bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular and serological responses in resistant and susceptible mice exposed to repeated infection with Heligmosomoides polygyrus bakeri
Authors: Behnke Jerzy M. , Lowe Anna , Clifford Simon , Wakelin Derek
Published: September 2003

An experiment was carried out to compare the parasitological and immunological responses of SWR and CBA mice to trickle (repeated) infection with Heligmosomoides polygyrus. Male mice were given 125 L3 once per week and were killed in groups, together with naïve control mice, weekly until week 8. Worm burdens accumulated in CBA, stabilizing in week 5 in excess of 400 worms and remaining high until week 8. In contrast in SWR worm burdens peaked in week 3 at a mean worm burden of 129 and then fell sharply so that by week 6, despite continuing re‐infection, no more worms were recovered from these mice. SWR mice showed a marked mast cell and mMCP‐1 response, peaking in weeks 2–3, whereas in CBA mice these responses were slower, and even at their height in week 8 still less intense than those in SWR mice. Both strains responded initially with a very similar goblet cell response, which declined in SWR mice as worms were eliminated, but was sustained in CBA mice until week 8. Serum TNFα concentrations were higher in SWR mice throughout the experiment. Infection elicited strong serological responses against adult and L4 antigens in both SWR and CBA mice, involving all the isotypes tested (IgG1, IgA and IgE). Anti‐L3 responses were examined only for IgG1. However, only two responses differed significantly between the strains: the IgE response to L4 antigens was more intense in SWR mice, and interestingly and unexpectedly, the IgG1 response to adult worm antigens was more intense in CBA mice. These results reflect the activation of predominantly Th2‐driven effector mechanisms, that may be associated with host‐protective immunity developing under the trickle infection protocol exploited in these experiments.
Mapping of chromosomal regions influencing immunological responses to gastrointestinal nematode infections in mice
Authors: Menge David M. , Behnke Jerzy M. , Lowe Anna , Gibson John P. , Iraqi Fuad A. , Baker R. Leyden , Wakelin Derek
Published: September 2003

This paper reports the results of a genome‐wide search for quantitative trait loci (QTL) influencing immunological responses to infection with the gastro‐intestinal nematode parasite Heligmosomoides polygyrus in an F2 population created by crossing the resistant SWR and the susceptible CBA inbred mouse strains. Following infections, intestinal granuloma score at post mortem, mucosal mast cell protease 1, and IgE and IgG1 titres were recorded. The susceptible CBA mice had significantly higher IgG1, but significantly lower IgE, mucosal mast cell protease 1 and granuloma scores than SWR mice. Significant QTL were mapped to chromosomes 4, 11, 13 and 17 for granuloma score; chromosomes 12 and 17 for IgE; chromosome 10, 17 and 18 for IgG1 and chromosomes 1, 9, 10, 11, 17 and 18 for mucosal mast cell protease 1. Chromosomes 10, 11, 17 and 18 had QTL affecting more than one trait, and these are most likely to represent single QTL with multiple effects rather than multiple QTL. Some of these QTL map to regions known to harbour genes responsible for the induction of immunological responses to intestinal worms.
Long‐term effects of mineral versus organic fertilizers on activity and structure of the methanotrophic community in agricultural soils
Authors: Seghers Dave , Top Eva M. , Reheul Dirk , Bulcke Robert , Boeckx Pascal , Verstraete Willy , Siciliano Steven D.
Published: September 2003

