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The two fungal human pathogens, Candida auris and Candida albicans, possess a variety of virulence mechanisms. Among them are the formation of biofilms to protect yeast against harsh conditions through the development of (pseudo)hyphae whilst also facilitating the invasion of host tissues. In recent years, increased rates of antifungal resistance have been associated with C. albicans and C. auris, posing a significant challenge for the effective treatment of fungal infections. In the course of our ongoing search for novel anti-infectives, six selected azaphilones were tested for their cytotoxicity and antimicrobial effects as well as for their inhibitory activity against biofilm and hyphal formation. This study revealed that rubiginosin C, derived from stromata of the ascomycete Hypoxylon rubiginosum, effectively inhibited the formation of biofilms, pseudohyphae, and hyphae in both C. auris and C. albicans without lethal effects. Crystal violet staining assays were utilized to assess the inhibition of biofilm formation, while complementary microscopic techniques, such as confocal laser scanning microscopy, scanning electron microscopy, and optical microscopy, were used to investigate the underlying mechanisms. Rubiginosin C is one of the few substances known to effectively target both biofilm formation and the yeast-to-hyphae transition of C. albicans and C. auris within a concentration range not affecting host cells, making it a promising candidate for therapeutic intervention in the future. [ABSTRACT FROM AUTHOR]

Basic processes of the fatty acid metabolism have an important impact on the function of intestinal epithelial cells (IEC). However, while the role of cellular fatty acid oxidation is well appreciated, it is not clear how de novo fatty acid synthesis (FAS) influences the biology of IECs. We report here that interfering with de novo FAS by deletion of the enzyme Acetyl-CoA-Carboxylase (ACC)1 in IECs results in the loss of epithelial crypt structures and a specific decline in Lgr5+ intestinal epithelial stem cells (ISC). Mechanistically, ACC1-mediated de novo FAS supports the formation of intestinal organoids and the differentiation of complex crypt structures by sustaining the nuclear accumulation of PPARδ/β-catenin in ISCs. The dependency of ISCs on cellular de novo FAS is tuned by the availability of environmental lipids, as an excess delivery of external fatty acids is sufficient to rescue the defect in crypt formation. Finally, inhibition of ACC1 reduces the formation of tumors in colitis-associated colon cancer, together highlighting the importance of cellular lipogenesis for sustaining ISC function and providing a potential perspective to colon cancer therapy. Here the authors report that inhibition of de novo fatty acid synthesis by deleting the enzyme Acetyl-CoA-Carboxylase 1 in the intestinal epithelium results in the loss of crypt structures and a specific decline in Lgr5+ intestinal epithelial stem cells. [ABSTRACT FROM AUTHOR]

Fungi are present in the most diverse environments including the interior of plant tissues, living as endophytes without causing apparent damage. These endophytes are producers of secondary metabolites, also known as natural products, such as fungicides. Here, we evaluated the ethyl acetate fractions obtained from endophytic fungi isolated from plants in the genus Begonia. The fractions were submitted to inhibitory test against the plant pathogens Diaporthe phaseolorum and Colletotrichum gloeosporioides. From the 88 ethyl acetate fractions evaluated, 14.7 % inhibited C. gloeosporioides and 11.3 % inhibited D. phaseolorum. One fungal isolate displaying an active fraction was selected for chemical investigation. The fungus identified as Neopestalotiopsis sp., produced a compound that was active against D. phaseolorum, with a MIC of 312 µg mL-1 (1,695.3 µM). The compound was identified by mass spectrometry and ¹H NMR as the known compound fumiquinone B. The results highlight that the endophytes are capable of producing compounds that may be used to control plant pathogens. The compound fumiquinone B is reported for the first time as an antifungal agent against D. phaseolorum, a relevant plant pathogen worldwide. This is also the first report of the production of fumiquinone B by the genus Neopestalotiopsis. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, is able to efficiently manipulate the host immune system establishing chronic infection, yet the underlying mechanisms of immune evasion are not fully understood. Evidence suggests that this pathogen interferes with host cell lipid metabolism to ensure its persistence. Fatty acid metabolism is regulated by acetyl-CoA carboxylase (ACC) 1 and 2; both isoforms catalyze the conversion of acetyl-CoA into malonyl-CoA, but have distinct roles. ACC1 is located in the cytosol, where it regulates de novo fatty acid synthesis (FAS), while ACC2 is associated with the outer mitochondrial membrane, regulating fatty acid oxidation (FAO). In macrophages, mycobacteria induce metabolic changes that lead to the cytosolic accumulation of lipids. This reprogramming impairs macrophage activation and contributes to chronic infection. In dendritic cells (DCs), FAS has been suggested to underlie optimal cytokine production and antigen presentation, but little is known about the metabolic changes occurring in DCs upon mycobacterial infection and how they affect the outcome of the immune response. We therefore determined the role of fatty acid metabolism in myeloid cells and T cells during Mycobacterium bovis BCG or Mtb infection, using novel genetic mouse models that allow cell-specific deletion of ACC1 and ACC2 in DCs, macrophages, or T cells. Our results demonstrate that de novo FAS is induced in DCs and macrophages upon M. bovis BCG infection. However, ACC1 expression in DCs and macrophages is not required to control mycobacteria. Similarly, absence of ACC2 did not influence the ability of DCs and macrophages to cope with infection. Furthermore, deletion of ACC1 in DCs or macrophages had no effect on systemic pro-inflammatory cytokine production or T cell priming, suggesting that FAS is dispensable for an intact innate response against mycobacteria. In contrast, mice with a deletion of ACC1 specifically in T cells fail to generate efficient T helper 1 responses and succumb early to Mtb infection. In summary, our results reveal ACC1-dependent FAS as a crucial mechanism in T cells, but not DCs or macrophages, to fight against mycobacterial infection. [ABSTRACT FROM AUTHOR]

