Mcc在脱色希瓦氏菌S12电极呼吸中的作用

【目的】研究脱色希瓦氏菌S12周质空间c型细胞色素Mcc的功能,进一步探索和补充微生物胞外电子传递过程的机制。【方法】借助自杀质粒敲除mcc基因,通过细胞浓度测定和激光共聚焦显微镜比较分析突变株和野生株之间的浮游细胞和生物膜的生长情况,并比较分析二者在微生物燃料电池电极还原、铁还原和胞外偶氮染料还原过程中的功能。【结果】Mcc缺失对铁还原和偶氮还原没有影响,但却造成电极呼吸活性下降34.1%;与野生株相比,mcc突变株的好氧生长和厌氧浮游细胞生长无明显影响,但却显著抑制了电极表面生物膜的形成。【结论】Mcc是希瓦氏菌S12电极呼吸过程中周质空间电子传递的重要组分之一,缺失会显著抑制其电极呼吸效率以及生物膜的形成。     

Characterization of electrochemical activity of a strain ISO2‐3 phylogenetically related to Aeromonas sp. isolated from a glucose‐fed microbial fuel cell

The microbial communities associated with electrodes in closed and open circuit microbial fuel cells (MFCs) fed with glucose were analyzed by 16S rRNA approach and compared. The comparison revealed that bacteria affiliated with the Aeromonas sp. within the Gammaproteobacteria constituted the major population in the closed circuit MFC (harvesting electricity) and considered to play important roles in current generation. We, therefore, attempted to isolate the dominant bacteria from the anode biofilm, successfully isolated a Fe (III)‐reducing bacterium phylogenetically related to Aeromonas sp. and designated as strain ISO2‐3. The isolated strain ISO2‐3 could grow and concomitantly produce current (max. 0.24 A/m²) via oxidation of glucose or hydrogen with an electrode serving as the sole electron acceptor. The strain could ferment glucose, but generate less electrical current. Cyclic voltammetry supported the strain ISO2‐3 was electrically active and likely to transfer electrons to the electrode though membrane‐associated compounds (most likely c‐type cytochrome). This mechanism requires intimate contact with the anode surface. Scanning electron microscopy revealed that the strain ISO2‐3 developed multiplayer biofilms on the anode surface and also produced anchor‐like filamentous appendages (most likely pili) that may promote long‐range electron transport across the thick biofilm. Biotechnol. Bioeng. 2009; 104: 901–910. © 2009 Wiley Periodicals, Inc.     

Understanding the Anodic Mechanism of a Seafloor Fuel Cell: Interactions Between Geochemistry and Microbial Activity

Seafloor fuel cells made with graphite electrodes generate electricity by promoting electron transfer in response to a natural voltage difference (−0.7 to −0.8 V) between anoxic sediments and overlying oxic seawater. Geochemical impacts of a seafloor fuel cell on sediment solids and porewaters were examined to identify the anodic mechanisms and substrates available for current production. In an estuarine environment with little dissolved sulfide, solid-phase acid volatile sulfide and Cr²⁺-reducible sulfur minerals decreased significantly toward the anode after 7 months of nearly continuous energy harvesting. Porewater iron and sulfate increased by millimolar amounts. Scanning electron microscope images showed a biofilm overcoating the anode, and electron microprobe analyses revealed accumulations of sulfur, iron, silicon and phosphorus at the electrode surface. Sulfur deposition was also observed on a laboratory fuel cell anode used to generate electricity with only dissolved sulfide as an electron donor. Moreover, current densities and voltages displayed by these purely chemical cells were similar to the values measured with field devices. These results indicate that electron transfer to seafloor fuel cells can readily result in the oxidation of dissolved and solid-phase forms of reduced sulfur producing mainly S⁰ which deposits at the electrode surface. This oxidation product is consistent with the observed enrichment of bacteria most closely related to Desulfobulbus/Desulfocapsa genera within the anode biofilm, and its presence is proposed to promote a localized biogeochemical cycle whereby biofilm bacteria regenerate sulfate and sulfide. This electron-shuttling mechanism may co-occur while these or other bacteria use the anode directly as a terminal electron acceptor.     

