Aerobic Heterotrophic Bacteria Indigenous to pH 2.8 Acid Mine Water: Predominant Slime-Producing Bacteria in Acid Streamers

Five gram-negative bacteria, two gram-positive bacteria, and one yeast were isolated from "acid streamers" taken from acid mine water. One gram-positive rod which has been tentatively identified as a Bacillus species appeared to be the predominant organism in the streamers. This isolate produced copious amounts of extracellular polymer at 10 C in the laboratory and was considered to be the primary source of polymer in the "acid streamer" slime matrix. The organism grew slowly at pH 2.8 in mine water media, but the optimal pH was approximately 7.0.     

Macroscopic streamer growths in acidic, metal-rich mine waters in north wales consist of novel and remarkably simple bacterial communities

The microbial composition of acid streamers (macroscopic biofilms) in acidic, metal-rich waters in two locations (an abandoned copper mine and a chalybeate spa) in north Wales was studied using cultivation-based and biomolecular techniques. Known chemolithotrophic and heterotrophic acidophiles were readily isolated from disrupted streamers, but they accounted for only <1 to 7% of the total microorganisms present. Fluorescent in situ hybridization (FISH) revealed that 80 to 90% of the microbes in both types of streamers were beta-Proteobacteria. Terminal restriction fragment length polymorphism analysis of the streamers suggested that a single bacterial species was dominant in the copper mine streamers, while two distinct bacteria (one of which was identical to the bacterium found in the copper mine streamers) accounted for about 90% of the streamers in the spa water. 16S rRNA gene clone libraries showed that the beta-proteobacterium found in both locations was closely related to a clone detected previously in acid mine drainage in California and that its closest characterized relatives were neutrophilic ammonium oxidizers. Using a modified isolation technique, this bacterium was isolated from the copper mine streamers and shown to be a novel acidophilic autotrophic iron oxidizer. The beta-proteobacterium found only in the spa streamers was closely related to the neutrophilic iron oxidizer Gallionella ferruginea. FISH analysis using oligonucleotide probes that targeted the two beta-proteobacteria confirmed that the biodiversity of the streamers in both locations was very limited. The microbial compositions of the acid streamers found at the two north Wales sites are very different from the microbial compositions of the previously described acid streamers found at Iron Mountain, California, and the Rio Tinto, Spain.     

Macroscopic Streamer Growths in Acidic, Metal-Rich Mine Waters in North Wales Consist of Novel and Remarkably Simple Bacterial Communities

The microbial composition of acid streamers (macroscopic biofilms) in acidic, metal-rich waters in two locations (an abandoned copper mine and a chalybeate spa) in north Wales was studied using cultivation-based and biomolecular techniques. Known chemolithotrophic and heterotrophic acidophiles were readily isolated from disrupted streamers, but they accounted for only <1 to 7% of the total microorganisms present. Fluorescent in situ hybridization (FISH) revealed that 80 to 90% of the microbes in both types of streamers were β-Proteobacteria. Terminal restriction fragment length polymorphism analysis of the streamers suggested that a single bacterial species was dominant in the copper mine streamers, while two distinct bacteria (one of which was identical to the bacterium found in the copper mine streamers) accounted for about 90% of the streamers in the spa water. 16S rRNA gene clone libraries showed that the β-proteobacterium found in both locations was closely related to a clone detected previously in acid mine drainage in California and that its closest characterized relatives were neutrophilic ammonium oxidizers. Using a modified isolation technique, this bacterium was isolated from the copper mine streamers and shown to be a novel acidophilic autotrophic iron oxidizer. The β-proteobacterium found only in the spa streamers was closely related to the neutrophilic iron oxidizer Gallionella ferruginea. FISH analysis using oligonucleotide probes that targeted the two β-proteobacteria confirmed that the biodiversity of the streamers in both locations was very limited. The microbial compositions of the acid streamers found at the two north Wales sites are very different from the microbial compositions of the previously described acid streamers found at Iron Mountain, California, and the Rio Tinto, Spain.     

