Roles of two Shewanella oneidensis MR-1 extracellular endonucleases

The dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 is capable of using extracellular DNA (eDNA) as the sole source of carbon, phosphorus, and nitrogen. In addition, we recently demonstrated that S. oneidensis MR-1 requires eDNA as a structural component during all stages of biofilm formation. In this study, we characterize the roles of two Shewanella extracellular endonucleases, ExeS and ExeM. While ExeS is likely secreted into the medium, ExeM is predicted to remain associated with the cell envelope. Both exeM and exeS are highly expressed under phosphate-limited conditions. Mutants lacking exeS and/or exeM exhibit decreased eDNA degradation; however, the capability of S. oneidensis MR-1 to use DNA as the sole source of phosphorus is only affected in mutants lacking exeM. Neither of the two endonucleases alleviates toxic effects of increased eDNA concentrations. The deletion of exeM and/or exeS significantly affects biofilm formation of S. oneidensis MR-1 under static conditions, and expression of exeM and exeS drastically increases during static biofilm formation. Under hydrodynamic conditions, a deletion of exeM leads to altered biofilms that consist of densely packed structures which are covered by a thick layer of eDNA. Based on these results, we hypothesize that a major role of ExeS and, in particular, ExeM of S. oneidensis MR-1, is to degrade eDNA as a matrix component during biofilm formation to improve nutrient supply and to enable detachment.     

Control of formation and cellular detachment from Shewanella oneidensis MR-1 biofilms by cyclic di-GMP

Stability and resilience against environmental perturbations are critical properties of medical and environmental biofilms and pose important targets for their control. Biofilm stability is determined by two mutually exclusive processes: attachment of cells to and detachment from the biofilm matrix. Using Shewanella oneidensis MR-1, an environmentally versatile, Fe(III) and Mn(IV) mineral-reducing microorganism, we identified mxdABCD as a new set of genes essential for formation of a three-dimensional biofilm. Molecular analysis revealed that mxdA encodes a cyclic bis(3',5')guanylic acid (cyclic di-GMP)-forming enzyme with an unusual GGDEF motif, i.e., NVDEF, which is essential for its function. mxdB encodes a putative membrane-associated glycosyl transferase. Both genes are essential for matrix attachment. The attachment-deficient phenotype of a DeltamxdA mutant was rescued by ectopic expression of VCA0956, encoding another diguanylate cyclase. Interestingly, a rapid cellular detachment from the biofilm occurred upon induction of yhjH, a gene encoding an enzyme that has been shown to have phosphodiesterase activity. In this way, it was possible to bypass the previously identified sudden depletion of molecular oxygen as an environmental trigger to induce biofilm dissolution. We propose a model for c-di-GMP as a key intracellular regulator for controlling biofilm stability by shifting the state of a biofilm cell between attachment and detachment in a concentration-dependent manner.     

NMR methods for in situ biofilm metabolism studies

Novel procedures and instrumentation are described for nuclear magnetic resonance (NMR) spectroscopy and imaging studies of live, in situ microbial films. A perfused NMR/optical microscope sample chamber containing a planar biofilm support was integrated into a recirculation/dilution flow loop growth reactor system and used to grow in situ Shewanella oneidensis strain MR-1 biofilms. Localized NMR techniques were developed and used to non-invasively monitor time-resolved metabolite concentrations and to image the biomass volume and distribution. As a first illustration of the feasibility of the methodology an initial 13C-labeled lactate metabolic pathway study was performed, yielding results consistent with existing genomic data for MR-1. These results represent progress toward our ultimate goal of correlating time- and depth-resolved metabolism and mass transport with gene expression in live in situ biofilms using combined NMR/optical microscopy techniques.     

Hypothesis for the role of nutrient starvation in biofilm detachment

A combination of experimental and theoretical approaches was used to investigate the role of nutrient starvation as a potential trigger for biofilm detachment. Experimental observations of detachment in a variety of biofilm systems were made with pure cultures of Pseudomonas aeruginosa. These observations indicated that biofilms grown under continuous-flow conditions detached after flow was stopped, that hollow cell clusters were sometimes observed in biofilms grown in flow cells, and that lysed cells were apparent in the internal strata of colony biofilms. When biofilms were nutrient starved under continuous-flow conditions, detachment still occurred, suggesting that starvation and not the accumulation of a metabolic product was responsible for triggering detachment in this particular system. A cellular automata computer model of biofilm dynamics was used to explore the starvation-dependent detachment mechanism. The model predicted biofilm structures and dynamics that were qualitatively similar to those observed experimentally. The predicted features included centrally located voids appearing in sufficiently large cell clusters, gradients in growth rate within these clusters, and the release of most of the biofilm with simulated stopped-flow conditions. The model was also able to predict biofilm sloughing resulting solely from this detachment mechanism. These results support the conjecture that nutrient starvation is an environmental cue for the release of microbes from a biofilm.     