The utility of Shewanella japonica for microbial fuel cells

Shewanella-containing microbial fuel cells (MFCs) typically use the fresh water wild-type strain Shewanella oneidensis MR-1 due to its metabolic diversity and facultative oxidant tolerance. However, S. oneidensis MR-1 is not capable of metabolizing polysaccharides for extracellular electron transfer. The applicability of Shewanella japonica (an agar-lytic Shewanella strain) for power applications was analyzed using a diverse array of carbon sources for current generation from MFCs, cellular physiological responses at an electrode surface, biofilm formation, and the presence of soluble extracellular mediators for electron transfer to carbon electrodes. Critically, air-exposed S. japonica utilizes biosynthesized extracellular mediators for electron transfer to carbon electrodes with sucrose as the sole carbon source.     

Cell surface properties of two differently virulent strains of Acinetobacter baumannii isolated from a patient

The aim of this study was to unravel, by focusing on cell surface properties, the underlying virulence factors contributing to the difference in the pathogenicity observed in two Acinetobacter baumannii strains isolated from the same patient. The two strains were phenotypically different: (i) a mucoid strain (AB-M), highly virulent in a mouse model of pneumonia, and (ii) a nonmucoid strain (AB-NM), moderately virulent in the same model. The study of the cell surface properties included the microbial adhesion to solvents method, the measurement of the electrophoretic mobility of bacteria, the analysis of biofilm formation by calcofluor white staining, the adherence to silicone catheters, and scanning electron microscopy. The AB-NM strain was more hydrophobic, more adherent to silicone catheters, and produced more biofilm than the AB-M strain. Scanning electron microscopy showed bacterial cells with a rough surface and the formation of large cell clusters for AB-NM whereas the AB-M strain had a smooth surface and formed only a few cell clusters. Contrary to the results of most previous studies, cell surface properties were not correlated to the virulence described in our experimental model, indicating that mechanisms other than adherence may be involved in the expression of A.?baumannii virulence.     

Cells dispersed from Marinobacter hydrocarbonoclasticus SP17 biofilm exhibit a specific protein profile associated with a higher ability to reinitiate biofilm development at the hexadecane-water interface

Biofilm formation by marine hydrocarbonoclastic bacteria is commonly observed and has been recognized as an important mechanism for the biodegradation of hydrocarbons. In order to colonize new oil-water interfaces, surface-attached communities of hydrocarbonoclastic bacteria must release cells into the environment. Here we explored the physiology of cells freshly dispersed from a biofilm of Marinobacter hydrocarbonoclasticus developing at the hexadecane-water interface, by combining proteomic and physiological approaches. The comparison of the dispersed cells' proteome with those of biofilm, logarithmic- and stationary-phase planktonic cells indicated that dispersed cells had lost most of the biofilm phenotype and expressed a specific proteome. Two proteins involved in cell envelope maturation, DsbA and CtpA, were exclusively detected in dispersed cells, suggesting a reshaping of the cell envelopes during biofilm dispersal. Furthermore, dispersed cells exhibited a higher affinity for hexadecane and initiated more rapidly biofilm formation on hexadecane than the reference planktonic cells. Interestingly, storage wax esters were rapidly degraded in dispersed cells, suggesting that their observed physiological properties may rely on reserve mobilization. Thus, by promoting oil surface colonization, cells emigrating from the biofilm could contribute to the success of marine hydrocarbonoclastic bacteria in polluted environments.     

Electron transfer from acetate to the surface of MnO2 particles by a marine bacterium

Summary Intact cells of marine pseudomonad strain BIII 88, grown in the presence of added MnSO₄ (induced cells), reduced MnO₂ aerobically and anaerobically with acetate. They did not reduce limonite (FeOOH) with acetate. Spectrophotometric evidence of respiratory pigments in the cell envelope and inhibition of MnO₂ reduction by antimycin A and NQNO indicated that a respiratory process was involved. Stimulation of MnO₂ reduction by the oxidative phosphorylation uncouplers CCCP and 2,4-DNP indicated energy conservation during the reduction. Intact cells of strain BIII 88 grown in the absence of added manganese (non-induced cells) showed marginal MnO₂-reducing activity. Cell envelope fractions from induced cells prepared with a French press exhibited higher specific MnO₂-reducing activity on average than those prepared by sonication. Cell envelope fractions from induced cells contained more manganese than cell envelope fractions from non-induced cells. Recombined cell fractions from induced cells were more active than recombined cell fractions from non-induced cells. MnO₂ reducing activity was correlated with manganese content in cell envelope fractions. Cell envelope fractions from two cultures that do not reduce. MnO₂ contained less manganese in their cell envelope fractions than similar fractions from non-induced strain BIII 88. Manganese in the cell envelope of strain BIII 88 appears to play a role in the transfer of reducing power from respiration on acetate across the cell envelope to the surface of MnO₂ particles.     