Testing the functional significance of tail streamers

Studies of the evolution of elaborate ornaments have concentrated on their role in increasing attractiveness to mates. The classic examples of such sexually selected structures are the elongated tails of some bird species. Elongated tails can be divided into three categories: graduated tails, pin tails and streamers. There seems to be little debate about whether graduated and pin tails are ornaments; i.e. costly signals used in mate choice. However, in the case of streamers there is considerable discussion about their function. It has been suggested that tail streamers could be (i) entirely naturally selected, (ii) entirely sexually selected, (iii) partly naturally and partly sexually selected. The prime example of a species with tail streamers is the swallow (Hirundo rustica) in which both sexes have tail streamers. In this paper we discuss the aerodynamic consequences of different types of manipulation of the streamer and/or outer tail feather. We make qualitative predictions about the aerodynamic performance of swallows with manipulated tail streamers; these predictions differ depending on whether streamers have a naturally or sexually selected function. We demonstrate that these hypotheses can only be separated if tail streamers are shortened and changes in aerodynamic performance measured during turning flight.     

Locked Nucleic Acid Pentamers as Universal PCR Primers for Genomic DNA Amplification

Background

Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping.

Results

We designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 species used in the assay, 28 species and 50 different genes were clearly identified using this method.

Conclusion

As a novel genomic DNA amplification, the use of locked nucleic acid pentamers as universal primer pairs in conjunction with suspension array genotyping, allows for the identification of multiple distinct genes or species with a single amplification procedure. This demonstrates that locked nucleic acid pentamer-based PCR can be utilized extensively in pathogen identification.     

The Ecology of the Acetic Acid Bacteria with Particular Reference to Cider Manufacture

The ecology of the acetic acid bacteria has been studied at various stages of their association with cider manufacture. Of the 278 strains of bacteria isolated during the survey, 255 proved to be representative of 6 species of acetic acid bacteria. The remaining 23 strains included one example of the spoilage organism, Zymomonas anaerobia , but they were mostly ubiquitous soil bacteria which could not survive the low pH of apple juice and were only found associated with the early stages of cider making. The acetic acid bacteria were isolated in a sequential type of pattern. Those species which preferentially oxidize sugars were found at the early stages of processing when sugars abound, but these were replaced by the relatively more acid-tolerant species, which are better equipped to oxidize alcohols, after the yeast fermentation had converted most of the sugar to ethanol.     

Protein phylogenies and signature sequences: evolutionary relationships within prokaryotes and between prokaryotes and eukaryotes

The evolutionary relationships within prokaryotes and between prokaryotes and eukaryotes is examined based on protein sequence data. Phylogenies and common signature sequences in some of the most conserved proteins point to a close evolutionary relationship between Archaebacteria and Gram-positive bacteria. The monophyletic nature and distinctness of the Archaebacterial domain is not supported by many of the phylogenies. Within Gram-negative bacteria, cyanobacteria are indicated as the deepest branching lineage, and a clade consisting of Archaebacteria, Gram-positive bacteria and cyanobacteria is supported by signature sequences in many proteins. However, the division within the prokaryotic species viz. Archaebacteria Gram-positive bacteria Cyanobacteria other groups of Gram-negative bacteria, is indicated to be not very rigid but, instead is an evolutionary continuum. It is expected that certain species will be found which represent intermediates in the above transitions. By contrast to the evolutionary relationships within prokaryotes, the eukaryotic species, which are structurally very different, appear to have originated by a very different mechanism. Protein phylogenies and signature sequences provide evidence that the eukaryotic nuclear genome is a chimera which has received major contributions from both an Archaebacterium and a Gram-negative bacterium. To explain these observations, it is suggested that the ancestral eukaryotic cell arose by a symbiotic fusion event between the above parents and that this fusion event led to the origin of both nucleus and endoplasmic reticulum. The monophyletic nature of all extant eukaryotic species further suggests that a 'successful primary fusion' between the prokaryotic species that gave rise to the ancestral eukaryotic cell took place only once in the history of this planet.     