Hypothesis for the Role of Nutrient Starvation in Biofilm Detachment

A combination of experimental and theoretical approaches was used to investigate the role of nutrient starvation as a potential trigger for biofilm detachment. Experimental observations of detachment in a variety of biofilm systems were made with pure cultures of Pseudomonas aeruginosa. These observations indicated that biofilms grown under continuous-flow conditions detached after flow was stopped, that hollow cell clusters were sometimes observed in biofilms grown in flow cells, and that lysed cells were apparent in the internal strata of colony biofilms. When biofilms were nutrient starved under continuous-flow conditions, detachment still occurred, suggesting that starvation and not the accumulation of a metabolic product was responsible for triggering detachment in this particular system. A cellular automata computer model of biofilm dynamics was used to explore the starvation-dependent detachment mechanism. The model predicted biofilm structures and dynamics that were qualitatively similar to those observed experimentally. The predicted features included centrally located voids appearing in sufficiently large cell clusters, gradients in growth rate within these clusters, and the release of most of the biofilm with simulated stopped-flow conditions. The model was also able to predict biofilm sloughing resulting solely from this detachment mechanism. These results support the conjecture that nutrient starvation is an environmental cue for the release of microbes from a biofilm.     

Initial Phases of biofilm formation in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative Fe(III)- and Mn(IV)-reducing microorganism and serves as a model for studying microbially induced dissolution of Fe or Mn oxide minerals as well as biogeochemical cycles. In soil and sediment environments, S. oneidensis biofilms form on mineral surfaces and are critical for mediating the metabolic interaction between this microbe and insoluble metal oxide phases. In order to develop an understanding of the molecular basis of biofilm formation, we investigated S. oneidensis biofilms developing on glass surfaces in a hydrodynamic flow chamber system. After initial attachment, growth of microcolonies and lateral spreading of biofilm cells on the surface occurred simultaneously within the first 24 h. Once surface coverage was almost complete, biofilm development proceeded with extensive vertical growth, resulting in formation of towering structures giving rise to pronounced three-dimensional architecture. Biofilm development was found to be dependent on the nutrient conditions, suggesting a metabolic control. In global transposon mutagenesis, 173 insertion mutants out of 15,000 mutants screened were identified carrying defects in initial attachment and/or early stages in biofilm formation. Seventy-one of those mutants exhibited a nonswimming phenotype, suggesting a role of swimming motility or motility elements in biofilm formation. Disruption mutations in motility genes (flhB, fliK, and pomA), however, did not affect initial attachment but affected progression of biofilm development into pronounced three-dimensional architecture. In contrast, mutants defective in mannose-sensitive hemagglutinin type IV pilus biosynthesis and in pilus retraction (pilT) showed severe defects in adhesion to abiotic surfaces and biofilm formation, respectively. The results provide a basis for understanding microbe-mineral interactions in natural environments.     

Agr-mediated dispersal of Staphylococcus aureus biofilms

The agr quorum-sensing system of Staphylococcus aureus modulates the expression of virulence factors in response to autoinducing peptides (AIPs). Recent studies have suggested a role for the agr system in S. aureus biofilm development, as agr mutants exhibit a high propensity to form biofilms, and cells dispersing from a biofilm have been observed displaying an active agr system. Here, we report that repression of agr is necessary to form a biofilm and that reactivation of agr in established biofilms through AIP addition or glucose depletion triggers detachment. Inhibitory AIP molecules did not induce detachment and an agr mutant was non-responsive, indicating a dependence on a functional, active agr system for dispersal. Biofilm detachment occurred in multiple S. aureus strains possessing divergent agr systems, suggesting it is a general S. aureus phenomenon. Importantly, detachment also restored sensitivity of the dispersed cells to the antibiotic rifampicin. Proteinase K inhibited biofilm formation and dispersed established biofilms, suggesting agr-mediated detachment occurred in an ica-independent manner. Consistent with a protease-mediated mechanism, increased levels of serine proteases were detected in detaching biofilm effluents, and the serine protease inhibitor PMSF reduced the degree of agr-mediated detachment. Through genetic analysis, a double mutant in the agr-regulated Aur metalloprotease and the SplABCDEF serine proteases displayed minimal extracellular protease activity, improved biofilm formation, and a strongly attenuated detachment phenotype. These findings indicate that induction of the agr system in established S. aureus biofilms detaches cells and demonstrate that the dispersal mechanism requires extracellular protease activity.     

