A single-cell sequencing approach to the classification of large, vacuolated sulfur bacteria

The colorless, large sulfur bacteria are well known because of their intriguing appearance, size and abundance in sulfidic settings. Since their discovery in 1803 these bacteria have been classified according to their conspicuous morphology. However, in microbiology the use of morphological criteria alone to predict phylogenetic relatedness has frequently proven to be misleading. Recent sequencing of a number of 16S rRNA genes of large sulfur bacteria revealed frequent inconsistencies between the morphologically determined taxonomy of genera and the genetically derived classification. Nevertheless, newly described bacteria were classified based on their morphological properties, leading to polyphyletic taxa. We performed sequencing of 16S rRNA genes and internal transcribed spacer (ITS) regions, together with detailed morphological analysis of hand-picked individuals of novel non-filamentous as well as known filamentous large sulfur bacteria, including the hitherto only partially sequenced species Thiomargarita namibiensis, Thioploca araucae and Thioploca chileae. Based on 128 nearly full-length 16S rRNA-ITS sequences, we propose the retention of the family Beggiatoaceae for the genera closely related to Beggiatoa, as opposed to the recently suggested fusion of all colorless sulfur bacteria into one family, the Thiotrichaceae. Furthermore, we propose the addition of nine Candidatus species along with seven new Candidatus genera to the family Beggiatoaceae. The extended family Beggiatoaceae thus remains monophyletic and is phylogenetically clearly separated from other related families.     

Sterilization by Cooling in Isochoric Conditions: The Case of Escherichia coli

High hydrostatic pressure (HHP) affects the structure, metabolism and survival of micro-organisms including bacteria. For this reason HHP is a promising treatment in the food industry. The aim of this work is to evaluate the effect of high pressure, under isochoric cooling conditions, on Escherichia coli, where such high pressure develops due to the fact water cannot expand. We combine survival curves obtained by spectrophotometry and images of atomic force microscopy in this study. Our results show that cooling at -20 and -30°C leads to a partial destruction of a Escherichia coli population. However, cooling at -15°C causes a total extermination of bacteria. This intriguing result is explained by the phase diagram of water. In the first case, the simultaneous formation of ice III and ice Ih crystals provides a safe environment for bacteria. In the second case (-15°C) Escherichia coli remains in a metastable and amorphous free-of-crystals liquid subjected to high pressure. Our work is the first experimental study carried out to inactivate Escherichia coli under isochoric cooling conditions. Unlike HHP, which is based on the application of an external load to augment the pressure, this technique only requires cooling. The method could be used for annihilation of other Escherichia coli strains and perhaps other micro-organisms.     

人工湿地不同季节与单元之间根际微生物多样性

采用选择性平板培养基法和PCR-DGGE法比较研究了三级垂直潜流人工湿地污水处理系统中不同季节与单元之间根际微生物数量与多样性,结果表明,随着“秋-冬-春-夏”的季节的变化,2种方法测定的微生物数量都呈现“高-低-高”季节性变化现象,但选择性平板培养基法显示秋季数量最多,PCR-DGGE法显示夏季数量最多.从三级处理单元的1-3级,两种方法测定的微生物数量都呈现“低-高-低”的规律.除土样11外,其余相同季节的土样均出现聚集在同一个分支下.得到的4个差异序列(YP1-YP 4),经BLAST比较,分别找到同源性最近的已知类群,分别为肠杆菌属、弧菌、欧文氏菌、产酸克雷伯菌.BLASTX比较发现YP1与细菌代谢分解有机物有关;YP2蜡样芽胞杆菌生命活动相关,具有解磷的作用;YP3与APOE载脂蛋白e功能相似,参与细菌的分解代谢;YP4功能可能与细菌去氮有关.     

The common bloom-forming cyanobacterium Microcystis is prone to a wide array of microbial antagonists

Many degraded waterbodies around the world are subject to strong proliferations of cyanobacteria – notorious for their toxicity, high biomass build-up and negative impacts on aquatic food webs – the presence of which puts serious limits on the human use of affected water bodies. Cyanobacterial blooms are largely regarded as trophic dead ends since they are a relatively poor food source for zooplankton. As a consequence, their population dynamics are generally attributed to changes in abiotic conditions (bottom-up control). Blooms however generally contain a vast and diverse community of micro-organisms of which some have shown devastating effects on cyanobacterial biomass. For Microcystis, one of the most common bloom-forming cyanobacteria worldwide, a high number of micro-organisms (about 120 taxa) including viruses, bacteria, microfungi, different groups of heterotrophic protists, other cyanobacteria and several eukaryotic microalgal groups are currently known to negatively affect its growth by infection and predation or by the production of allelopathic compounds. Although many of these specifically target Microcystis, sharp declines of Microcystis biomass in nature are only rarely assigned to these antagonistic microbiota. The commonly found strain specificity of their interactions may largely preclude strong antagonistic effects on Microcystis population levels but may however induce compositional shifts that can change ecological properties such as bloom toxicity. These highly specific interactions may form the basis of a continuous arms race (co-evolution) between Microcystis and its antagonists which potentially limits the possibilities for (micro)biological bloom control.     