Mathematical Model of Interaction Between Bacteriocin-Producing Lactic Acid Bacteria and Listeria. Part 1: Steady States and Thresholds

Mathematical modeling is an important tool to assessing quantitative conjectures and to answer specific questions. In the modeling, we assume that a competitor represented by a lactic acid bacterium produces antimicrobial compounds (substances that kill microorganisms or inhibit their growth), such as lactic acid and bacteriocins, with some cost to its own growth. Bacteriocins are protein compounds with antimicrobial effect against related species and bacteria such as Listeria monocytogenes, which is foodborne pathogen that cause listeriosis. From the analysis of the model, we found the thresholds which determine the existence of multiple equilibria and we studied their stability, in order to evaluate the interaction between lactic acid bacteria and L. monocytogenes.     

Growth-inhibiting effects of concentrations of fusaric acid on the growth of Bacillus mojavensis and other biocontrol Bacillus species

AIMS: To determine the effects of concentrations of fusaric acid on the growth of several strains of the biocontrol bacterial endophyte Bacillus mojavensis and other species within the Bacillus subtilis group, as well as the genetic relationships within this small group of Gram-positive bacteria, and their antagonisms to Fusarium verticillioides, which produce fusaric acid. METHODS AND RESULTS: The growth of 50 Bacillus strains and species were tested at two concentrations of fusaric acid determined in maize infected by an isolate of F. verticillioides. Molecular characterizations of the strains and species of bacteria were determined with an automated ribotyper. The growth of bacteria measured under both concentrations with an automated turbidometer, Bioscreen, indicated that fusaric acid was toxic to most strains of the bacterial endophyte B. mojavensis. However, the effects of these two concentrations on other Bacillus species varied in that fusaric acid was either bacteriocidal or bacteriostatic to most species. CONCLUSIONS: These data indicate that the concentrations of fusaric acid are inhibitory to the growth of most Bacillus species, some of which are used as biocontrol agents. This suggests that the endophytic and saprophytic states of F. verticillioides and other Fusarium species cannot be controlled by fusaric-acid-sensitive Bacillus species. SIGNIFICANCE AND IMPACT OF STUDY: Mycotoxic Fusarium species, such as F. verticillioides, are competitive because all produce fusaric acid, which is inhibitory to biocontrol bacteria, and mutants tolerant to fusaric acid must be developed in order to be effective on biocontrol bacteria.     

Comparative studies of bacterial biofilms on steel surfaces using atomic force microscopy and environmental scanning electron microscopy

Environmental scanning electron microscopy (ESEM) and atomic force microscopy (AFM) were compared as tools for the observation of bacterial biofilms developed on carbon steel and AISI 316 stainless steel surfaces under stagnant conditions. Biofilms were generated in batch cultures of two different isolates of marine sulphate reducing bacteria (SRB) and in cultures consisting of mixed populations of acidophilic bacteria, known as "acid streamers";. Imaging of single SRB cells on mica was also carried out to reveal the surface topography of individual bacterial cells at nanometre resolution. Following the removal of biofilms, the stainless steel surfaces were profiled using AFM to determine the degree of steel deterioration. ESEM and AFM studies of bacterial biofilms in-situ, gave both qualitative and quantitative information on biofilm structure at high resolution. The use of AFM image analysis software allowed estimation of the width and height of bacterial cells, the thickness and width of exopolymeric (EPS) capsule and bacterial flagella, as well as characterisation of the surface roughness of the steel, including measurements of depth and diameter of individual pits. Exposure of stainless steel specimens to acid streamers resulted in a significant increase in the surface roughness of the steel, compared to specimens placed in sterile medium.     