Eremophilanes are sesquiterpenes with a rearranged carbon skeleton formed both by plants and fungi, however, almost no plant eremophilanes are found in fungi. These eremophilanes possess mainly phytotoxic, antimicrobial, anticancer and immunomodulatory properties and in this review fungal eremophilanes with bioactivities of potential medicinal applications are reviewed and discussed. A special focus is set on natural products bearing highly functionalized fatty acids at C-1 or C-3 position of the eremophilane backbone. Many of these fatty acids seem to contribute to the bioactivity of the metabolites enhancing the activity of the sesquiterpene moieties. Several approaches for optimization of these natural products for clinical needs and testing of the resulting derivatives are presented and discussed. The combination of identification of bioactive natural products with their subsequent improvement using a variety of genetical or chemical tools and the pharmacokinetic assessment of the products is presented here as a promising approach to new drugs. [ABSTRACT FROM AUTHOR]

Treating infections organized in biofilms is a challenge due to the resistance of the pathogens against antibiotics and host immune cells. Many fungi grow in a wet environment, favorable for the growth of bacterial biofilms, and we speculated that fungi possess some strategies to control these bacterial biofilms. A fungus identified as Hypoxylon fragiforme, was collected in the Harz Mountains, Germany, and its mycelial culture was fermented in different culture media for 67 days to test its biological potential against bacterial biofilms. Sclerin, sclerin diacid and its 3-methyl monoester (methyl 1-(5-hydroxy-6-carboxylic-2,3,4-trimethylphenyl) propionate) are here described for the first time from this fungus. Sclerin and its diacid interfered with the biofilm formation of the pathogen Staphylococcus aureus, inhibiting 86% and 80% of the biofilm at 256 μg mL-1, respectively, but not killing the bacterium. Interestingly, the monomethylester of sclerin diacid was inactive. Although these compounds did not possess any activity against a pre-formed biofilm, they prevented its formation at subtoxic concentrations. Furthermore, sclerin and its diacid displayed a high specificity against Staphylococcus aureus, indicating a good strategy against pathogenic biofilms when combined with antibiotics. [ABSTRACT FROM AUTHOR]

Pathogens embedded in biofilms are involved in many infections and are very difficult to treat with antibiotics because of higher resistance compared with planktonic cells. Therefore, new approaches for their control are urgently needed. One way to search for biofilm dispersing compounds is to look at defense strategies of organisms exposed to wet environments, which makes them prone to biofilm infections. It is reasonable to assume that mushrooms have developed mechanisms to control biofilms on their sporocarps (fruiting bodies). A preliminary screening for biofilms on sporocarps revealed several species with few or no bacteria on their sporocarps. From the edible mushroom Coprinus comatus where no bacteria on the sporocarp could be detected (3R,4S)-2-methylene-3, 4-dihydroxypentanoic acid 1,4-lactone, named coprinuslactone, was isolated. Coprinuslactone interfered with quorum-sensing and dispersed biofilms of Pseudomonas aeruginosa, where it also reduced the formation of the pathogenicity factors pyocyanin and rhamnolipid B. Coprinuslactone also damaged Staphylococcus aureus cells in biofilms at subtoxic concentrations. Furthermore, it inhibited UDP-Nacetylglucosamine enolpyruvyl transferase (MurA), essential for bacterial cell wall synthesis. These two modes of action ensure the inhibition of a broad spectrum of pathogens on the fruiting body but may also be useful for future clinical applications. [ABSTRACT FROM AUTHOR]

γ-Hexachlorocyclohexane (γ-HCH) persists in the environment and is recalcitrant to microbial degradation. To determine the extent of the microbial potential for the degradation of γ-HCH the diversity of bacteria from 12 soil samples collected around insecticide- and pesticide-producing factories in Egypt were assessed and compared with biofilm communities grown on γ-HCH microcrystals. From all samples, highly diverse microbes were isolated, able to grow on γ-HCH as sole source of carbon. The same soil samples were used to inoculate γ-HCH microcrystals on a substratum in microcosms to grow biofilm communities. All soil samples formed multispecies biofilms on γ-HCH. Biofilms stained with Nile Red showed distinct cell clusters of high hydrophobicity, and it is speculated that these aggregates have a substantial role in the degradation of the hydrophobic substrate. While many Bacillus species were isolated, this group was almost absent in the different biofilm communities. The finding of cells with highly hydrophobic envelopes together with the differences in species composition between isolates and interacting microbial communities points to fundamental differences in the interaction with hydrophobic substrates of single strains and microbial communities. [ABSTRACT FROM AUTHOR]

Most bacteria attach to surfaces where they form a biofilm, cells embedded in a complex matrix of polymers. Cells in biofilms are much better protected against noxious agents than free-living cells. As a consequence it is very difficult to control pathogens with antibiotics in biofilm infections and novel targets are urgently needed. One approach aims at the communication between cells to form and to maintain a biofilm, a process called quorum-sensing. Water soluble small-sized molecules mediate this process and a number of antagonists of these compounds have been found. In this review natural compounds and synthetic drugs which do not interfere with the classical quorum-sensing compounds are discussed. For some of these compounds the targets are still not known, but others interfere with the formation of exopolysaccharides, virulence factors, or cell wall synthesis or they start an internal program of biofilm dispersal. Some of their targets are more conserved among pathogens than the receptors for quorum sensing autoinducers mediating quorum-sensing, enabling a broader application of the drug. The broad spectrum of mechanisms, the diversity of bioactive compounds, their activity against several targets, and the conservation of some targets among bacterial pathogens are promising aspects for several clinical applications of this type of biofilm-controlling compound in the future. [ABSTRACT FROM AUTHOR]