Airborne Bacteria in an Urban Environment

Samples were taken at random intervals over a 2-year period from urban air and tested for viable bacteria. The number of bacteria in each sample was determined, and each organism isolated was identified by its morphological and biochemical characteristics. The number of bacteria found ranged from 0.013 to 1.88 organisms per liter of air sampled. Representatives of 19 different genera were found in 21 samples. The most frequently isolated organisms and their percent of occurence were Micrococcus (41%), Staphylococcus (11%), and Aerococcus (8%). The bacteria isolated were correlated with various weather and air pollution parameters using the Pearson product-moment correlation coefficient method. Statistically significant correlations were found between the number of viable bacteria isolated and the concentrations of nitric oxide (−0.45), nitrogen dioxide (+0.43), and suspended particulate pollutants (+0.56). Calculated individually, the total number of Micrococcus, Aerococcus, and Staphylococcus, number of rods, and number of cocci isolated showed negative correlations with nitric oxide and positive correlations with nitrogen dioxide and particulates. Statistically significant positive correlations were found between the total number of rods isolated and the concentration of nitrogen dioxide (+0.54) and the percent relative humidity (+0.43). The other parameters tested, sulfur dioxide, hydrocarbons, and temperature, showed no significant correlations.     

A genomic view on syntrophic versus non-syntrophic lifestyle in anaerobic fatty acid degrading communities

In sulfate-reducing and methanogenic environments complex biopolymers are hydrolyzed and degraded by fermentative micro-organisms that produce hydrogen, carbon dioxide and short chain fatty acids. Degradation of short chain fatty acids can be coupled to methanogenesis or to sulfate-reduction. Here we study from a genome perspective why some of these micro-organisms are able to grow in syntrophy with methanogens and others are not. Bacterial strains were selected based on genome availability and upon their ability to grow on short chain fatty acids alone or in syntrophic association with methanogens. Systematic functional domain profiling allowed us to shed light on this fundamental and ecologically important question. Extra-cytoplasmic formate dehydrogenases (InterPro domain number; IPR006443), including their maturation protein FdhE (IPR024064 and IPR006452) is a typical difference between syntrophic and non-syntrophic butyrate and propionate degraders. Furthermore, two domains with a currently unknown function seem to be associated with the ability of syntrophic growth. One is putatively involved in capsule or biofilm production (IPR019079) and a second in cell division, shape-determination or sporulation (IPR018365). The sulfate-reducing bacteria Desulfobacterium autotrophicum HRM2, Desulfomonile tiedjei and Desulfosporosinus meridiei were never tested for syntrophic growth, but all crucial domains were found in their genomes, which suggests their possible ability to grow in syntrophic association with methanogens. In addition, profiling domains involved in electron transfer mechanisms revealed the important role of the Rnf-complex and the formate transporter in syntrophy, and indicate that DUF224 may have a role in electron transfer in bacteria other than Syntrophomonas wolfei as well. This article is a part of a Special Issue entitled: 18th European Bioenergetics Conference (Biochim. Biophys. Acta, Volume 1837, Issue 7, July 2014).     

Effect of an Extract from Saprozoic Nematode-Infested Compost on the Mycelial Growth of Agaricus brunnescens

Extracts from compost infested with Caenorhabditis elegans suppressed mycelial growth of Agaricus brunnescens. An extract from uninfested compost also inhibited mycelial growth but to a lesser degree. The critical role of compost bacteria and/or other compost micro-organisms is implicated by these results.     

Isolation of Aerobic Anoxygenic Photosynthetic Bacteria from Black Smoker Plume Waters of the Juan de Fuca Ridge in the Pacific Ocean

A strain of the aerobic anoxygenic photosynthetic bacteria was isolated from a deep-ocean hydrothermal vent plume environment. The in vivo absorption spectra of cells indicate the presence of bacteriochlorophyll a incorporated into light-harvesting complex I and a reaction center. The general morphological and physiological characteristics of this new isolate are described.     

