Ecological characteristics of anaerobic ammonia oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is the microbial conversion of ammonium and nitrite to dinitrogen gas. The functional microbes of anammox reaction are anammox bacteria, which were discovered in a wastewater treatment system for nitrogen removal. Anammox bacteria are prevalent in anoxic ecosystems and play an important role in both biological nitrogen cycle and nitrogen pollution control. In this paper, we reviewed the investigation on ecological characteristics of anammox bacteria, and tried to figure out their complicated intraspecies and interspecies relationships. As for intraspecies relationship, we focused on the quorum sensing system, a cell density-dependent phenomenon. As for interspecies relationship, we focused on the synergism and competition of anammox bacteria with other microorganisms for substrate and space. Finally, we discussed the great influence of environmental factors (e.g., dissolved oxygen, organic matters) on the constitution, structure and function of anammox bacteria community.     

Detection of ingested bacteria in benthic ciliates using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was applied to detect ingested natural bacteria within the food vacuoles of ciliates harvested from the natural sediment. In addition to this important qualitative aspect, FISH was also successfully used to measure the bacterivory of a culture of the ciliate Tetrahymena pyriformis on natural field sediment bacteria. In this feeding experiment, we compared the FISH technique with the only available alternative technique using fluorescently stained sediment (FS-sediment). The ingestion rate of unstained sediment bacteria determined by FISH was 4.6 bacteria per ciliate and hour. In contrast, Tetrahymena pyriformis cells that fed on bacteria from FS-sediment ingested 12.7 bacteria per ciliate and hour. Bacterial abundances in the sediment were equal in both sediment types (4 x 10(8) cells g sediment dry weight(-1)) when determined by DAPI counts. However, when analyzed using DTAF-counts, the number of bacteria in the FS-sediment increased to 9.7 x 10(8) cells g sediment dry weight(-1). From our findings we conclude that bacterivory by ciliates is overestimated when FS-sediment is used because DTAF stains bacteria as well as protein-containing detritus particles, which are also ingested by many ciliates. In contrast, FISH is a direct, a posteriori method that specifically stains phylogenetic lineages, e.g. eubacteria, after ingestion and thereby avoids a false determination of the number of ingested bacteria. Thus this method can also be used for the study of natural ciliate bacterivory in benthic systems.     

Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere

Extremophiles - Genome analyses are being used to characterize plant growth-promoting (PGP) bacteria living in different plant compartiments. In this context, we have recently isolated bacteria...     

Distribution of capsulated bacterioplankton in the North Atlantic and North Sea

In laboratory experiments, bacterioplankton were incubated under different nutrient conditions, and the percentage of bacteria exhibiting a polysaccharidic capsule (capsulated bacteria) and that of CTC (cyanotetrazolium chloride)-positive and therefore metabolically highly active bacteria were determined. In these seawater cultures amended with nutrients more than 95% of the CTC-positive cells exhibited a capsule. During two cruises, one to the North Atlantic and one to the North Sea, we investigated the distribution of capsulated bacteria throughout the water column. Capsulated bacteria were generally more abundant in eutrophic surface waters than in deeper layers or more oligotrophic regions. In the upper 100 m of the North Atlantic, about 6–14% of the total bacterioplankton community was capsulated, while in the layers below 100 m depth, 97% of the bacteria lacked a visible capsule. The percentage of capsulated bacteria correlated with bacterial abundance and production, and chlorophyll a concentration. Also, the bioavailability of DOC (dissolved organic carbon), estimated by the ratio between bacterial production and DOC concentration, significantly correlated with the percentage of capsulated bacteria. In the North Sea, the contribution of capsulated bacteria to the total number of bacteria decreased from the surface (3 m depth) to the near-bottom (25–35 m) layers from 20% to 14% capsulated bacteria. In the nearshore area of the North Sea, about 27% of the bacteria exhibited a capsule. Overall, a pronounced decrease in the contribution of capsulated bacteria to the total bacterial abundance was detectable from the eutrophic coastal environment to the open North Atlantic. Using this epifluorescence-based technique to enumerate capsulated bacterioplankton thus allowed us to routinely assess the number of capsulated bacteria even in the oceanic water column. Based on the data obtained in this study we conclude that almost all metabolically highly active bacteria exhibit a capsule, but also some of the metabolically less active cells express a polysaccharide capsule detectable with this method.     