Abundance of the Multiheme c-Type Cytochrome OmcB Increases in Outer Biofilm Layers of Electrode-Grown Geobacter sulfurreducens

When Geobacter sulfurreducens utilizes an electrode as its electron acceptor, cells embed themselves in a conductive biofilm tens of microns thick. While environmental conditions such as pH or redox potential have been shown to change close to the electrode, less is known about the response of G. sulfurreducens to growth in this biofilm environment. To investigate whether respiratory protein abundance varies with distance from the electrode, antibodies against an outer membrane multiheme cytochrome (OmcB) and cytoplasmic acetate kinase (AckA) were used to determine protein localization in slices spanning ∼25 µm-thick G. sulfurreducens biofilms growing on polished electrodes poised at +0.24 V (vs. Standard Hydrogen Electrode). Slices were immunogold labeled post-fixing, imaged via transmission electron microscopy, and digitally reassembled to create continuous images allowing subcellular location and abundance per cell to be quantified across an entire biofilm. OmcB was predominantly localized on cell membranes, and 3.6-fold more OmcB was detected on cells 10–20 µm distant from the electrode surface compared to inner layers (0–10 µm). In contrast, acetate kinase remained constant throughout the biofilm, and was always associated with the cell interior. This method for detecting proteins in intact conductive biofilms supports a model where the utilization of redox proteins changes with depth.     

Architecture of a nascent Sphingomonas sp. biofilm under varied hydrodynamic conditions

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 x 10(5) microm(2)) or microcolony size (1 x 10(6) microm(2)) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 mum above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-a-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.     

Horizontal transfer of nonconjugative plasmids in a colony biofilm of Escherichia coli

We tested the possibility of nonconjugative lateral DNA transfer in a colony biofilm of mixed Escherichia coli strains. By simply coculturing a plasmid-free F(-) strain and another F(-) strain harboring a nonconjugative plasmid in a colony biofilm on antibiotic-free agar media, transformed cells were produced within 24-48 h at the frequency of 10(-10)-10(-9) per recipient cell. PCR analysis of the transformed cells demonstrated the occurrence of lateral plasmid transfer. These cells survived until at least day 7 under antibiotic-free conditions. Liquid cultures of the same strains in Luria-Bertani broth produced no or few transformants, suggesting the importance of colony-biofilm formation for plasmid transfer. This is a novel line of evidence indicating that nonconjugative, nonviral horizontal gene transfer can occur between E. coli cells.     

Assembly and mechanisms of bacterial type IV secretion machines

Type IV secretion occurs across a wide range of prokaryotic cell envelopes: Gram-negative, Gram-positive, cell wall-less bacteria and some archaea. This diversity is reflected in the heterogeneity of components that constitute the secretion machines. Macromolecules are secreted in an ATP-dependent process using an envelope-spanning multi-protein channel. Similar to the type III systems, this apparatus extends beyond the cell surface as a pilus structure important for direct contact and penetration of the recipient cell surface. Type IV systems are remarkably versatile in that they mobilize a broad range of substrates, including single proteins, protein complexes, DNA and nucleoprotein complexes, across the cell envelope. These machines have broad clinical significance not only for delivering bacterial toxins or effector proteins directly into targeted host cells, but also for direct involvement in phenomena such as biofilm formation and the rapid horizontal spread of antibiotic resistance genes among the microbial community.     

Monitoring of microbial adhesion and biofilm growth using electrochemical impedancemetry

Electrochemical impedance spectroscopy was tested to monitor the cell attachment and the biofilm proliferation in order to identify characteristic events induced on the metal surface by Gram-negative (Pseudomonas aeruginosa PAO1) and Gram-positive (Bacillus subtilis) bacteria strains. Electrochemical impedance spectra of AISI 304 electrodes during cell attachment and initial biofilm growth for both strains were obtained. It can be observed that the resistance increases gradually with the culture time and decreases with the biofilm detachment. So, the applicability of electric cell-substrate impedance sensing (ECIS) for studying the attachment and spreading of cells on a metal surface has been demonstrated. The biofilm formation was also characterized by the use of scanning electron microscopy and confocal laser scanning microscopy and COMSTAT image analysis. The electrochemical results roughly agree with the microscope image observations. The ECIS technique used in this study was used for continuous real-time monitoring of the initial bacterial adhesion and the biofilm growth. It provides a simple and non-expensive electrochemical method for in vitro assessment of the presence of biofilms on metal surfaces.     