Detection of Arboviruses and Other Micro-Organisms in Experimentally Infected Mosquitoes Using Massively Parallel Sequencing

Human disease incidence attributed to arbovirus infection is increasing throughout the world, with effective control interventions limited by issues of sustainability, insecticide resistance and the lack of effective vaccines. Several promising control strategies are currently under development, such as the release of mosquitoes trans-infected with virus-blocking Wolbachia bacteria. Implementation of any control program is dependent on effective virus surveillance and a thorough understanding of virus-vector interactions. Massively parallel sequencing has enormous potential for providing comprehensive genomic information that can be used to assess many aspects of arbovirus ecology, as well as to evaluate novel control strategies. To demonstrate proof-of-principle, we analyzed Aedes aegypti or Aedes albopictus experimentally infected with dengue, yellow fever or chikungunya viruses. Random amplification was used to prepare sufficient template for sequencing on the Personal Genome Machine. Viral sequences were present in all infected mosquitoes. In addition, in most cases, we were also able to identify the mosquito species and mosquito micro-organisms, including the bacterial endosymbiont Wolbachia. Importantly, naturally occurring Wolbachia strains could be differentiated from strains that had been trans-infected into the mosquito. The method allowed us to assemble near full-length viral genomes and detect other micro-organisms without prior sequence knowledge, in a single reaction. This is a step toward the application of massively parallel sequencing as an arbovirus surveillance tool. It has the potential to provide insight into virus transmission dynamics, and has applicability to the post-release monitoring of Wolbachia in mosquito populations.     

Thioautotrophic ectosymbiosis in Pseudovorticella sp., a peritrich ciliate species colonizing wood falls in marine mangrove

Ciliates represent a diversified group of protists known to establish symbioses with prokaryotic micro-organisms. They are mainly phagotrophs and symbiotic relationships with bacteria can give them an important advantage in chemosynthetic environments. The aim of this study is to describe the thiotrophic association that occurs between the peritrich ciliate Pseudovorticella sp. and potential sulfur-oxidizing bacteria. Investigations at microscopic scale (LM, SEM, TEM) showed ectosymbiotic bacteria covering the surface of the body of Pseudovorticella sp. According to 16S rDNA phylogenetic analysis, these ectosymbiotic bacteria belong to γ-proteobacteria and are phylogenetically close to the symbiont of the recently described Zoothamnium ignavum, which inhabits shallow-water wood falls. FISH experiments, using symbiont specific probes, clearly indicate that these ectosymbiotic bacteria are also ingested into food vacuoles. Electron lucent granules observed in TEM in the cytoplasm of the ectosymbiotic bacteria have been identified as sulfur granules by Raman microspectrometry analyses. Raman microspectrometry analyses confirmed the thiotrophic nature of this relationship already suggested by the results obtained by TEM and phylogeny. A complete sulfur map was then performed to investigate the sulfur distribution in the zooid. Results show that the relationship between this protist and its bacterial partner is a thiotrophic ectosymbiosis.     

Interactions of Candida albicans with other Candida spp. and bacteria in the biofilms

AIMS: To study the interactions between Candida albicans and 12 other species of Candida and bacteria in biofilms. METHODS AND RESULTS: The number of cells within growing biofilms in a polystyrene tube model was measured after adding C. albicans to preformed biofilms of other micro-organisms and vice versa. It was also measured after simultaneous biofilm formation of C. albicans and other micro-organisms. The number of cells of C. albicans within the growing biofilms decreased significantly (P < 0.05) when the fungus was added to preformed biofilms of Candida spp. and bacteria except, with C. parapsilosis, Torulopsis glabrata and the glycocalyx producer Pseudomonas aeruginosa. When C. parapsilosis, Staphylococcus epidermidis (nonglycocalyx producer) or Serratia marcescens was added to preformed biofilms of C. albicans, the number of cells of these micro-organisms increased in the growing biofilms. CONCLUSIONS: Biofilms of C. albicans are capable of holding other micro-organisms and more likely to be heterogeneous with other bacteria and fungi in the environment and on medical devices. SIGNIFICANCE AND IMPACT OF THE STUDY: Recognition of the heterogeneity of biofilm-associated organisms can influence treatment decisions, particularly in patients who do not respond to initial appropriate therapy.     