Validation of heavy-water stable isotope probing for the characterization of rapidly responding soil bacteria

Rapid responses of bacteria to sudden changes in their environment can have important implications for the structure and function of microbial communities. In this study, we used heavy-water stable isotope probing (H2(18)O-SIP) to identify bacteria that respond to soil rewetting. First, we conducted experiments to address uncertainties regarding the H2(18)O-SIP method. Using liquid chromatography-mass spectroscopy (LC-MS), we determined that oxygen from H2(18)O was incorporated into all structural components of DNA. Although this incorporation was uneven, we could effectively separate 18O-labeled and unlabeled DNAs derived from laboratory cultures and environmental samples that were incubated with H2(18)O. We found no evidence for ex vivo exchange of oxygen atoms between DNA and extracellular H2O, suggesting that 18O incorporation into DNA is relatively stable. Furthermore, the rate of 18O incorporation into bacterial DNA was high (within 48 to 72 h), coinciding with pulses of CO2 generated from soil rewetting. Second, we examined shifts in the bacterial composition of grassland soils following rewetting, using H2(18)O-SIP and bar-coded pyrosequencing of 16S rRNA genes. For some groups of soil bacteria, we observed coherent responses at a relatively course taxonomic resolution. Following rewetting, the relative recovery of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria increased, while the relative recovery of Chloroflexi and Deltaproteobacteria decreased. Together, our results suggest that H2(18)O-SIP is effective at identifying metabolically active bacteria that influence soil carbon dynamics. Our results contribute to the ecological classification of soil bacteria while providing insight into some of the functional traits that influence the structure and function of microbial communities under dynamic soil moisture regimes.     

Tylosin-resistant bacteria cultivated from agricultural soil

In this study we analyzed the numbers and types of cultivable tylosin-resistant bacteria from six agricultural soils that differed with respect to their association with subtherapeutic antibiotic use. The proportion of tylosin-resistant bacteria to the total number of bacteria cultivated was significantly higher (7.2-16.5%) at three sites affected by subtherapeutic antibiotic use compared to three sites unaffected by subtherapeutic antibiotic use (0.7-2.5%). We also detected differences in the types of cultivable tylosin-resistant bacteria. At a site affected by subtherapeutic antibiotic use, we detected tylosin-resistant bacteria from the alpha- and beta-subdivisions of Proteobacteria. In contrast, at a site unaffected by subtherapeutic use, we detected only Streptomyces-like (high-G+C Gram-positive) tylosin-resistant bacteria. Our results suggest a link between subtherapeutic use of antibiotics and the numbers and types of antibiotic-resistant bacteria in nearby soil. However, other factors, such as soil type and temporal variation, may have also contributed to the differences observed.     

革兰氏阴性菌AcrAB-TolC多药外排泵结构数据对抑制剂研发的启示

革兰氏阴性菌的多重耐药性已成为全球广泛聚焦的问题。近年研究发现,耐药结节细胞分化(resistance-nodulation-cell division,RND)家族外排泵的过表达,与革兰氏阴性菌的多重耐药性密切相关。在RND家族中,广泛存在于革兰氏阴性菌中的AcrAB-TolC外排泵被认为是导致多重耐药性的主要原因之一。为了开发有效的抑制剂,需要对AcrAB-TolC外排泵的结构有一个清晰的认识。以往对该外排泵结构的研究主要局限于体外采用X射线晶体学技术或冷冻电镜单颗粒分析技术来解析其单个组分或全泵的结构。细胞冷冻电子断层扫描技术为揭示AcrAB-TolC外排泵在天然细胞膜环境中的组装和运行机制提供了新的见解,本文综述了AcrAB-TolC不同层级的结构数据在研发外排泵抑制剂方面的贡献。     

Finite time blow-up in some models of chemotaxis

We consider a class of models of chemotactic bacterial populations, introduced by Keller-Segel. For those models, we investigate the possibility of chemotactic collapse, in other words, the possibility that in finite time the population of predators aggregates to form a delta-function. To study this phenomenon, we construct self-similar solutions, which may or may not blow-up (in finite time), depending on the relative strength of three mechanisms in competition: (i) the chemotactic attraction of bacteria towards regions of high concentration in substrate (ii) the rate of consumption of the substrate by the bacteria and (iii) (possibly) the diffusion of bacteria. The solutions we construct are radially symmetric, and therefore have no relation with the classical traveling wave solutions. Our scaling can be justified by a dimensional analysis. We give some evidence of numerical stability.     

Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus

In this study we investigated the viable but non-culturable (VBNC) state of Aeromonas hydrophila and its virulence in goldfish. Aeromonas hydrophila cultured in a 0.35% NaCl solution at pH 7.5 and at 25 degrees C for 50 days showed the VBNC state. In the VBNC state we were unable to detect viable bacteria by the plate count method but we did find 10(4) cells/ml by the direct viable count microscopical method after staining with fluorescein diacetate and ethidium bromide. The virulence comparison in goldfish showed that bacteria cultured at 25 degrees C for 1 day in a 0.35% NaCl solution were more virulent than bacteria cultured for 28 days. VBNC bacteria showed lower virulence in goldfish compared to 28-day-cultured bacteria by intraperitoneal injection. The results from the study suggest that A. hydrophila can remain in the aquatic environment for prolonged periods in the VBNC state but those cells are not pathogenic to goldfish.     

Using Liquid Crystals to Reveal How Mechanical Anisotropy Changes Interfacial Behaviors of Motile Bacteria

Bacteria often inhabit and exhibit distinct dynamical behaviors at interfaces, but the physical mechanisms by which interfaces cue bacteria are still poorly understood. In this work, we use interfaces formed between coexisting isotropic and liquid crystal (LC) phases to provide insight into how mechanical anisotropy and defects in LC ordering influence fundamental bacterial behaviors. Specifically, we measure the anisotropic elasticity of the LC to change fundamental behaviors of motile, rod-shaped Proteus mirabilis cells (3 μm in length) adsorbed to the LC interface, including the orientation, speed, and direction of motion of the cells (the cells follow the director of the LC at the interface), transient multicellular self-association, and dynamical escape from the interface. In this latter context, we measure motile bacteria to escape from the interfaces preferentially into the isotropic phase, consistent with the predicted effects of an elastic penalty associated with strain of the LC about the bacteria when escape occurs into the nematic phase. We also observe boojums (surface topological defects) present at the interfaces of droplets of nematic LC (tactoids) to play a central role in mediating the escape of motile bacteria from the LC interface. Whereas the bacteria escape the interface of nematic droplets via a mechanism that involved nematic director-guided motion through one of the two boojums, for isotropic droplets in a continuous nematic phase, the elasticity of the LC generally prevented single bacteria from escaping. Instead, assemblies of bacteria piled up at boojums and escape occurred through a cooperative, multicellular phenomenon. Overall, our studies show that the dynamical behaviors of motile bacteria at anisotropic LC interfaces can be understood within a conceptual framework that reflects the interplay of LC elasticity, surface-induced order, and topological defects.     

微生物VBNC状态形成及复苏机制

99%以上的微生物因处于活的但非可培养(viable but non-culturable,VBNC)状态而无法分离培养。复苏促进因子(resuscitation-promoting factors,Rpfs)是培养获取VBNC菌的最重要突破。结合课题组近十余年从环境功能视角利用Rpf复苏培养VBNC菌的研究,本文在阐述微生物VBNC状态的形成及复苏进展的基础上,从VBNC菌形成及复苏过程出发,探究"探索因子"与群体感应的内在关系。并总结了课题组利用Rpf所复苏培养的具有潜在环境功能的VBNC菌种。本论文将为揭示微生物VBNC状态的形成及复苏机制提供新的思路,并为认识和重新评价Rpf法复苏培养VBNC菌在污染环境微生物修复中的作用提供理论依据。     

Invasive and adherent bacterial pathogens co-Opt host clathrin for infection

Infection by the bacterium Listeria monocytogenes depends on host cell clathrin. To determine whether this requirement is widespread, we analyzed infection models using diverse bacteria. We demonstrated that bacteria that enter cells following binding to cellular receptors (termed "zippering" bacteria) invade in a clathrin-dependent manner. In contrast, bacteria that inject effector proteins into host cells in order to gain entry (termed "triggering" bacteria) invade in a clathrin-independent manner. Strikingly, enteropathogenic Escherichia coli (EPEC) required clathrin to form actin-rich pedestals in host cells beneath adhering bacteria, even though this pathogen remains extracellular. Furthermore, clathrin accumulation preceded the actin rearrangements necessary for Listeria entry. These data provide evidence for a clathrin-based entry pathway allowing internalization of large objects (bacteria and ligand-coated beads) and used by "zippering" bacteria as part of a general mechanism to invade host mammalian cells. We also revealed a nonendocytic role for clathrin required for extracellular EPEC infections.     