Biofilm development and the dynamics of preferential flow paths in porous media

A two-dimensional pore-scale numerical model was developed to evaluate the dynamics of preferential flow paths in porous media caused by bioclogging. The liquid flow and solute transport through the pore network were coupled with a biofilm model including biomass attachment, growth, decay, lysis, and detachment. Blocking of all but one flow path was obtained under constant liquid inlet flow rate and biomass detachment caused by shear forces only. The stable flow path formed when biofilm detachment balances growth, even with biomass weakened by decay. However, shear forces combined with biomass lysis upon starvation could produce an intermittently shifting location of flow channels. Dynamic flow pathways may also occur when combined liquid shear and pressure forces act on the biofilm. In spite of repeated clogging and unclogging of interconnected pore spaces, the average permeability reached a quasi-constant value. Oscillations in the medium permeability were more pronounced for weaker biofilms.     

Growth and detachment of cell clusters from mature mixed-species biofilms.

Detachment from biofilms is an important consideration in the dissemination of infection and the contamination of industrial systems but is the least-studied biofilm process. By using digital time-lapse microscopy and biofilm flow cells, we visualized localized growth and detachment of discrete cell clusters in mature mixed-species biofilms growing under steady conditions in turbulent flow in situ. The detaching biomass ranged from single cells to an aggregate with a diameter of approximately 500 microm. Direct evidence of local cell cluster detachment from the biofilms was supported by microscopic examination of filtered effluent. Single cells and small clusters detached more frequently, but larger aggregates contained a disproportionately high fraction of total detached biomass. These results have significance in the establishment of an infectious dose and public health risk assessment.     

Growth and Detachment of Cell Clusters from Mature Mixed-Species Biofilms

Detachment from biofilms is an important consideration in the dissemination of infection and the contamination of industrial systems but is the least-studied biofilm process. By using digital time-lapse microscopy and biofilm flow cells, we visualized localized growth and detachment of discrete cell clusters in mature mixed-species biofilms growing under steady conditions in turbulent flow in situ. The detaching biomass ranged from single cells to an aggregate with a diameter of approximately 500 μm. Direct evidence of local cell cluster detachment from the biofilms was supported by microscopic examination of filtered effluent. Single cells and small clusters detached more frequently, but larger aggregates contained a disproportionately high fraction of total detached biomass. These results have significance in the establishment of an infectious dose and public health risk assessment.     

Extracellular polymeric substances from Shewanella sp. HRCR-1 biofilms: characterization by infrared spectroscopy and proteomics

The composition of extracellular polymeric substances (EPS) from Shewanella sp. HRCR-1 biofilms was investigated using infrared spectroscopy and proteomics to provide insight into potential ecophysiological functions and redox activity of the EPS. Both bound and loosely associated EPS were extracted from Shewanella sp. HRCR-1 biofilms prepared using a hollow-fibre membrane biofilm reactor. Fourier transform infrared spectra revealed the presence of proteins, polysaccharides, nucleic acids, membrane lipids and fatty acids in the EPS fractions. Using a global proteomic approach, a total of 58 extracellular and outer membrane proteins were identified in the EPS. These included homologues of multiple Shewanella oneidensis MR-1 proteins that potentially contribute to key physiological biofilm processes, such as biofilm-promoting protein BpfA, surface-associated serine protease, nucleotidases (CpdB and UshA), an extracellular lipase, and oligopeptidases (PtrB and a M13 family oligopeptidase lipoprotein). In addition, 20 redox proteins were found in extracted EPS. Among the detected redox proteins were the homologues of two S. oneidensis MR-1 c-type cytochromes, MtrC and OmcA, which have been implicated in extracellular electron transfer. Given their detection in the EPS of Shewanella sp. HRCR-1 biofilms, c-type cytochromes may contribute to the possible redox activity of the biofilm matrix and play important roles in extracellular electron transfer reactions.     