Phylogeny of Microorganisms Populating a Thick, Subaerial, Predominantly Lithotrophic Biofilm at an Extreme Acid Mine Drainage Site

An unusually thick (~1 cm) slime developed on a slump of finely disseminated pyrite ore within an extreme acid mine drainage site at Iron Mountain, near Redding, Calif. The slime was studied over the period of 1 year. The subaerial form of the slime distinguished it from more typical submerged streamers. Phylogenetic analysis of 16S rRNA genes revealed a diversity of sequences that were mostly novel. Nearest relatives to the majority of sequences came from iron-oxidizing acidophiles, and it appears that iron oxidation is the predominant metabolic characteristic of the organisms in the slime. The most abundant of the 16S rRNA genes detected were from organisms related to Leptospirillum species. The dominant sequence (71% of clones) may represent a new genus. Sequences within the Archaea of the Thermoplasmales lineage were detected. Most of these were only distantly related to known microorganisms. Also, sequences affiliating with Acidimicrobium were detected. Some of these were closely related to “Ferromicrobium acidophilus,” and others were affiliated with a lineage only represented by environmental clones. Unexpectedly, sequences that affiliated within the delta subdivision of the Proteobacteria were detected. The predominant metabolic feature of bacteria of this subdivision is anaerobic sulfate or metal reduction. Thus, microenvironments of low redox potential possibly exist in the predominantly oxidizing environments of the slime. These results expand our knowledge of the biodiversity of acid mine drainage environments and extend our understanding of the ecology of extremely acidic systems.     

Formation of bacterial streamers during filtration in microfluidic systems

Bacterial behavior during filtration is complex and is influenced by numerous factors. The aim of this paper is to report on experiments designed to make progress in the understanding of bacterial transfer in filters and membranes. Polydimethylsiloxane (PDMS) microsystems were built to allow direct dynamic observation of bacterial transfer across different microchannel geometries mimicking filtration processes. When filtering Escherichia coli suspensions in such devices, the bacteria accumulated in the downstream zone of the filter forming long streamers undulating in the flow. Confocal microscopy and 3D reconstruction of streamers showed how the streamers are connected to the filter and how they form in the stream. Streamer development was found to be influenced by the flow configuration and the presence of connections or tortuosity between channels. Experiments showed that streamer formation was greatest in a filtration system composed of staggered arrays of squares 10?μm apart.     

Formation of bacterial streamers during filtration in microfluidic systems

Bacterial behavior during filtration is complex and is influenced by numerous factors. The aim of this paper is to report on experiments designed to make progress in the understanding of bacterial transfer in filters and membranes. Polydimethylsiloxane (PDMS) microsystems were built to allow direct dynamic observation of bacterial transfer across different microchannel geometries mimicking filtration processes. When filtering Escherichia coli suspensions in such devices, the bacteria accumulated in the downstream zone of the filter forming long streamers undulating in the flow. Confocal microscopy and 3D reconstruction of streamers showed how the streamers are connected to the filter and how they form in the stream. Streamer development was found to be influenced by the flow configuration and the presence of connections or tortuosity between channels. Experiments showed that streamer formation was greatest in a filtration system composed of staggered arrays of squares 10 μm apart.     

Secondary flow as a mechanism for the formation of biofilm streamers

In most environments, such as natural aquatic systems, bacteria are found predominantly in self-organized sessile communities known as biofilms. In the presence of a significant flow, mature multispecies biofilms often develop into long filamentous structures called streamers, which can greatly influence ecosystem processes by increasing transient storage and cycling of nutrients. However, the interplay between hydrodynamic stresses and streamer formation is still unclear. Here, we show that suspended thread-like biofilms steadily develop in zigzag microchannels with different radii of curvature. Numerical simulations of a low-Reynolds-number flow around these corners indicate the presence of a secondary vortical motion whose intensity is related to the bending angle of the turn. We demonstrate that the formation of streamers is directly proportional to the intensity of the secondary flow around the corners. In addition, we show that a model of an elastic filament in a two-dimensional corner flow is able to explain how the streamers can cross fluid streamlines and connect corners located at the opposite sides of the channel.     