Previous work by our group described that human β-defensin-2 induces accumulation of extracellular adenosine (Ado) in E. coli cultures through a non-lytic mechanism causing severe plasmolysis. Here, we investigate the presence of AMP as a direct precursor and the involvement of a bacterial enzyme in the generation of extracellular Ado by treated bacteria. Following hBD-2 treatment, metabolites were quantified in the supernatants using targeted HPLC-MS/MS analysis. Microbial growth was monitored by optical density and cell viability was determined by colony forming units counts. Phosphatase activity was measured using chromogenic substrate pNPP. The results demonstrate that defensin-treated E. coli strain W releases AMP in the extracellular space, where it is converted to Ado by a bacterial soluble factor. An increase in phosphatase activity in the supernatant was observed after peptide treatment, similar to the effect of sucrose-induced osmotic stress, suggesting that the periplasmic 5'nucleotidase (5'-NT) is released following the plasmolysis event triggered by the peptide. Ado accumulation was enhanced in the presence of Co2+ ion and inhibited by EDTA, further supporting the involvement of a metallo-phosphatase such as 5’-NT in extracellular AMP conversion into Ado. The comparative analysis of hBD-induced Ado accumulation in different E. coli strains and in Pseudomonas aeruginosa revealed that the response is not correlated to the peptide's effect on cell viability, but indicates it might be dependent on the subcellular distribution of the nucleotidase. Taken together, these data shed light on a yet undescribed mechanism of host-microbial interaction: a human antimicrobial peptide inducing selective release of a bacterial enzyme (E. coli 5'-NT), leading to the formation of a potent immunomodulator metabolite (Ado). [ABSTRACT FROM AUTHOR]

Interleukin-17 (IL-17)-secreting T cells of the T helper 17 (TH17) lineage play a pathogenic role in multiple inflammatory and autoimmune conditions and thus represent a highly attractive target for therapeutic intervention. We report that inhibition of acetyl-CoA carboxylase 1 (ACC1) restrains the formation of human and mouse TH17 cells and promotes the development of anti-inflammatory Foxp3+ regulatory T (Treg) cells. We show that TH17 cells, but not Treg cells, depend on ACC1-mediated de novo fatty acid synthesis and the underlying glycolytic-lipogenic metabolic pathway for their development. Although TH17 cells use this pathway to produce phospholipids for cellular membranes, Treg cells readily take up exogenous fatty acids for this purpose. Notably, pharmacologic inhibition or T cell-specific deletion of ACC1 not only blocks de novo fatty acid synthesis but also interferes with the metabolic flux of glucose-derived carbon via glycolysis and the tricarboxylic acid cycle. In vivo, treatment with the ACC-specific inhibitor soraphen A or T cell-specific deletion of ACC1 in mice attenuates TH17 cell-mediated autoimmune disease. Our results indicate fundamental differences between TH17 cells and Treg cells regarding their dependency on ACC1-mediated de novo fatty acid synthesis, which might be exploited as a new strategy for metabolic immune modulation of TH17 cell-mediated inflammatory diseases. [ABSTRACT FROM AUTHOR]

Some intracellular bacteria are known to cause long-term infections that last decades without compromising the viability of the host. Although of critical importance, the adaptations that intracellular bacteria undergo during this long process of residence in a host cell environment remain obscure. Here, we report a novel experimental approach to study the adaptations of mycobacteria imposed by a long-term intracellular lifestyle. Selected M ycobacterium bovis BCG through continuous culture in macrophages underwent an adaptation process leading to impaired phenolic glycolipids ( PGL) synthesis, improved usage of glucose as a carbon source and accumulation of neutral lipids. These changes correlated with increased survival of mycobacteria in macrophages and mice during re-infection and also with the specific expression of stress- and survival-related genes. Our findings identify bacterial traits implicated in the establishment of long-term cellular infections and represent a tool for understanding the physiological states and the environment that bacteria face living in fluctuating intracellular environments. [ABSTRACT FROM AUTHOR]

Probing the interactions between microbes and their environment with stable isotopes became a powerful technique over the last years. While quadruple mass spectrometry or isotope ratio mass spectrometry (IRMS) require at least 300,000 bacterial cells, analysis at the single-cell level is possible with secondary ion mass spectrometry (SIMS) or Raman microspectrometry. While SIMS needs enrichments of more than 0.1 and Raman microscopy of more than 25 at.-%, IRMS can deal with 0.0001 at.-%. To find out who eats what, one has to discern between the different species in a community. Several methods have been introduced to discern between the different taxa in microbial communities, e.g., by using fatty acids as biomarkers, density centrifugation of DNA/RNA, or fluorescent in situ hybridization (FISH) with phylogenetic probes. While the biomarker approach can be coupled with the high sensitivity of the IRMS, the DNA approach gives in general a better phylogenetic resolution of the metabolic active microbes. A combination of both is the separation via coupling of FISH-probes to magnetic beads or fluorescent assisted cell sorting (FACS) of stained cells leading to fractions which can be analyzed by IRMS. Applying these techniques over a time course can reveal the metabolic kinetics and food webs. In this review, the different methods are presented with examples and their advantages and disadvantages are discussed. An outlook on the combination of the various techniques and their applications in microbial ecology is given. [ABSTRACT FROM AUTHOR]

Microbial lipases are widely used in industrial applications due to their versatility, and the characterization of new lipase-producing microorganisms could provide new sources of these enzymes, with different specificities and better activities. In this context, we have improved lipase production by Luteibacter sp. by using basal medium supplemented with 2 % olive oil, a pH of 6 and a growth temperature of 37 °C. The enzyme extraction process with the addition of 0.25 % Tween 80 increased lipase activity. Implementation of these modifications increased lipase activity by approximately 430 %. The lipase activities produced in the culture supernatant (LCS) and extracted with Tween 80 (LCST80) were characterized. Both extracts hydrolyzed ρ-nitrophenyl (ρNP) esters with different acyl chain lengths, with a preference for short acyl lengths, and had optimum activity at 45 °C. The LCS was stable at acidic and alkaline pH, but LCST80 was only stable at alkaline pH. Methanol, SDS, Triton X-100, EDTA, and EGTA did not affect lipase activity, while divalent cations (Ca, Zn, Mg) - with the exception of Co- increased lipase activity. Both extracts showed transesterification activity on ρNP ester substrates, and both were able to hydrolyze different natural lipids. The characterization of lipase produced by Luteibacter sp. introduces this recently described genus as a new source of lipases with great biotechnological potential. [ABSTRACT FROM AUTHOR]