Antibacterial activity of cyclodextrins against <Emphasis Type="Italic">Bacillus</Emphasis> strains

Growth of alkaliphilic Bacillus halodurans C-125 both on agar plates and in liquid culture was inhibited by methyl-β-cyclodextrin (CD). Furthermore, resting cells of the strain were lysed by contact with methyl-β-CD higher than 10 mM. α-CD also showed lysis activity against Bacillus and related strains. The activity was not observed with Gram-negative and Gram-positive bacteria except for Bacillus strains. Fluorescence staining and scanning electron microscopy of cells revealed that methyl-β-CD disrupted cell membranes, and consequently, the cells were lysed. This is a novel physiological property of CDs.     

Enrichment of electrochemically active bacteria using a three-electrode electrochemical cell

Electrochemically active bacteria were successfully enriched in an electrochemical cell using a positively poised working electrode. The positively poised working electrode (+0.7 V vs. Ag/AgCl) was used as an electron acceptor for enrichment and growth of electrochemically active bacteria. When activated sludge and synthetic wastewater were fed to the electrochemical cell, a gradual increase in amperometric current was observed. After a period of time in which the amperometric current was stabilized (generally 8 days), linear correlations between the amperometric signals from the electrochemical cell and added BOD (biochemical oxygen demand) concentrations were established. Cyclic voltammetry of the enriched electrode also showed prominent electrochemical activity. When the enriched electrodes were examined with electron microscopy and confocal scanning laser microscopy, a biofilm on the enriched electrode surface and bacterium-like particles were observed. These experimental results indicate that the electrochemical system in this study is a useful tool for the enrichment of an electrochemically active bacterial consortium and could be used as a novel microbial biosensor.     

Listeria monocytogenes LO28: surface physicochemical properties and ability to form biofilms at different temperatures and growth phases

The surface physicochemical properties of Listeria monocytogenes LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents. The effect of these parameters on adhesion and biofilm formation by L. monocytogenes LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene [PTFE]) surfaces was assessed. The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature. The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature. However, biofilm formation was faster on the hydrophilic substratum. After 5 days at 37 or 20 degrees C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37 degrees C. At 8 degrees C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE. The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation. The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.     

Characterization and comparison of biofilm development by pathogenic and commensal isolates of Histophilus somni

Histophilus somni (Haemophilus somnus) is an obligate inhabitant of the mucosal surfaces of bovines and sheep and an opportunistic pathogen responsible for respiratory disease, meningoencephalitis, myocarditis, arthritis, and other systemic infections. The identification of an exopolysaccharide produced by H. somni prompted us to evaluate whether the bacterium was capable of forming a biofilm. After growth in polyvinyl chloride wells a biofilm was formed by all strains examined, although most isolates from systemic sites produced more biofilm than commensal isolates from the prepuce. Biofilms of pneumonia isolate strain 2336 and commensal isolate strain 129Pt were grown in flow cells, followed by analysis by confocal laser scanning microscopy and scanning electron microscopy. Both strains formed biofilms that went through stages of attachment, growth, maturation, and detachment. However, strain 2336 produced a mature biofilm that consisted of thick, homogenous mound-shaped microcolonies encased in an amorphous extracellular matrix with profound water channels. In contrast, strain 129Pt formed a biofilm of cell clusters that were tower-shaped or distinct filamentous structures intertwined with each other by strands of extracellular matrix. The biofilm of strain 2336 had a mass and thickness that was 5- to 10-fold greater than that of strain 129Pt and covered 75 to 82% of the surface area, whereas the biofilm of strain 129Pt covered 35 to 40% of the surface area. Since H. somni is an obligate inhabitant of the bovine and ovine host, the formation of a biofilm may be crucial to its persistence in vivo, and our in vitro evidence suggests that formation of a more robust biofilm may provide a selective advantage for strains that cause systemic disease.     

