PIN7, as well as the expression of genes that maintain the identity of the root meristem, were analyzed in Arabidopsis thaliana wild-type (WT) seedlings and in a mutant affected in the SOLITARY ROOT (SLR1/IAA14) locus, which is required for root response to Cr(VI). We show that primary root inhibition, auxin transporter levels, and expression of meristem identity genes were maintained in the slr-1 mutants but not in WT plants in response to Cr(VI) in a time- and concentration-dependent manner. Notably, the outermost single cell layer of the lateral root cap, which normally dies and tends to peel off, remains viable and increases in size following exposure of WT plants, but not slr-1 mutants, to Cr(VI). Our results suggest that (1) the primary root tip senses Cr(VI), (2) the external lateral root cap may play a protective role during Cr(VI) exposure, and (3) Cr(VI) impacts cell division in root meristems via auxin redistribution and SLR1/IAA14 function, influencing the expression of root meristem genes.     

Flagella-Mediated Adhesion and Extracellular DNA Release Contribute to Biofilm Formation and Stress Tolerance of Campylobacter jejuni

Campylobacter jejuni is a leading cause of foodbourne gastroenteritis, despite fragile behaviour under standard laboratory conditions. In the environment, C. jejuni may survive within biofilms, which can impart resident bacteria with enhanced stress tolerance compared to their planktonic counterparts. While C. jejuni forms biofilms in vitro and in the wild, it had not been confirmed that this lifestyle confers stress tolerance. Moreover, little is understood about molecular mechanisms of biofilm formation in this pathogen. We previously found that a ΔcprS mutant, which carries a deletion in the sensor kinase of the CprRS two-component system, forms enhanced biofilms. Biofilms were also enhanced by the bile salt deoxycholate and contained extracellular DNA. Through more in-depth analysis of ΔcprS and WT under conditions that promote or inhibit biofilms, we sought to further define this lifestyle for C. jejuni. Epistasis experiments with ΔcprS and flagellar mutations (ΔflhA, ΔpflA) suggested that initiation is mediated by flagellum-mediated adherence, a process which was kinetically enhanced by motility. Lysis was also observed, especially under biofilm-enhancing conditions. Microscopy suggested adherence was followed by release of eDNA, which was required for biofilm maturation. Importantly, inhibiting biofilm formation by removal of eDNA with DNase decreased stress tolerance. This work suggests the biofilm lifestyle provides C. jejuni with resilience that has not been apparent from observation of planktonic bacteria during routine laboratory culture, and provides a framework for subsequent molecular studies of C. jejuni biofilms.     

Potential of Marine-Derived Fungi to Remove Hexavalent Chromium Pollutant from Culture Broth

Chromium (Cr) released from industrial units such as tanneries, textile and electroplating industries is detrimental to the surrounding ecosystems and human health. The focus of the present study was to check the Cr(VI) removal efficiency by marine-derived fungi from liquid broth. Amongst the three Cr(VI) tolerant isolates, #NIOSN-SK56-S19 (Aspergillus sydowii) showed Cr-removal efficiency of 0.01 mg Cr mg^?1 biomass resulting in 26% abatement of total Cr with just 2.8 mg of biomass produced during the growth in 300 ppm Cr(VI). Scanning Electron Microscopy revealed aggregation of mycelial biomass with exopolysaccharide, while Electron Dispersive Spectroscopy showed the presence of Cr₂O₃ inside the biomass indicating presence of active Cr(VI) removal mechanisms. This was further supported when the Cr(VI) removal was monitored using DPC (1,5-diphenylcarbazide) method. The results of this study point to the potential of marine-derived fungal isolates for Cr(VI) removal.