Agricultural practices, such as mineral nitrogen fertilization, have an impact on the soil's ability to oxidize methane, but little is known about the shifts in the methanotrophic community composition associated with these practices. Therefore, the long‐term effect of both mineral (NH4NO3) and organic (manure and GFT‐compost) fertilizer applications on the soil methanotrophic community activity and structure were investigated. Both high and low affinity methane oxidation rates were lower in the soil treated with mineral fertilizer compared to the other soils. An enhanced nitrate concentration was observed in the mineral fertilized soil but nitrate did not show a direct affect on the high affinity methane oxidation. In contrast, the low affinity methane oxidation was slowed down by increased nitrate concentrations, which suggests a direct effect of nitrate on low affinity methane oxidation. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments specific for methanotrophs revealed a distinct community between the mineral and organic fertilized soils as extra Type I methanotrophic bands (phylotypes) became visible in the organic fertilized soils. These phylotypes were not visible in the patterns of the added organic fertilizers suggesting an indirect effect of the organic fertilizers on the methanotrophic community. Additionally, a molecular analysis was performed after the low affinity methane oxidation test. The enhanced methane concentrations used in the test enriched certain low affinity methanotrophs in the organic fertilized soils but not in the mineral fertilized soil. Supporting the molecular and functional observations, fatty acids characteristic for methanotrophs were less abundant in the soil treated with mineral fertilizer compared to the soil treated with compost. In conclusion, the function and molecular and chemical composition of the methanotrophic community are all altered in soil fertilized with mineral fertilizer.
Effect of integration of a GFP reporter gene on fitness of Ralstonia eutropha during growth with 2,4‐dichlorophenoxyacetic acid
Authors: Füchslin Hans Peter , Rüegg Irene , Van Der Meer Jan Roelof , Egli Thomas
Published: September 2003

Green fluorescent proteins (GFPs) are frequently used as marker and reporter systems to assess the fate and activity of microbial strains with the ability to degrade xenobiotic compounds. To evaluate the potential of this tool for tracking herbicide‐degrading microorganisms in the environment a promoterless gfp was linked to the tfd C promoter, which is activated during degradation of the herbicide 2,4‐dichlorophenoxyacetic acid (2,4‐D), and integrated into the chromosome of the 2,4‐D–degrading strain Ralstonia eutropha JMP 134. The effects of the inserted gfp gene on the kinetics of 2,4‐D degradation by R. eutropha in batch and chemostat culture were compared to those of the wild‐type strain. In batch culture with 2,4‐D as the only carbon and energy source the maximum specific growth rate of the gfp‐marked strain did not differ significantly from the wild type. However, compared to the wild type, the 2,4‐D steady‐state concentration in 2,4‐D‐limited chemostat cultures of the gfp‐marked strain was higher at all dilution rates tested. The reduced competitiveness of the gfp‐marked strain at low substrate concentrations was confirmed in a competition experiment for 2,4‐D in continuous culture at a dilution rate of 0.075 h−1. Reproducibly, the gfp‐marked strain was displaced by the wild‐type strain. The study clearly demonstrates that fitness of constructs cannot be assessed by measuring µmax with selected substrates in batch cultures only but that a thorough kinetic analysis is needed, which also considers slow, carbon‐limited growth conditions as they occur in the environment.
Temporal change in culturable phenanthrene degraders in response to long‐term exposure to phenanthrene in a soil column system
Authors: Bodour Adria A. , Wang Jiann‐Ming , Brusseau Mark L. , Maier Raina M.
Published: September 2003

Widespread environmental contamination by polycyclic aromatic hydrocarbons (PAH) has led to increased interest in the use of natural attenuation as a clean‐up strategy. However, few bioremediation studies have investigated the behaviour of the indigenous PAH‐degrading community after long‐term exposure to a PAH. In this study, a column packed with sandy loam soil was exposed to a solution saturated with phenanthrene (≈ 1.2 mg l−1) for a 6‐month period to examine the temporal response of the indigenous phenanthrene‐degrading community. Initial soil, effluent, and final soil samples were collected and analysed for phenanthrene concentration and culturable phenanthrene degraders. Phenanthrene‐degrading isolates were grouped by colony morphology. For each unique group, 16S rDNA polymerase chain reaction was performed, and then sequencing analysis was used to identify the isolate at the genus level. Twenty‐five phenanthrene‐degrading isolates, potentially representing 19 genera, were obtained from this analysis. Of these, eight genera have not been reported previously to degrade phenanthrene, including Afipia, Janthinobacterium, Leptothrix, Massilia, Methylobacterium, Rhizobium, Sinorhizobium and Thiobacillus. Results indicate that the dominant phenanthrene‐degrading population changed over the course of this 6‐month experiment. Specifically, the isolates obtained initially from the soil were not subsequently found in either effluent samples or the soil at the end of the experiment. Furthermore, several isolates that were found in the soil at the end of the experiment were not observed in the soil initially or in the effluent samples. This study confirms earlier findings indicating that a diverse community participates in phenanthrene degradation in the environment, and also suggests that the composition of this community is temporally variable.
New primers for the class Actinobacteria: application to marine and terrestrial environments
Authors: Stach James E. M. , Maldonado Luis A. , Ward Alan C. , Goodfellow Michael , Bull Alan T.
Published: September 2003