The significance of the incorporation of [14C]leucine into different protein fractions by isolated ox heart mitochondria

The problem of whether isolated mitochondria are able to synthesize specific proteins was investigated, particular consideration being paid to the possible contribution of micro-organisms to this activity. With ox heart mitochondria it was shown that: (1) The medium used for the incubations inhibits the exponential phase of bacterial growth for at least 8h either in the absence or the presence of fresh mitochondria, but the inhibition disappears after 4h when mitochondria damaged by freezing and thawing are used. (2) The incorporation of [¹⁴C]leucine into total proteins is linear up to at least 8h, although part of the radioactivity at the later periods might be due to some incorporation by resting-phase bacteria. (3) A contamination by as little as 800 cells/mg of mitochondrial protein is enough to contribute substantially to the total radioactivity incorporated by the mitochondrial preparations. (4) Purified cytochrome b and cytochrome oxidase are labelled even under conditions of minimal contamination by micro-organisms (less than 60 cells/mg of mitochondrial protein) and the contribution of bacterial proteins to the radioactivity found in cytochromes is negligible, as shown by double-labelling experiments. (5) At 4h the specific radioactivities of cytochrome b and cytochrome oxidase are seven- and 16-fold lower respectively than that of a structural protein-rich fraction, suggesting that the labelling of cytochromes is due to a residual contamination by these proteins.     

Enhanced photosynthetic output via dichroic beam-sharing

Microbial solar biofuels offer great promise for future sustainable food, fuels and chemicals but are limited by low productivities and a requirement for large land areas to harvest sunlight. A 71?% increase in combined photosynthetic activity was achieved by illuminating both Rhodobacter sphaeroides and Arthrospira (Spirulina) platensis from a single beam of simulated sunlight, divided using a dichroic mirror. Therefore, this technique is termed ‘dichroic beam-sharing’, in which the complementary action spectra of two different useful micro-organisms, belonging to green and purple groups, is exploited and allows a single beam of sunlight to be shared efficiently between separate photobioreactors. Because the action spectra of these two organisms are typical of large groups, this novel method could increase the productivity of photosynthetic micro-organisms in the production of diverse commodities.     

<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> antibiotic resistance identified by atomic force microscopy

In the last decade the detection of the resistance of bacteria to antibiotics treatment, developed by different kind of bacteria, is becoming a huge problem. We hereby present a different approach to the current problem of detection of bacteria resistance to antibiotics. Our aims were to use the atomic force microscopy (AFM) to investigate bacteria morphological changes in response to antibiotics treatment and explore the possibility of reducing the time required to obtain information on their resistance. In particular, we studied Klebsiella pneumoniae bacteria provided by the Lavagna Hospital ASL4 Liguria (Italy), where there are cases linked with antibiotics resistance of the Klebsiella pneumoniae. By comparing AFM images of bacteria strains treated with different antibiotics is possible to identify unambiguously the Klebsiella pneumoniae strains resistant to antibiotics. In fact, the analysis of the AFM images of the antibiotic-sensitive bacteria shows clearly the presence of morphological alterations in the cell wall. While in the case of the antibiotic-resistant bacteria morphological alterations are not present. This approach is based on an easy and potentially rapid AFM analysis.     

Genetic and Morphological Variation of Two Rare Species of Lepidium (Brassicaceae) on Mangere Island,Chatham Islands,and Implications for Their Conservation Management

The genus Lepidium in New Zealand comprises a number of threatened and endemic species. Two recently described species, Lepidium panniforme and L. oblitum, have a restricted distribution on the remote and difficult to access Mangere Island, Chatham Islands. We examined the genetic and morphological variation of L. panniforme and L. oblitum on Mangere Island to assess the best conservation management options. Amplified fragment length polymorphism (AFLP) data and morphological data were used to detect finer scale variation in L. panniforme and L. oblitum. Bayesian analysis of AFLP genotypes detected up to four genetic clusters on Mangere Island. The morphological data were also variable, although better reflected the taxonomic treatment of the Mangere Island plants. The conservation implications of such high genetic and morphological variation over such a small spatial scale are discussed, to propose the best method for ensuring the ongoing survival of these island endemics.     