Mathematical Model of Interaction Between Bacteriocin-Producing Lactic Acid Bacteria and Listeria. Part 2: Bifurcations and Applications

The big challenge for the food industry is the attending to demands for minimally processed foods, avoiding intense heat treatments and reducing the addition of chemical preservatives, but at the same time ensuring microbiological safety of these products. Lactic acid bacteria are traditionally used in the production of fermented foods. They are responsible for the production of antimicrobial compounds, such as organic acids and bacteriocins, which are protein compounds with bactericidal effect against related species and bacteria such as Listeria monocytogenes and Staphylococcus aureus. Aiming to study quantitatively the biological control as a technique of conservation, we developed a mathematical model to describe the interaction between lactic acid bacteria and Listeria in the food. The steady state and dynamical trajectories analyses of the model permit us to study the suitability of including lactic acid bacteria in order to reduce the growth of Listeria in food.     

Engineered cell-to-cell signalling within growing bacterial cellulose pellicles

Bacterial cellulose is a strong and flexible biomaterial produced at high yields by Acetobacter species and has applications in health care, biotechnology and electronics. Naturally, bacterial cellulose grows as a large unstructured polymer network around the bacteria that produce it, and tools to enable these bacteria to respond to different locations are required to grow more complex structured materials. Here, we introduce engineered cell-to-cell communication into a bacterial cellulose-producing strain of Komagataeibacter rhaeticus to enable different cells to detect their proximity within growing material and trigger differential gene expression in response. Using synthetic biology tools, we engineer Sender and Receiver strains of K. rhaeticus to produce and respond to the diffusible signalling molecule, acyl-homoserine lactone. We demonstrate that communication can occur both within and between growing pellicles and use this in a boundary detection experiment, where spliced and joined pellicles sense and reveal their original boundary. This work sets the basis for synthetic cell-to-cell communication within bacterial cellulose and is an important step forward for pattern formation within engineered living materials.     

Phylogeny of microorganisms populating a thick, subaerial, predominantly lithotrophic biofilm at an extreme acid mine drainage site

An unusually thick ( approximately 1 cm) slime developed on a slump of finely disseminated pyrite ore within an extreme acid mine drainage site at Iron Mountain, near Redding, Calif. The slime was studied over the period of 1 year. The subaerial form of the slime distinguished it from more typical submerged streamers. Phylogenetic analysis of 16S rRNA genes revealed a diversity of sequences that were mostly novel. Nearest relatives to the majority of sequences came from iron-oxidizing acidophiles, and it appears that iron oxidation is the predominant metabolic characteristic of the organisms in the slime. The most abundant of the 16S rRNA genes detected were from organisms related to Leptospirillum species. The dominant sequence (71% of clones) may represent a new genus. Sequences within the Archaea of the Thermoplasmales lineage were detected. Most of these were only distantly related to known microorganisms. Also, sequences affiliating with Acidimicrobium were detected. Some of these were closely related to "Ferromicrobium acidophilus," and others were affiliated with a lineage only represented by environmental clones. Unexpectedly, sequences that affiliated within the delta subdivision of the Proteobacteria were detected. The predominant metabolic feature of bacteria of this subdivision is anaerobic sulfate or metal reduction. Thus, microenvironments of low redox potential possibly exist in the predominantly oxidizing environments of the slime. These results expand our knowledge of the biodiversity of acid mine drainage environments and extend our understanding of the ecology of extremely acidic systems.     