In this study, we redesigned and evaluated primers for the class Actinobacteria. In silico testing showed that the primers had a perfect match with 82% of genera in the class Actinobacteria, representing a 26–213% improvement over previously reported primers. Only 4% of genera that displayed mismatches did so in the terminal three bases of the 3′ end, which is most critical for polymerase chain reaction success. The primers, designated S‐C‐Act‐0235‐a‐S‐20 and S‐C‐Act‐0878‐a‐A‐19, amplified an ≈ 640 bp stretch of the 16S rRNA gene from all actinobacteria tested (except Rubrobacter radiotolerans) up to an annealing temperature of 72°C. An Actinobacteria Amplification Resource (http://microbe2.ncl.ac.uk/MMB/AAR.htm) was generated to provide a visual guide to aid the amplification of actinobacterial 16S rDNA. Application of the primers to DNA extracted from marine and terrestrial samples revealed the presence of actinobacteria that have not been described previously. The use of 16S rDNA similarity and DNA–DNA pairing correlations showed that almost every actinomycete clone represented either a new species or a novel genus. The results of this study reinforce the proposition that current culture‐based techniques drastically underestimate the diversity of Actinobacteria in the environment and highlight the need to evaluate taxon‐specific primers regularly in line with improvements in databases holding 16S rDNA sequences.
Comparative 16S rDNA and 16S rRNA sequence analysis indicates that Actinobacteria might be a dominant part of the metabolically active bacteria in heavy metal‐contaminated bulk and rhizosphere soil
Authors: Gremion Fabienne , Chatzinotas Antonis , Harms Hauke
Published: September 2003

Bacterial diversity in 16S ribosomal DNA and reverse‐transcribed 16S rRNA clone libraries originating from the heavy metal‐contaminated rhizosphere of the metal‐hyperaccumulating plant Thlaspi caerulescens was analysed and compared with that of contaminated bulk soil. Partial sequence analysis of 282 clones revealed that most of the environmental sequences in both soils affiliated with five major phylogenetic groups, the Actinobacteria, α‐Proteobacteria, β‐Proteobacteria, Acidobacteria and the Planctomycetales. Only 14.7% of all phylotypes (sequences with similarities> 97%), but 45% of all clones, were common in the rhizosphere and the bulk soil clone libraries. The combined use of rDNA and rRNA libraries indicated which taxa might be metabolically active in this soil. All dominant taxa, with the exception of the Actinobacteria, were relatively less represented in the rRNA libraries compared with the rDNA libraries. Clones belonging to the Verrucomicrobiales, Firmicutes, Cytophaga–Flavobacterium–Bacteroides and OP10 were found only in rDNA clone libraries, indicating that they might not represent active constituents in our samples. The most remarkable result was that sequences belonging to the Actinobacteria dominated both bulk and rhizosphere soil libraries derived from rRNA (50% and 60% of all phylotypes respectively). Seventy per cent of these clone sequences were related to the Rubrobacteria subgroups 2 and 3, thus providing for the first time evidence that this group of bacteria is probably metabolically active in heavy metal‐contaminated soil.
Molecular investigation on strain genetic relatedness and population structure of Beauveria bassiana
Authors: Wang Chengshu , Shah Farooq A. , Patel Nita , Li Zengzhi , Butt Tariq M.
Published: September 2003

Triplicate molecular methods, i.e. polymerase chain reaction‐restriction fragment length polymorphism of the pr1 gene, microsatellite markers and 28S rDNA haplotyping by detecting the presence or absence of group I introns, were used for population study of the entomopathogenic fungus, Beauveria bassiana. The findings showed that the average genetic diversity index of geographical populations was significantly smaller than that of populations derived from insect host orders, indicating that the genetic relatedness of B. bassiana strains was highly associated with geographical locality rather than insect host species. The reproductive style of all the B. bassiana populations was found to be non‐clonal. Population structure analysis revealed that the average divergent coefficient among populations of B. bassiana was far below 1 (0.1112), which indicated that there was no significant genetic differentiation between populations, and that the overall genetic diversity mainly resulted from the genetic variations within geographical populations. Statistically, genetic distances between populations were positively correlated with geographical distances, suggesting that geographical separation poses an obstacle to the possibility and frequency of genetic exchanges between populations. On the other hand, gene flow was indirectly established to occur between B. bassiana populations.
Effect of thorium on the growth and capsule morphology of Bradyrhizobium
Authors: Santamaría Mónica , Díaz‐Marrero Ana R. , Hernández Jairo , Gutiérrez‐Navarro Angel M. , Corzo Javier
Published: September 2003