ExperimentalEscherichia coli pyelonephritis: Formation of spheroplasts in the kidney of the untreated rat

An experimental hematogenous pyelonephritis produced in the rat byEscherichia coli was studied from the point of view of the possible rôle of L-forms of bacteria in chronic renal infection. Osmotically fragile cells were shown in the kidneys from the 3rd day after inoculation, and their percentage of the totalE. coli cell number increased during the course of the infection. In order to distinguish bacteria and spheroplasts of theE. coli strain used, two more methods were elaborated: 1) a bacteriological method based on the different sensitivity of bacteria and spheroplasts to erythromycin; this method confirmed the results obtained with osmotic shock; 2) an immunological method, using the indirect fluorescent antibody technique and an antiserum specifically labelling the spheroplasts; in kidneys removed 10–15 days after the inoculation, spherical bodies with morphological and antigenic characteristics similar to those of spheroplasts became visible.     

Comparison of different DNA-extraction techniques to investigate the bacterial community of marine copepods

Marine zooplanktic organisms, such as copepods, are usually associated with large numbers of bacteria. Some of these bacteria live attached to copepods’ exoskeleton, while others prevail in their intestine and faecal pellets. Until now, general conclusions concerning the identity of these bacteria are problematic since the majority of previous studies focused on cultivable bacteria only. Hence, to date little is known on whether copepod genera or species harbour distinct bacterial populations and about the nature of this association. To shed more light on these copepod/bacteria consortia, the focus of this study was the development and evaluation of a suitable approach to extract bacterial DNA from different North Sea copepod genera. Furthermore, the bacterial DNA was analysed by PCR-DGGE and subsequent sequencing of excised bands. The result of this work was an appropriate extraction method for batches of ten to one copepod specimens and offered first insights as to which bacteria are attached to the copepods Acartia sp. and Temora sp. from Helgoland Roads (German Bight) and a laboratory-grown Acartia tonsa culture. It revealed the prevalence of Alphaproteobacteria.     

Persistence and Growth of Fecal Bacteroidales Assessed by Bromodeoxyuridine Immunocapture

Extraintestinal growth of fecal bacteria can impair accurate assessment of watershed health. Anaerobic fecal bacteria belonging to the order Bacteroidales are attractive candidates for fecal source tracking because they have host-specific distributions and do not grow well in the presence of high oxygen concentrations. Growth of general and human-specific fecal Bacteroidales marker organisms in environmental samples (sewage) and persistence of the corresponding genetic markers were investigated using bromodeoxyuridine (BrdU) DNA labeling and immunocapture, followed by PCR detection. Background amplification of unlabeled controls occasionally occurred when a high number of PCR cycles was used. By using fluorescent detection of PCR products obtained after 15 cycles, which was determined to be quantitative, we enriched for BrdU-labeled DNA and did not detect unlabeled DNA. By using pure cultures of Bacteroides vulgatus, the ability of Bacteroidales bacteria to take up and incorporate BrdU into nascent DNA was confirmed. Fecal Bacteroidales organisms took up and incorporated BrdU into DNA during growth. In sewage incubated aerobically at the in situ temperature, Bacteroidales genetic marker sequences persisted for at least 24 h and Bacteroidales fecal bacteria grew for up to 24 h as well. Detection by PCR using a low, quantitative cycle number decreased the sensitivity of the assay such that we were unable to detect fecal Bacteroidales human-specific marker sequences in unlabeled or BrdU-labeled fractions, even when fluorescent detection was used. Using 30 PCR cycles with unlabeled fractions, human-specific Bacteroidales sequences were detected, and they persisted for up to 24 h in sewage. These data support the utility of BrdU labeling and immunocapture followed by length heterogeneity PCR or fluorescent detection using low numbers of PCR cycles. However, this method may not be sensitive enough to identify cells that are present at low densities in aquatic environments.     

Viruses in bottom sediments of a Mesotrophic Reservoir (Rybinsk Reservoir,Upper Volga)

The total number and morphological and size composition of viriobenthos, number of bacteria infected by viruses, and burst size, as well as virus-induced mortality, abundance, and production of bacteriobenthos, have been estimated in bottom sediments of the Rybinsk Reservoir. The total number of viriobenthos in the reservoir varies within (1.1–10.9) × 10⁹ (on average, (5.9 ± 0.6) × 10⁹) particles/cm³; the total number of virus-to-bacteria ratio ranges within 0.2–2.1 (on average 0.9 ± 0.3). A weak positive correlation is found between the abundance of benthic viruses and the abundance and production of benthic bacteria. In most surveyed parts of the reservoir, infected benthic bacteria were not found or the portion of visibly infected bacterial cells (FVIC) did not exceed 0.5% of the total abundance of bacteriobenthos (N _B). A comparatively high infection of bacteria by viruses was recorded in bottom sediments only at one deep-water station, where FVIC was 2.5 of N _B.