Endoplasmic Reticulum Forms a Dynamic Continuum for Lipid Diffusion between Contiguous Soybean Root Cells

Intercellular communication between plant cells for low molecular weight hydrophilic molecules occurs through plasmodesmata. These tubular structures are embedded in the plant cell wall in association with the plasmalemma and endoplasmic reticulum (ER). Transmission electron microscopy has provided strong evidence to support the view that both the ER and plasmalemma are structurally continuous across the wall at these sites. In experiments to be described, the technique of fluorescence redistribution after photobleaching was used to examine the lateral mobility and intercellular transport capability of a number of fluorescent lipid and phospholipid analogs. These probes were shown by confocal fluorescence microscopy to partition in either the ER or plasmalemma. Results from these measurements provide evidence for cell communication between contiguous cells for probes localized predominantly in the ER. In contrast, no detectable intercellular communication was observed for probes residing exclusively in the plasmalemma. It was of particular interest to note that when 1-acyl-2-(N-4-nitrobenzo-2-oxa-l,3-diazole)aminoacylphosphatidylcholine was utilized as a potential reporter molecule for phospholipids in the plasmalemma, it was quickly degraded to 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)aminoacyldiglyceride (NBD-DAG), which then appeared predominantly localized to the ER and nuclear envelope. This endogenously synthesized NBD-DAG was found to be capable of transfer between cells, as was exogenously incorporated NBD-DAG. Results from these investigations provide support for the following conclusions: (1) ER, but apparently not the plasmalemma, can form dynamic communication pathways for lipids across the cell wall between connecting plant cells; (2) the plasmodesmata appear to form a barrier for lipid diffusion through the plasmalemma; and (3) lipid signaling molecules such as diacylglycerol are capable of transfer between contiguous plant cells through the ER. These observations speak to issues of plant cell autonomy for lipid synthesis and mechanisms of intercellular signaling and communication.     

Electroactive bacteria—molecular mechanisms and genetic tools

In nature, different bacteria have evolved strategies to transfer electrons far beyond the cell surface. This electron transfer enables the use of these bacteria in bioelectrochemical systems (BES), such as microbial fuel cells (MFCs) and microbial electrosynthesis (MES). The main feature of electroactive bacteria (EAB) in these applications is the ability to transfer electrons from the microbial cell to an electrode or vice versa instead of the natural redox partner. In general, the application of electroactive organisms in BES offers the opportunity to develop efficient and sustainable processes for the production of energy as well as bulk and fine chemicals, respectively. This review describes and compares key microbiological features of different EAB. Furthermore, it focuses on achievements and future prospects of genetic manipulation for efficient strain development.     

Peptide pheromone-dependent regulation of antimicrobial peptide production in Gram-positive bacteria: a case of multicellular behavior

Quorum sensing enables unicellular organisms to behave in a multicellular way by allowing population-wide synchronized adaptive responses that involve modulation of a wide range of physiological responses in a cell density-, cell proximity- or growth phase-dependent manner. Examples of processes modulated by quorum sensing are the development of genetic competence, conjugative plasmid transfer, sporulation and cell differentiation, biofilm formation, virulence response, production of antibiotics, antimicrobial peptides and toxins, and bioluminescence (for reviews see [38]). The cell-to-cell communication strategies involved in these processes are based on the utilization of small signal molecules produced and released into the environment by the microorganisms. These communication molecules are referred to as pheromones and act as chemical messengers that transmit information across space. The extracellular pheromones accumulate in the environment and trigger a response in the target cells when its concentration reaches a certain threshold value. Elucidation of the chemical nature of the pheromones modulating the processes mentioned above reveals that most of them are unmodified peptides, post-translationally modified peptides, N-acyl homoserine lactones, or butyrolactones. Lactone-based pheromones are the preferred communication signals in Gram-negative bacteria (for review see [47,48]), whereas peptide-based pheromones are the predominant extracellular signals among Gram-positive bacteria (for review see [37,61]). However, lactone-based pheromones are utilized as signals that modulate differentiation and secondary metabolism production in Streptomyces (for review see [20]).This review focuses on the major advances and current views of the peptide-pheromone dependent regulatory circuits involved in production of antimicrobial peptides in Gram-positive bacteria.