The thorium effect on Bradyrhizobium growth was assayed in liquid media. Th4+ inhibited the growth of Bradyrhizobium (Chamaecytisus) BGA‐1, but this effect decreased in the presence of suspensions of live or dead bacterial cells. Th4+ induced the formation of a gel‐like precipitate when added to a dense suspension of B. (Chamaecytisus) BGA‐1 cells. Viable Bradyrhizobium cells remained in suspension after precipitate formation. Thorium was recovered in the precipitate, in which polysaccharide, lipopolysaccharide and proteins were also found. After Th4+ addition, the morphology of B. (Chamaecytisus) BGA‐1 or Bradyrhizobium japonicum USDA 110 sedimented cells studied by scanning electron microscopy changed from an entangled network of capsulated bacteria to uncapsulated individual cells and an amorphous precipitate. Energy‐dispersive X‐ray spectroscopy showed that thorium was mainly in the amorphous fraction. Precipitate was also formed between B. (Chamaecytisus) BGA‐1 and Al3+, which was also toxic to this bacterium. Precipitate induced by Th4+ or Al3+ was found in all Bradyrhizobium and Sinorhizobium strains tested, but not in Rhizobium, Salmonella typhimurium, Aerobacter aerogenes or Escherichia coli. These results suggest a specific defence mechanism based on metal precipitation by extracellular polymers.
Stationary phase mutagenesis: mechanisms that accelerate adaptation of microbial populations under environmental stress
Authors: Kivisaar Maia
Published: September 2003

Microorganisms are exposed to constantly changing environmental conditions. In a growth‐restricting environment (e.g. during starvation), mutants arise that are able to take over the population by a process known as stationary phase mutation. Genetic adaptation of a microbial population under environmental stress involves mechanisms that lead to an elevated mutation rate. Under stressful conditions, DNA synthesis may become more erroneous because of the induction of error‐prone DNA polymerases, resulting in a situation in which DNA repair systems are unable to cope with increasing amounts of DNA lesions. Transposition may also increase genetic variation. One may ask whether the rate of mutation under stressful conditions is elevated as a result of malfunctioning of systems responsible for accuracy or are there specific mechanisms that regulate the rate of mutations under stress. Evidence for the presence of mutagenic pathways that have probably been evolved to control the mutation rate in a cell will be discussed.
Marine prosthecate bacteria involved in the ennoblement of stainless steel
Authors: Baker Paul W. , Ito Kimio , Watanabe Kazuya
Published: September 2003

Ennoblement, a phenomenon in which open‐circuit potential is elevated to a noble value, triggers metal corrosion in the environment and is considered to be biologically catalysed. This study investigated the involvement of marine microorganisms in the ennoblement of stainless steel coupons in sea water pumped from Kamaishi Bay. Scanning electron microscopy (SEM) showed significant attachment of prosthecate bacteria on the surfaces of stainless steel coupons in the course of ennoblement. In denaturing gradient gel electrophoresis (DGGE) analyses of polymerase chain reaction‐amplified bacterial 16S rDNA fragments, several major bands were detected from the surface of the ennobled coupons, including those affiliated with the α and γ subclasses of the Proteobacteria. After these observations, bacterial strains were isolated from the surface of the ennobled coupon. The 16S rDNA analysis revealed that a bacterial isolate (designated PWB3) corresponded to a major DGGE band representing an α‐Proteobacterial population; a database analysis showed that its closest relative was Rhodobium spp., albeit with low homology (≈ 89%). SEM indicated that this bacterium was a prosthecate bacterium that was morphologically similar to those observed on the ennobled coupons. In pure culture of strain PWB3, stainless steel coupons were ennobled when the culture was supplemented with MnCl2. Manganese was recovered from the surface of the ennobled coupons after treatment with a reducing agent. These results suggest that the attachment of manganese‐oxidizing prosthecate bacteria triggered the ennoblement of stainless steel in Kamaishi Bay sea water.
Validation of a more sensitive method for using spotted oligonucleotide DNA microarrays for functional genomics studies on bacterial communities
Authors: Denef Vincent J. , Park Joonhong , Rodrigues Jorge L. M. , Tsoi Tamara V. , Hashsham Syed A. , Tiedje James M.
Published: September 2003