Pelagic redoxclines of the central Baltic Sea are dominated by the epsilonproteobacterial group Sulfurimonas GD17, considered to be the major driver of chemolithoautotrophic denitrification in this habitat. Autecological investigations of a recently isolated representative of this environmental group, Sulfurimonas gotlandica str. GD1T, demonstrated that the bacterium grows best under sulfur-oxidizing, denitrifying conditions. However, in the presence of bicarbonate, this strain is able to use pyruvate as both an additional carbon source and an alternative electron donor. These observations suggested that the environmental group GD17 actively metabolizes organic substrates in situ. To examine this possibility, we used RNA-based stable isotope probing ( RNA- SIP) on a natural redoxcline community provided with 13C-labeled pyruvate. While in this experiment, we were able to identify putative heterotrophic microorganisms, the uptake of 13C-pyruvate in GD17 nucleic acids could not be established. To resolve these contradictory findings, combined incorporation experiments with 14C- and 13C-labeled pyruvate were carried out in cells of strain GD1T cultivated under chemolithoautotrophic conditions, which favor pyruvate uptake rather than oxidation. An analysis of the labeled biomolecules revealed that pyruvate was mostly incorporated in cellular components such as amino acids, whose synthesis requires only minimal transformation. Carbon transfer into nucleic acids was not observed, explaining the inability of RNA- SIP to detect pyruvate incorporation by strain GD1T and the environmental group GD17. Together, these findings suggest that by integrating organic compounds such as pyruvate into cellular components S. gotlandica GD1T is able to replenish chemolithoautotrophic growth and thus ensure its survival in nutrient-limited habitats such as marine pelagic redoxclines. [ABSTRACT FROM AUTHOR]

The cattle tick Rhipicephalus ( Boophilus) microplus lays eggs in the soil near the roots of grass, or in similar highly moist environments that are prone to biofilm formation. Tick eggs have a protective wax coating that may be a source of nutrients for microorganisms. However, as the eggs remain viable and show no visible signs of microbial colonization, we hypothesized that the coating might have anti-biofilm properties. We show here that the coating inhibits biofilm formation by both Gram-negative and Gram-positive bacteria, though by different mechanisms. We have identified the anti-biofilm molecule as N-(3-sulfooxy-25-cholest-5-en-26-oyl)- L-isoleucine (boophiline), and we show that it inhibits the expression of fliC (flagellin) and cdrA (biofilm scaffold), whose products are necessary for biofilm formation in Pseudomonas aeruginosa. Boophiline is a novel biofilm inhibitor being also effective against Staphylococcus epidermidis biofilm. In our study we show evidences of the boophiline mode of action in the protection of arthropod eggs against biofilm colonization. [ABSTRACT FROM AUTHOR]

BACKGROUND: Asymptomatic bacterial colonisation of medical implants is increasingly recognised. The role of the often unusual bacteria is usually unknown however. OBJECTIVE AND METHODS: During a three-year period, patients presenting with clinically evident CIED lead endocarditis and/or pocket infections were included in a prospective database. Using 16s DNA analysis and sequencing, devices and pockets of consecutive patients with evident CIED infection were examined for the presence of bacterial species in addition to the ones grown in conventional culture in order to characterise their clinical role. RESULTS: During the study 77 of a total of 409 consecutive operations involving the explantation of a CIED were performed for clinically evident infection. 71 were included in the study and conventional extended culture and 16s DNA analysis performed and compared. In 42.3% of the patients bacteria were identified by DNA analysis in addition to the culture of the supposedly causative organisms. CONCLUSION: DNA analysis is more sensitive than conventional culture of swabs in detecting any, or indeed multiple bacterial strains in CIED infection. This may in future influence treatment strategies as e.g. the cohabitation of different strains seems common and is not reflected by classical culture results. Spread of infection along the leads was clearly demonstrated and this strongly supports the recommendation of complete removal of all CIED components in every form of CIED infection. The more unusual bacteria demonstrated here do not appear to play a significant clinical role as suspected earlier. [ABSTRACT FROM AUTHOR]

Fungi can be found in almost all sorts of habitats competing with an even higher number of other organisms. As a consequence fungi developed a number of strategies for protection and communication with other organisms. This review focuses on the increasing number of volatile sesquiterpenes found to be produced by fungal species. The remarkable diversity of this type of volatile organic compound (VOC) within the kingdom fungi is presented and their benefits for the fungi are discussed. The majority of these compounds are hydrocarbons comprising several dozens of carbon skeletons. Together with oxygenated sesquiterpenes they include compounds unique to fungi. Only in recent years the interest shifted from a mere detection and characterization of compounds to their biological function. This review reveals highly diverse ecological functions including interactions with bacteria, other fungi, insects and plants. VOCs act as autoinducer, defend against competing species and play essential roles in attracting pollinators for spreading fungal spores. For many sesquiterpene VOCs sophisticated responses in other organisms have been identified. Some of these interactions are complex involving several partners or transformation of the emitted sesquiterpene. A detailed description of ecological functions of selected sesquiterpenes is given as well as their potential application as marker molecules for detection of mould species. Structures of all described sesquiterpenes are given in the review and the biosynthetic routes of the most common skeletons are presented. Summarizing, this article provides a detailed overview over the current knowledge on fungal sesquiterpene VOCs and gives an outlook on the future developments. [ABSTRACT FROM AUTHOR]

Poor sanitation, poor treatments of waste water, as well as catastrophic floods introduce pathogenic bacteria into rivers, infecting and killing many people. The goal of clean water for everyone has to be achieved with a still growing human population and their rapid concentration in large cities, often megacities. How long introduced pathogens survive in rivers and what their niches are remain poorly known but essential to control water-borne diseases in megacities. Biofilms are often niches for various pathogens because they possess high resistances against environmental stress. They also facilitate gene transfers of antibiotic resistance genes which become an increasing health problem. Beside biofilms, amoebae are carriers of pathogenic bacteria and niches for their survival. An overview about our current understanding of the fate and niches of pathogens in rivers, the multitude of microbial community interactions, and the impact of severe flooding, a prerequisite to control pathogens in polluted rivers, is given. [ABSTRACT FROM AUTHOR]