Using Liquid Crystals to Reveal How Mechanical Anisotropy Changes Interfacial Behaviors of Motile Bacteria

Bacteria often inhabit and exhibit distinct dynamical behaviors at interfaces, but the physical mechanisms by which interfaces cue bacteria are still poorly understood. In this work, we use interfaces formed between coexisting isotropic and liquid crystal (LC) phases to provide insight into how mechanical anisotropy and defects in LC ordering influence fundamental bacterial behaviors. Specifically, we measure the anisotropic elasticity of the LC to change fundamental behaviors of motile, rod-shaped Proteus mirabilis cells (3 μm in length) adsorbed to the LC interface, including the orientation, speed, and direction of motion of the cells (the cells follow the director of the LC at the interface), transient multicellular self-association, and dynamical escape from the interface. In this latter context, we measure motile bacteria to escape from the interfaces preferentially into the isotropic phase, consistent with the predicted effects of an elastic penalty associated with strain of the LC about the bacteria when escape occurs into the nematic phase. We also observe boojums (surface topological defects) present at the interfaces of droplets of nematic LC (tactoids) to play a central role in mediating the escape of motile bacteria from the LC interface. Whereas the bacteria escape the interface of nematic droplets via a mechanism that involved nematic director-guided motion through one of the two boojums, for isotropic droplets in a continuous nematic phase, the elasticity of the LC generally prevented single bacteria from escaping. Instead, assemblies of bacteria piled up at boojums and escape occurred through a cooperative, multicellular phenomenon. Overall, our studies show that the dynamical behaviors of motile bacteria at anisotropic LC interfaces can be understood within a conceptual framework that reflects the interplay of LC elasticity, surface-induced order, and topological defects.     

Construction of Plasmid Vectors for Screening Replicons from Gram-Positive Bacteria and Their Use as Shuttle Cloning Vectors

Plasmids play a central role in engineering recombinant bacteria because they are the primary vehicles used to manipulate targeted sequences. In some cases, bacteria of interest are poorly provided with suitable tools for these molecular or genetic manipulations. In this context, we constructed from two shuttle cloning vectors, pUCB2871 and pUCB2872, the basic vectors pUCB30 and pUCB31, which could represent suitable tools to isolate replicons from Gram-positive bacteria. These plasmid vectors are characterized by the following after-features: (a) the pUC origin of replication is unable to replicate in Gram-positive bacteria; (b) an erythromycin-resistance encoding gene that is functional in both Gram-negative and -positive bacteria; (c) the pUC19 multiple cloning site (MCS) within the lacZα reporter gene; and (4) an additional multiple cloning site (MCS). Cloning replicons from Gram-positive bacteria in this additional MCS would allow the derivative vectors to function directly as shuttle cloning vectors.     

Stimulatory Effect of Xenobiotics on Oxidative Electron Transport of Chemolithotrophic Nitrifying Bacteria Used as Biosensing Element

Electron transport chain (ETCh) of ammonium (AOB) and nitrite oxidizing bacteria (NOB) participates in oxidation of ammonium to nitrate (nitrification). Operation of ETCh may be perturbed by a range of water-soluble xenobiotics. Therefore, consortia of nitrifying bacteria may be used as a biosensor to detect water contamination. A surprising feature of this system is an increase of oxygen consumption, detected in the presence of certain inhibitors of ETCh. Thus, to shed light on the mechanism of this effect (and other differences between inhibitors) we monitored separately respiration of the bacteria of the first (AOB - Nitrosomonas) and second (NOB -Nitrobacter) stages of nitrification. Furthermore, we measured plasma membrane potential and the level of reduction of NAD(P)H. We propose a novel model of ETCh in NOB to explain the role of reverse electron transport in the stimulation of oxygen consumption (previously attributed to hormesis).     

Preliminary selection study of potential probiotic bacteria from aquacultural area in Tunisia

In order to test the ability to produce antibacterial substances within marine bacteria, prior to select potential probiotics for use in shellfish farming, we targeted a large collection of bacterial isolates (132 strains), brought from the clamRuditapes decussatus and 37 reference strains. First, we proceeded to their biochemical identification and the screening of antibiotic resistance profiles. Else, we investigated their inhibitory activityin vitro against several fish and shellfish pathogens, using two methods: the double-layer agar and the direct simultaneous antagonism methods. The results showed high frequencies of inhibitory producing bacteria (IPB) within the isolates. These bacteria (25%) were aerobic mesophylic bacteria belonging to various bacterial groups: 33.7% oxidase-positive Gram-negative bacteria, 7.4%Enterobacteriaceae and 28% lactic acid bacteria. Besides this group, nine strains produced strong inhibition effect. These bacteria belonged to:Aeromonas hydrophila, Aeromonas sobria, Pseudomonas cepacia, Vibrio sp,Serratia liquefaciens andLactobacillus rhamnosus. They were active against pathogenic bacteria belonging to the genera:Aeromonas, Pseudomonas andVibrio. These potential probiotics were submitted to further investigations prior to their introduction in larval shellfish farming.     