Evaluation of the effectiveness of light streamer tori-lines and characteristics of bait attacks by seabirds in the western North Pacific

To improve the effectiveness of tori-lines it is necessary to evaluate the ability of tori-lines to mitigate seabird bycatch and determine what kind of seabird species gather during line settings, attack the bait and are incidentally caught. We conducted two experiments in the western North Pacific and examined the effectiveness for seabird mitigation of light streamer tori-lines which have no long streamers but many light (short) streamers and are mainly used in the North Pacific area. Firstly, the effectiveness of two different types of tori-line (light streamer (1 m) and long streamer (up to 7 m) tori-line) and of two different colors (yellow and red) of light streamers for seabird bycatch avoidance was evaluated using 567 sets based on data from 20 offshore surface commercial longliners. No significant difference in the bycatch number between the different tori-line types and streamer colors was found. Secondly, we investigated the characteristics of the seabird bycatch in the North Pacific and the effectiveness of three different types of streamers (light, hybrid and modified light types) by detailed observations of seabird attacks using a chartered longline vessel. Although the appearance rate of albatrosses and shearwaters were 40.9% and 27.7%, Laysan albatross was the main seabird species that followed the vessel but shearwaters seldom followed the vessel and did not aggregate during line setting. In all attacks on bait observed during line settings, 81% and 7% were by albatrosses and shearwaters, respectively. In the number of primary attacks by Laysan albatrosses which attacked most aggressively of all seabirds, there were no significant differences among the tori-line types. No individuals of shearwater were caught. The results of both experiments indicated that light streamer tori-lines were as effective as tori-lines with long streamers for mitigating seabird bycatch in the North Pacific.     

An Evolutionary Perspective on Linoleic Acid Synthesis in Animals

The diet of organisms generally provides a sufficient supply of energy and building materials for healthy growth and development, but should also contain essential nutrients. Species differ in their exogenous requirements, but it is not clear why some species are able to synthesize essential nutrients, while others are not. The unsaturated fatty acid, linoleic acid (LA; 18:2n-6) plays an important role in functions such as cell physiology, immunity, and reproduction, and is an essential nutrient in diverse organisms. LA is readily synthesized in bacteria, protozoa and plants, but it was long thought that all animals lacked the ability to synthesize LA de novo and thus required a dietary source of this fatty acid. Over the years, however, an increasing number of studies have shown active LA synthesis in animals, including insects, nematodes and pulmonates. Despite continued interest in LA metabolism, it has remained unclear why some organisms can synthesize LA while others cannot. Here, we review the mechanisms by which LA is synthesized and which biological functions LA supports in different organisms to answer the question why LA synthesis was lost and repeatedly gained during the evolution of distinct invertebrate groups. We propose several hypotheses and compile data from the available literature to identify which factors promote LA synthesis within a phylogenetic framework. We have not found a clear link between our proposed hypotheses and LA synthesis; therefore we suggest that LA synthesis may be facilitated through bifunctionality of desaturase enzymes or evolved through a combination of different selective pressures.     

Freon 11 Extraction of Volatile Metabolites Formed by Certain Lactic Acid Bacteria

The volatile metabolites formed by 18 lactic acid bacteria, representing three genera, were extracted from a complex medium by using a Freon 11 extraction method. The Freon extracts were then analyzed by capillary gas chromatography, and certain extracts were analyzed by gas chromatography-mass spectrometry. A total of 35 major peaks, of which 20 were positively identified, were used to differentiate between the various strains. On the basis of the results obtained, it was possible to differentiate between the members of the genera Lactobacillus, Pediococcus, and Leuconostoc, as well as between various species within the genus Leuconostoc. Of the 10 Leuconostoc oenos strains included in this study, 9 yielded similar results, but it was still possible to differentiate between the various strains. L. oenos B66 differed from the other L. oenos strains. Use of the Freon 11 extraction technique to determine volatile metabolites formed by lactic acid bacteria was shown to be highly reproducible and of great value. Furthermore, certain compounds not previously known to be formed by lactic acid bacteria were found.