Marine pelagic redoxclines are zones of high dark CO2 fixation rates, which can correspond up to 30% of the surface primary production. However, despite this significant contribution to the pelagic carbon cycle, the identity of most chemolithoautotrophic organisms is still unknown. Therefore, the aim of this study was to directly link the dark CO2 fixation capacity of a pelagic redoxcline in the central Baltic Sea (Landsort Deep) with the identity of the main chemolithoautotrophs involved. Our approach was based on the analysis of natural carbon isotope signatures in fatty acid methyl esters (FAMEs) and on measurements of CO2 incorporation in 13C-bicarbonate pulse experiments. The incorporation of 13C into chemolithoautotrophic cells was investigated by rRNA-based stable isotope probing (RNA-SIP) and FAME analysis after incubation for 24 and 72 h under in situ conditions. Our results demonstrated that fatty acids indicative of Proteobacteria were significantly enriched in 13C slightly below the chemocline. RNA-SIP analyses revealed that two different Gammaproteobacteria and three closely related Epsilonproteobacteria of the Sulfurimonas cluster were active dark CO2-fixing microorganisms, with a time-dependent community shift between these groups. Labelling of Archaea was not detectable, but after 72 h of incubation the 13C-label had been transferred to a potentially bacterivorous ciliate related to Euplotes sp. Thus, RNA-SIP provided direct evidence for the contribution of chemolithoautotrophic production to the microbial food web in this marine pelagic redoxcline, emphasizing the importance of dark CO2-fixing Proteobacteria within this habitat. [ABSTRACT FROM AUTHOR]

Forest ecosystems assimilate more CO2 from the atmosphere and store more carbon in woody biomass than most nonforest ecosystems, indicating strong potential for afforestation to serve as a carbon management tool. However, converting grasslands to forests could affect ecosystem–atmosphere exchanges of other greenhouse gases, such as nitrous oxide and methane (CH4), effects that are rarely considered. Here, we show that afforestation on a well-aerated grassland in Siberia reduces soil CH4 uptake by a factor of 3 after 35 years of tree growth. The decline in CH4 oxidation was observed both in the field and in laboratory incubation studies under controlled environmental conditions, suggesting that not only physical but also biological factors are responsible for the observed effect. Using incubation experiments with 13CH4 and tracking 13C incorporation into bacterial phospholipid fatty acid (PLFA), we found that, at low CH4 concentrations, most of the 13C was incorporated into only two PLFAs, 18 : 1ω7 and 16 : 0. High CH4 concentration increased total 13C incorporation and the number of PLFA peaks that became labeled, suggesting that the microbial assemblage oxidizing CH4 shifts with ambient CH4 concentration. Forests and grasslands exhibited similar labeling profiles for the high-affinity methanotrophs, suggesting that largely the same general groups of methanotrophs were active in both ecosystems. Both PLFA concentration and labeling patterns indicate a threefold decline in the biomass of active methanotrophs due to afforestation, but little change in the methanotroph community. Because the grassland consumed CH4 at a rate five times higher than forest soils under laboratory conditions, we concluded that not only biomass but also cell-specific activity was higher in grassland than in afforested plots. While the decline in biomass of active methanotrophs can be explained by site preparation (plowing), inorganic N (especially NH4+) could be responsible for the change in cell-specific activity. Overall, the negative effect of afforestation of upland grassland on soil CH4 uptake can be largely explained by the reduction in biomass and to a lesser extent by reduced cell-specific activity of CH4-oxidizing bacteria. [ABSTRACT FROM AUTHOR]

Aims: Recent work has been focused on causes of and risk factors for rhythm management device infections. The aim of this study was to elucidate whether patients may be asymptomatic carriers of bacteria on their rhythm management device, possibly allowing later manifestation of infection. [ABSTRACT FROM PUBLISHER]

Methanotrophs in the rhizosphere play an important role in global climate change since they attenuate methane emission from rice field ecosystems into the atmosphere. Most of the CH4 is emitted via transport through the plant gas vascular system. We used this transport for stable isotope probing (SIP) of the methanotrophs in the rhizosphere under field conditions and pulse-labelled rice plants in a Chinese rice field with CH4 (99% 13C) for 7 days. The rate of 13CH4 loss rate during 13C application was comparable to the CH4 oxidation rate measured by the difluoromethane inhibition technique. The methanotrophic communities on the roots and in the rhizospheric soil were analyzed by terminal-restriction fragment length polymorphism (T-RFLP), cloning and sequencing of the particulate methane monooxygenase (pmoA) gene. Populations of type I methanotrophs were larger than those of type II. Both methane oxidation rates and composition of methanotrophic communities suggested that there was little difference between urea-fertilized and unfertilized fields. SIP of phospholipid fatty acids (PLFA-SIP) and rRNA (RNA-SIP) were used to analyze the metabolically active methanotrophic community in rhizospheric soil. PLFA of type I compared with type II methanotrophs was labelled more strongly with 13C, reaching a maximum of 6.8 atom-%. T-RFLP analysis and cloning/sequencing of 16S rRNA genes showed that methanotrophs, especially of type I, were slightly enriched in the ‘heavy’ fractions. Our results indicate that CH4 oxidation in the rice rhizosphere under in situ conditions is mainly due to type I methanotrophs.The ISME Journal (2008) 2, 602–614; doi:10.1038/ismej.2008.34; published online 3 April 2008 [ABSTRACT FROM AUTHOR]

Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH4 into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH4 flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using 13C-labelled CH4 directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to 13CH4 for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms. [ABSTRACT FROM AUTHOR]

The article presents a study that assesses the occurrence and resistance of pathogenic bacteria along the Tietê River Downstream of Sâo Paolo in Brazil. High concentrations of pathogens were found in the Tietê River near the city of Sâo Paolo, which were absent 30 km downstream. The antibiotic resistances observed in the river were rather low and decreased after the major input in Sâo Paolo to lower levels about 30 km downstream. It is observed that the Brazilian isolates were more susceptible to ampicillin. The reverse was observed for gentamycin.