The Role of Secretory Immunoglobulin A in the Natural Sensing of Commensal Bacteria by Mouse Peyer's Patch Dendritic Cells

The mammalian gastrointestinal (GI) tract harbors a diverse population of commensal species collectively known as the microbiota, which interact continuously with the host. From very early in life, secretory IgA (SIgA) is found in association with intestinal bacteria. It is considered that this helps to ensure self-limiting growth of the microbiota and hence participates in symbiosis. However, the importance of this association in contributing to the mechanisms ensuring natural host-microorganism communication is in need of further investigation. In the present work, we examined the possible role of SIgA in the transport of commensal bacteria across the GI epithelium. Using an intestinal loop mouse model and fluorescently labeled bacteria, we found that entry of commensal bacteria in Peyer''s patches (PP) via the M cell pathway was mediated by their association with SIgA. Preassociation of bacteria with nonspecific SIgA increased their dynamics of entry and restored the reduced transport observed in germ-free mice known to have a marked reduction in intestinal SIgA production. Selective SIgA-mediated targeting of bacteria is restricted to the tolerogenic CD11c⁺CD11b⁺CD8⁻ dendritic cell subset located in the subepithelial dome region of PPs, confirming that the host is not ignorant of its resident commensals. In conclusion, our work supports the concept that SIgA-mediated monitoring of commensal bacteria targeting dendritic cells in the subepithelial dome region of PPs represents a mechanism whereby the host mucosal immune system controls the continuous dialogue between the host and commensal bacteria.     

Degradation of BTX by dissimilatory iron-reducing cultures

The ability of indigenous bacteria to anaerobically degrade monoaromatic hydrocarbons has received attention as a potential strategy to remediate polluted aquifers. Despite the fact that iron-reducing conditions are often dominating in contaminated sediment, most of the studies have focussed on degradation of this class of pollutants with other terminal acceptors. In this work, we enriched bacteria from an iron-reducing aquifer in which a plume of pollution has developed over several decades and we show that benzene, toluene, meta- and para-xylene (BTX) could be degraded by the enriched cultures containing intrinsic iron-reducing microorganisms. To our knowledge, this is the first time that para-xylene degradation by dissimilatory iron-reducing bacteria has been reported in sediment free enrichment cultures. BTX degradation rates in enrichment cultures progressively increased in time and were found in good agreement with theoretical values calculated assuming complete BTX oxidation with Fe(II) as final electron acceptor. In addition, using labelled ((13)C(1)) benzene and toluene we could unambiguously identify intermediates of their respective degradation pathways. We provide evidence for benzene degradation via phenol formation under iron-reducing conditions, whereas toluene and meta-xylene were transformed into the corresponding benzylsuccinates.     

Investigation of the first events leading to loss of culturability during Escherichia coli starvation: future nonculturable bacteria form a subpopulation

In previous experiments we were able to separate, using a nondestructive separation technique, culturable and nonculturable bacteria, from a Luria-Bertani (LB) medium culture of Escherichia coli incubated for 48 h. We observed in the nonculturable bacterial population an increase in oxidative damage and up-induction of most defenses against reactive oxygen species (ROS), along with a decrease in cytoplasmic superoxide dismutases. In this study, using the same separation technique, we separated into two subpopulations a 10-h LB medium culture containing only culturable bacteria. For the first time, we succeeded in associating physical separation with physiological differences. Although the levels of defense against ROS (RpoS, RpoH, OxyR, and SoxRS regulons) and oxidative damage (carbonyl contents) were apparently the same, we found that bacteria in one subpopulation were more sensitive to LB medium starvation and to various stresses, such as phosphate buffer starvation, heat shock, and hydrogen peroxide exposure. Based on these results, we suggest that these physiological differences reflect uncharacterized bacterial modifications which do not directly involve defenses against ROS.