Biofilm dispersion in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

In recent decades, many researchers have written numerous articles about microbial biofilms. Biofilm is a complex community of microorganisms and an example of bacterial group behavior. Biofilm is usually considered a sessile mode of life derived from the attached growth of microbes to surfaces, and most biofilms are embedded in self-produced extracellular matrix composed of extracellular polymeric substances (EPSs), such as polysaccharides, extracellular DNAs (eDNA), and proteins. Dispersal, a mode of biofilm detachment indicates active mechanisms that cause individual cells to separate from the biofilm and return to planktonic life. Since biofilm cells are cemented and surrounded by EPSs, dispersal is not simple to do and many researchers are now paying more attention to this active detachment process. Unlike other modes of biofilm detachment such as erosion or sloughing, which are generally considered passive processes, dispersal occurs as a result of complex spatial differentiation and molecular events in biofilm cells in response to various environmental cues, and there are many biological reasons that force bacterial cells to disperse from the biofilms. In this review, we mainly focus on the spatial differentiation of biofilm that is a prerequisite for dispersal, as well as environmental cues and molecular events related to the biofilm dispersal. More specifically, we discuss the dispersal-related phenomena and mechanisms observed in Pseudomonas aeruginosa, an important opportunistic human pathogen and representative model organism for biofilm study.     

A kinetic model describing Shewanella oneidensis MR-1 growth, substrate consumption, and product secretion

Aerobic growth of Shewanella oneidensis MR-1 in minimal lactate medium was studied in batch cultivation. Acetate production was observed in the middle of the exponential growth phase and was enhanced when the dissolved oxygen (DO) concentration was low. Once the lactate was nearly exhausted, S. oneidensis MR-1 used the acetate produced during growth on lactate with a similar biomass yield as lactate. A two-substrate Monod model, with competitive and uncompetitive substrate inhibition, was devised to describe the dependence of biomass growth on lactate, acetate, and oxygen and the acetate growth inhibition across a broad range of concentrations. The parameters estimated for this model indicate interesting growth kinetics: lactate is converted to acetate stoichiometrically regardless of the DO concentration; cells grow well even at low DO levels, presumably due to a very low K(m) for oxygen; cells metabolize acetate (maximum specific growth rate, micro(max,A) of 0.28 h(-1)) as a single carbon source slower than they metabolize lactate (micro(max,L) of 0.47 h(-1)); and growth on acetate is self-inhibiting at a concentration greater than 10 mM. After estimating model parameters to describe growth and metabolism under six different nutrient conditions, the model was able to successfully estimate growth, oxygen and lactate consumption, and acetate production and consumption under entirely different growth conditions.     

vpsA- and luxO-independent biofilms of Vibrio cholerae

The natural life cycle of Vibrio cholerae involves the transitioning of cells between different environmental surfaces such as the chitinous shell of Crustaceae and the epithelial layer of the human intestine. Previous studies using static biofilm systems showed a strict dependence of biofilm formation on the vps and lux genes, which are essential for exopolysaccharide formation and cell-cell signaling, respectively. The authors' report here that in biofilms grown under hydrodynamic conditions, DeltavpsA and DeltaluxO mutants of V. cholerae do form pronounced, three-dimensional biofilms that resemble all aspects of wild-type biofilms. By genetic experiments, it was shown that in hydrodynamically grown biofilms this independence of vpsA is due to the expression of rpoS, which is a negative regulator of vpsA expression. Biofilms also underwent substantial dissolution after 96 h that could be induced by a simple stop of medium flow. The studies indicate that metabolic conditions control the reversible attachment of cells to the biofilm matrix and are key in regulating biofilm cell physiology via RpoS. Furthermore, the results redefine the roles of vps and quorum-sensing in V. cholerae biofilms.     

Two-dimensional model of biofilm detachment caused by internal stress from liquid flow