The purpose of this work was to determine the extent of microbial metabolic potential for polychlorinated biphenyls (PCBs) in soils that have had no previous exposure to this class of xenobiotic pollutants. Soil and sediment samples of distinct characteristics from six sites in Germany were used to inoculate PCB oil (Aroclor 1242) microdroplets. All samples yielded multispecies biofilms, as revealed by single-strand conformation polymorphism (SSCP) analyses of polymerase chain reaction (PCR) analysis of 16S rRNA genes, and sequence analysis of the main amplicons. Microbes representing 20 different operational taxonomic units (OTUs) were identified in the biofilms, but only a few were common to all biofilms, namely those closely related to Aquabacterium sp., Caulobacter sp., Imtechium assamiensis, Nevskia ramosa, Parvibaculum lavamentivorans and Burkholderia sp. The PCB biofilm communities were always distinct from control biofilms developing from the same samples in the absence of PCB. All PCB droplet-grown biofilms degraded multiple PCB congeners but differed in the congener spectra they degraded. These findings reveal that microbial potential to degrade PCBs is widespread in soils that have not been subjected to PCB contamination, and that this potential is characteristic of consortia of very diverse phylogenetic composition. [ABSTRACT FROM AUTHOR]

This report is part of a serial study applying stable isotope labelling to rice microcosms to track the utilization of recently photosynthesized carbon by active microbiota in the rhizosphere. The objective of the present study was to apply phospholipid fatty acid-based stable isotope probing (PLFA-SIP) to detect the spatial variation of active microorganisms associated with rhizosphere carbon flow. In total, 49 pulses of 13CO2 were applied to rice plants in a microcosm over a period of 7 days. Rhizosphere soil was separated from bulk soil by a root bag. Soil samples were taken from rhizosphere and bulk soil, and the bulk soil samples were further partitioned both vertically (up layer and down layer) and horizontally with increasing distance to the root bag. Incorporation of 13C into PLFAs sharply decreased with distance to the roots. The labelling of 16:1ω9, 18:1ω7, 18:1ω9, 18:2ω6,9 and i14:0 PLFAs was relatively stronger in the rhizosphere while that of i15:0 and i17:0 increased in the bulk soil. The microorganisms associated with 16:1ω9 were active in both up- and down-layer soils. The microorganisms represented by i14:0, 18:1ω7 and 18:2ω6,9 exhibited a relatively higher activity in up-layer soil, whereas those represented by i15:0 and i17:0 were more active in down-layer soil. These results suggest that in the rhizosphere Gram-negative and eukaryotic microorganisms were most actively assimilating root-derived C, whereas Gram-positive microorganisms became relatively more important in the bulk soil. The active populations apparently differed between up- and down-layer soil and in particular changed with distance to the roots, demonstrating systematic changes in the activity of the soil microbiota surrounding roots. [ABSTRACT FROM AUTHOR]

Abstract: The naphthalene-degrading activity of a Pseudomonas sp. strain isolated from a creosote-contaminated soil was shown to be encoded by the IncP9 plasmid pNF142 by transfer to Pseudomonas putida KT2442. The effects of the inoculant strain KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community were studied in microcosms with the following treatments: (I) soil, (II) soil with naphthalene, (III) soil with naphthalene and inoculated with KT2442 (pNF142). The inoculant became the dominant bacterial population in treatment (III) as evidenced by cultivation and denaturing gradient gel electrophoresis (DGGE) analysis. The bacterial DGGE profiles revealed drastically reduced complexity due to the numerical dominance of the inoculant. However, group-specific fingerprints (β-proteobacteria, actinobacteria) that excluded KT2442 (pNF142) showed less severe changes in the bacterial community patterns. A major effect of naphthalene on the soil bacterial community was observed in treatment (II) after 21 days. Two dominant bands appeared whose sequences showed the highest similarity to those of Burkholderia sp. RP007 and Nocardia vinaceae based on 16S rRNA gene sequencing. These bands were less intense in treatment (III). The increased abundance of RP007-like populations due to naphthalene contamination was also confirmed by PCR amplification of the phnAc gene. The nahAc and nahH genes were detected in DNA and cDNA only in treatment III. Although the inoculant strain KT2442 (pNF142) showed good survival and expression of genes involved in naphthalene degradation, this study suggests that KT2442 (pNF142) suppressed the enrichment of indigenous naphthalene degraders. [Copyright &y& Elsevier]

The flow of new and native plant-derived C in the rhizosphere of an agricultural field during one growing season was tracked, the ratios in different soil C pools were quantified, and the residence times ( τs) were estimated. For this the natural differences in 13C abundances of: (1) C4 soil (with a history of C4 plant, Miscanthus sinensis, cultivation), (2) C3 soil (history of C3 plant cultivation), and (3) C4/3 soil (C4 soil, planted with a C3 plant, Triticum aestivum) were used. Total amounts and δ13C values of total soil C, non-hydrolysable C, light fraction C, water-soluble C, microbial biomass C, and phospholipid fatty acids (PLFA) were determined. Using the δ13C values of soil C in a mixing and a 1-box model enabled the quantification of relative contributions of C3 plant and C4 plant C to the total amount of the respective C pools in the C4/3 soil and their τs. Compared to early spring (March), the percentage of C3 plant C increased in all pools in June and August, showing the addition of new C to the different soil C fractions. In August the contribution of new C to microbial biomass C and water-soluble C reached 64 and 89%, respectively. The τs of these pools were 115 and 147 days. The δ13C values of the dominant soil PLFA, 18:1ω7c, cy19:0, 18:1ω9c, 16:0, and 10Me16:0, showed wide ranges (−35.1 to −13.0‰) suggesting that the microbial community utilized different pools as C sources during the season. The δ13C values of PLFA, therefore, enabled the analysis of the metabolically active populations. The majority of δ13C values of PLFA from the C4/3 soil were closely related to those of PLFA from the C3 soil when T. aestivum biomass contributions to the soil were high in June and August. Specific populations reacted differently to changes in environmental conditions and supplies of C sources, which reflect the high functional diversity of soil microorganisms. [ABSTRACT FROM AUTHOR]

Abstract: The active bacteria of a biofilm community grown directly on polychlorinated biphenyl (PCB) droplets were analyzed by 16S rRNA fingerprinting, identified by their 16S rRNA gene sequences and fatty acid profiling, and compared with isolates from the biofilm. Although, the multi-species biofilm degraded di- and trichlorinated PCB-congeners these substrates were not attacked by its individual isolated members, which suggests that a metabolic network is responsible for PCB degradation in the biofilm. The community metabolized [U-13C]-2,2′-dichlorobiphenyl incorporating the label into certain phospholipid fatty acids matching those found in Burkholderia species. In contrast, abundant biofilm community members, like Methylobacterium species, did not incorporate the label. These results provide prima faciae evidence for Burkholderia species as the main degraders of PCBs in this type of aerobic soils. [Copyright &y& Elsevier]

The salt composition of 14 acidic lakes was analysed, and their microbial communities were compared by their 16S rDNA-based single-strand conformation polymorphism (SSCP) fingerprints. The lakes were grouped into three chemically distinct types, and operational taxonomic units (OTUs) were identified in the 16S rDNA SSCP fingerprints. Discriminant analysis between these groups using a multivariate approach showed that not a single organism, but rather a set of seven OTUs (Ferromicrobium acidophilum,Bacillus pumilus,Acidophilium organovorum,Paenibacillus chibensis,Acidocellasp. and two different alpha-Proteobacteria), was found to be indicative for a given lake type. We conclude that, for these lakes, not single indicator strains but indicator groups exist that are controlled by the ionic composition of the lakes. [ABSTRACT FROM AUTHOR]

The isotope fractionation of carbon from substrates possessing different isotope ratios into fatty acids of polar lipids and amino acids was determined for four different fungi (Rhizopus arrhizus, Mortierella isabellina, Fusarium solani, Aspergillus niger). Carbon isotope ratios of fungi closely followed that of the substrates. Palmitic acid (C16:0), derived from phospholipids, did not display a large carbon isotope fractionation against the substrate. Stearic acid (C18:0), however, was depleted in 13C against C16:0 in all strains. The desaturation of C18:0 to oleic acid (C18:1ω9) had little effect on the carbon isotope ratio. The subsequent desaturation of C18:1ω9 to linolic acid (C18:2ω6,9) enriched the resulting C18:2ω6,9 by +3.9‰ and varied little among strains. This result is important because C18:2ω6,9 is often used as a biomarker in environmental studies. Most amino acids were enriched in 13C compared to the substrates, but isoleucine and lysine were close to the isotope ratio of the substrate and phenylalanine and leucine were depleted. Interestingly, the carbon isotope ratios of many amino acids differed significantly among different species. A discriminant analysis based on the isotope ratio of four amino acids (Thr, Ile, Phe, Val) resolved the two phyla in the first discriminant function and all four strains in the first two discriminant functions and confirmed a taxon-specific manner of isotope fractionation. The derived rules provide the basis for the use of stable isotopes in environmental studies to elucidate the role of fungi in the carbon flow in the environment. [Copyright &y& Elsevier]

A moorland soil site polluted with PCB showed a high diversity of metabolically active bacteria. Beside frequent types of 16S rRNA sequences similar to those of the species of Sphingomonas and the Acidobacterium phylum an unusual high number of sequences from the genus Burkholderia were found. Burkholderia was also the main genus in isolates enriched on biphenyl or various chlorobenzoates. In microcosm experiments sterilized surfaces exposed to PCB polluted soil always showed the presence of clay aggregates formed by bacteria attached to the substratum. The bacteria use the PCB loaded clay colloids as transport medium for the water insoluble substrate to get access to the carbon source. This is a novel mechanism of how bacteria deal with hydrophobic substrates. [ABSTRACT FROM AUTHOR]

Microbial consortia of composite biofilms, grown in surface water of acidic mining lakes near Lauchhammer, Germany, were investigated. The red-brown colored lake water was acidic (pH 2.5), had high concentrations of Fe(III), Al(III), and sulphate and low concentrations of dissolved organic matter. As a result the abundance of bacteria in the lake is with 104 cells mL-1 rather low. One input of organic material into the lake are autumnal leaves from trees, growing in the lakeside area. From aliquots of unfixed birch leave biofilms the 16S rRNA genes were amplified by PCR and community fingerprints were determined by single-strand conformation polymorphism (SSCP) analysis. Specific bands within the fingerprints were extracted from SSCP gels and sequenced for the taxonomical affiliation.These results were compared with those from the second type of biofilms which were grown on sterile substrata, floating submersed in surface waters of the lakes. By excising the bands from the gel and sequencing the individual bands bacterial taxa, common to both types of biofilms, were found but also some, which were only present in one type of biofilm. Ultrathin sectioned biofilms often showed bacteria associated with electron dense particles as main inorganic constituents. Elemental microanalysis by energy dispersive X-ray analysis (EDX) revealed them to contain iron, sulfur and oxygen as main elemental fractions and electron diffraction ring pattern analysis classified them to be schwertmannite. These bacteria and their interactions with each other as well as with the inorganic minerals formed in this lake generally is of great interest, in order to use these results for bioremediation applications. [ABSTRACT FROM AUTHOR]