A two-dimensional model for biofilm growth and detachment was used to evaluate the effect of detachment on biofilm structures. The detachment process is considered to be due to internal stress created by moving liquid past the biofilm. This model generated a variety of realistic biofilm-formation patterns. It was possible to model in a unified way two different biofilm detachment processes, erosion (small-particle loss), and sloughing (large-biomass-particle removal). The distribution of the fraction from total biomass detached as a function of detached particle mass, gives indications about which of the two mechanisms is dominant. Model simulations indicate that erosion makes the biofilm surface smoother. Sloughing, in contrast, leads to an increased biofilm-surface roughness. Faster growing biofilms have a faster detachment rate than slow-growing biofilms, under similar hydrodynamic conditions and biofilm strength. This is in perfect accordance with the experimental evidence showing that detachment is dependent on both shear- and microbial-growth rates. High growth rates trigger instability in biofilm accumulation and abrupt biomass loss (sloughing). Massive sloughing can be avoided by high liquid shear, combined with low biomass growth rates. As the modeling results show, the causes for sloughing must be sought not only in the biofilm strength, but also in its shape. Several "mushroom-like" biofilm structures like those repeatedly reported in the literature occurred, due to a combined effect of nutrient depletion and breaking at the colony base. A rough carrier surface promotes biofilm development in hydrodynamic conditions in which the biofilm on a flat surface would not form. Although biofilm patches filled completely the cavity in which they started to grow, they were unable to spill over the carrier peaks and to fully colonize the substratum.     

Shear‐induced detachment of biofilms from hollow fiber silicone membranes

A suite of techniques was utilized to evaluate the correlation between biofilm physiology, fluid‐induced shear stress, and detachment in hollow fiber membrane aerated bioreactors. Two monoculture species biofilms were grown on silicone fibers in a hollow fiber membrane aerated bioreactors (HfMBR) to assess detachment under laminar fluid flow conditions. Both physiology (biofilm thickness and roughness) and nutrient mass transport data indicated the presence of a steady state mature biofilm after 3 weeks of development. Surface shear stress proved to be an important parameter for predicting passive detachment for the two biofilms. The average shear stress at the surface of Nitrosomonas europaea biofilms (54.5 ± 3.2 mPa) was approximately 20% higher than for Pseudomonas aeruginosa biofilms (45.8 ± 7.7 mPa), resulting in higher biomass detachment. No significant difference in shear stress was measured between immature and mature biofilms of the same species. There was a significant difference in detached biomass for immature vs. mature biofilms in both species. However, there was no difference in detachment rate between the two species. Biotechnol. Bioeng. 2013; 110: 525–534. © 2012 Wiley Periodicals, Inc.     

Cell density related H2 consumption in relation to anoxic Fe(0) corrosion and precipitation of corrosion products by Shewanella oneidensis MR-1

In the absence of oxygen, a protective H2 film is formed around an Fe(0) surface, inhibiting the electron flow from this surface. Our study of anoxic corrosion of Fe(0) beads revealed that, in the presence of Shewanella oneidensis MR-1, H2 removal and precipitation of Fe mineral particles on the cell surface are determining processes for corrosion. These two biologically mediated processes were governed by cell density. H2 removal by Shewanella oneidensis was detected at cell concentrations of 1.0 x 10(6) live cells ml-1 and higher and H2 was electron donor for denitrification of NO3-. The removal of the protective H2 layer from Fe(0) beads by Shewanella oneidensis, resulted in an increase of Fe release out of the Fe(0) beads from 153 +/- 25 mg l(-1) to 196 +/- 7 mg l-1 after 20 h. When the cell concentration exceeded 1.0 x 10(8) live cells ml-1, precipitation of iron minerals on the cell surface was characteristic for the greatest percentage of MR-1 cells, whereas micrometre-scale iron precipitates not associated with culturable cell biomass significantly decreased in number. Addition of supernatant of a corrosion assay with high cell concentration induced metabolic activity in a corrosion assay with low cell concentration, resulting in increased H2 consumption and Fe release from Fe(0) beads. Homoserine lactone-like molecules were detected in the supernatant by a bio-assay, suggesting the involvement of a quorum-sensing regulatory mechanism.     

Microbial iron respiration can protect steel from corrosion

Microbiologically influenced corrosion (MC) of steel has been attributed to the activity of biofilms that include anaerobic microorganisms such as iron-respiring bacteria, yet the mechanisms by which these organisms influence corrosion have been unclear. To study this process, we generated mutants of the iron-respiring bacterium Shewanella oneidensis strain MR-1 that were defective in biofilm formation and/or iron reduction. Electrochemical impedance spectroscopy was used to determine changes in the corrosion rate and corrosion potential as a function of time for these mutants in comparison to the wild type. Counter to prevailing theories of MC, our results indicate that biofilms comprising iron-respiring bacteria may reduce rather than accelerate the corrosion rate of steel. Corrosion inhibition appears to be due to reduction of ferric ions to ferrous ions and increased consumption of oxygen, both of which are direct consequences of microbial respiration.