Microcosms were inoculated with sediments from both a petroleum-hydrocarbon (PHC)-contaminated aquifer and from a nearby pristine aquifer and incubated under anoxic denitrifying conditions with [methyl-13C]toluene. These microcosms served as a laboratory model system to evaluate the combination of isotope (13C-labeling of polar-lipid-derived fatty acids) and molecular techniques (16S rRNA-targeting gene probes) to identify the toluene-metabolizing population. After total depletion of toluene, the following bacterial phospholipid fatty acids (PLFA) were 13C-enriched: 16:1ω7c, 16:1ω7t, 16:0, cy17:0, and 18:1ω7c. Pure culture experiments demonstrated that these compounds were also found in PLFA profiles of PHC-degrading Azoarcus spp. (β-Proteobacteria) and related species. The origin of the CO2 evolved in the microcosms was determined by measurements of stable carbon isotope ratios. Toluene represented 11% of the total pool of mineralized substrates in the contaminated sediment and 54% in the pristine sediment. The microbial community in the microcosm incubations was characterized by using DAPI staining and whole-cell hybridization with specific fluorescently labeled 16S rRNA-targeted oligonucleotide probes. Results revealed that 6% of the DAPI-stained cells in the contaminated sediment and 32% in the pristine sediment were PHC-degrading Azoarcus spp. In biotic control microcosms (incubated under denitrifying conditions, no toluene added), Azoarcus spp. cells remained at less than 1% of the DAPI-stained cells. The results show that isotope analysis in combination with whole-cell hybridization is a promising approach to identify and to quantify denitrifying toluene degraders within microbial communities. [ABSTRACT FROM AUTHOR]

The presumptive metabolically active members of a bacterial community in a moorland soil in Germany, highly polluted with polychlorinated biphenyls (PCBs), were identified by sequencing of cloned reverse transcription–polymerase chain reaction (RT–PCR) amplification products of 16S rRNA generated from total RNA extracts. Analysis of the 16S rRNA clone library revealed a considerable diversity of metabolically active bacteria in the soil, despite the acidic pH and high concentrations of PCBs. Cloned sequence types clustered within the Proteobacteria (34% alpha-, 33% beta- and 7% gamma-subclasses), the Holophaga –Acidobacterium phylum (14%), the Actinobacteria (6.5%) and the Planctomycetales (2%). Three cloned sequence types were not affiliated to any described phylogenetic group. An unusual feature of this soil was the abundance of sequence types within the beta-subclass of the Proteobacteria , most of which were similar to the 16S rRNA gene sequences of species from only two genera, Burkholderia and Variovorax . Three other numerous 16S rRNA sequence types were similar to the sequences of Sphingomonas species, members of the Rhodopila globiformis group and Acidobacterium capsulatum . Some of the sequence types retrieved were similar to the 16S rRNA sequences of bacterial isolates able to degrade a variety of organic pollutants, including PCBs. As the PCB contamination is the major source of measurable carbon in this soil, some of the 16S rRNA sequence types detected and presumed to represent the metabolically active members of the community indicate the organisms likely to be involved, directly or indirectly, in the utilization of the PCBs as carbon and energy sources. [ABSTRACT FROM AUTHOR]

Although much information on metabolic pathways within individual organisms is available, little is known about the pathways operating in natural communities in which extensive sharing of nutritional resources is the rule. In order to analyse such a consortium pathway, we have investigated the flow of 4chlorosalicylate as carbon substrate within a simple chemostat microbial community using [sup 13C-labelled metabolites and isotopic ratio mass spectrometric analysis of label enrichment in immunocaptured member populations of the community. A complex pathway network of carbon sharing was thereby revealed, involving two different metabolic routes, one of which is completely novel and involves the toxic metabolite protoanemonin. The high stability of the community results, at least in part, from interdependencies based on carbon sharing and the rapid removal of toxic metabolites. [ABSTRACT FROM AUTHOR]

Fusarium solani strains DSM 62416 and 62413 were found to hydrate trans-nerolidol or geranylacetone at the inner double bond. The stereochemical requirements for the substrate are rather narrow. Only E-configurated double bonds were accepted by the hydratases and no hydration was observed at the terminal isoprenyl unit of various substrates. The hydratases differ in their substrate specificity. While trans-nerolidol is accepted by the hydratase of strain DSM 62416 and geranylacetone is not, the contrary is found for the hydratase of strain DSM 62413. Higher yields of hydrated products under anaerobic conditions revealed that oxygen is not required as a cofactor. [ABSTRACT FROM AUTHOR]

Monoterpenoids with 1-p-menthene-structure can be transformed by Corynespora cassiicola DSM 62 474, DSM 62 475, and Diplodia gossypina ATCC 10 936 to chiral 1,2-trans-diols in yields of more than 60% with only minor amounts of side-products. Whereas the substrates (S)-(-)-limonene, α-terpinene, γ-terpinene, and terpinolene are converted to the (1R,2R)-p-menthene-1,2-diols, (R)-(+)-limonene and (R)-(-)-phellandrene yield the (1S,2S)-1,2-diols. For the transformation of (S)-α-terpineol and (S)-α-terpinene-4-ol to the (1S,2S)-1,2-diols Gibberella cyanea DSM 62 719 can be used, which however oxidizes parallel at the 6- and 7-position. [ABSTRACT FROM AUTHOR]

Microorganisms which efficiently oxidize limonene 1, do not attack 3,3,5,5-tetramethyllimonene 4. Gibberella cyanea which converts limonene 1 into 8-p-menthene-1,2-diol 2, transforms 4 into 8,9-epoxy-3,3,5,5-tetramethyl-1-menthenol-6 5 as major product. Although epoxidation is not completely stereoselective, the substituents at C-4 and C-6 are always trans to each other. [ABSTRACT FROM AUTHOR]

Quorum sensing is a type of chemical communication by which bacterial populations control expression of their genes in a coordinated manner. This regulatory mechanism is commonly used by pathogens to control the expression of genes encoding virulence factors and that of genes involved in the bacterial adaptation to variations in environmental conditions. In phytopathogenic bacteria, several mechanisms of quorum sensing have been characterized. In this review, we describe the different quorum sensing systems present in phytopathogenic bacteria, such as those using the signal molecules named N-acyl-homoserine lactone (AHL), diffusible signal factor (DSF), and the unknown signal molecule of the virulence factor modulating (VFM) system. We focus on studies performed on phytopathogenic bacteria of major importance, including Pseudomonas, Ralstonia, Agrobacterium, Xanthomonas, Erwinia, Xylella,Dickeya, and Pectobacterium spp. For each system, we present the mechanism of regulation, the functions targeted by the quorum sensing system, and the mechanisms by which quorum sensing is regulated. [ABSTRACT FROM AUTHOR]