Spotted oligonucleotide microarrays potentially offer a wide scope of applications for microbial ecology, especially as they improve the flexibility of design and the specificity of detection compared to PCR product based microarrays. Sensitivity, however, was expected to be problematic, as studies with the more sensitive PCR‐based cDNA microarrays indicate that only genes from populations contributing to more than 5% of the community DNA can be detected. We evaluated several parameters to increase sensitivity and then tested applicability for bacterial functional genomics. The optimal parameters were the use of 5′‐C6‐amino‐modified 70‐mers printed on CMT‐GAPS II substrates at a 40 µM concentration combined with the use of Tyramide Signal Amplification labelling. This protocol allowed detection of single copy genes belonging to an organism contributing to 1% or more of the total community. To demonstrate its application, we detected the specific aromatic oxygenase genes in a soil community degrading polychlorinated biphenyls (PCBs). This increase in sensitivity is important if oligonucleotide microarrays are to be used for simultaneous monitoring of a range of functions performed by different microorganisms in the environment.
Effect of N‐fertilization, plant genotype and environmental conditions on nifH gene pools in roots of rice
Authors: Tan Zhiyuan , Hurek Thomas , Reinhold‐Hurek Barbara
Published: September 2003

Terminal restriction fragment length polymorphism (T‐RFLP) analysis of PCR‐amplified nitrogenase gene (nifH) fragments is a rapid technique for profiling of diazotrophic microbial communities without the necessity of cultures for study. Here, we examined the impact of N‐fertilization, plant genotype and environmental conditions on diazotrophic microbial populations in association with roots of rice (Oryza species) by T‐RFLP community profiling and found marked effects on the composition of the microbial community. We found a rapid change of the diazotrophic population structure within 15 days after application of nitrogen fertilizer and a strong effect of environmental conditions and plant genotype. Control experiments revealed that phylogenetically distantly related nifH genes were proportionately amplified, and that signal strength reflected the relative abundance of nifH genes in the sample within a 10‐fold range of template concentrations. These results clearly demonstrated that our T‐RFLP method was suitable to reflect compositional differences in the diazotrophic community in a semiquantitative manner and that the diazotrophic rhizosphere communities of rice are not static but presumably rather highly dynamic.
A novel pathway for mineralization of the thiocarbamate herbicide molinate by a defined bacterial mixed culture
Authors: Barreiros Luísa , Nogales Balbina , Manaia Célia M. , Ferreira António C. Silva , Pieper Dietmar H. , Reis Maria A. , Nunes Olga C.
Published: September 2003

A bacterial mixed culture able to mineralize molinate was established, through enrichment, using mineral medium with molinate as the only carbon, nitrogen and energy source. The combination of five cultivable isolates, purified from the enrichment culture, permitted the reconstitution of a degrading consortium. Both enrichment and defined cultures were able to mineralize molinate without accumulation of degradation products by the end of the growth. Among the five isolates constituting the defined mixed culture, an actinomycete, strain ON4, was essential for biodegradation, being involved in the cleavage of the thioester bond of molinate, the initial step of the degradation pathway. Isolate ON4 was able to grow on molinate at concentrations below 2 mM, with the accumulation of ethanethiol and diethyl disulphide. These sulphur compounds were toxic to strain ON4 when accumulating at higher concentrations. However, this inhibitory effect was avoided by the presence of other members of the mixed culture, out of which isolates ON1 and ON2 were observed to consume ethanethiol and diethyl disulphide. In this way, interactions among defined mixed culture members involve metabolic and detoxifying association.
Novel Proteorhodopsin variants from the Mediterranean and Red Seas
Authors: Sabehi Gazalah , Massana Ramon , Bielawski Joseph P. , Rosenberg Mira , Delong Edward F. , Béjà Oded
Published: September 2003

Proteorhodopsins, ubiquitous retinylidene photoactive proton pumps, were recently found in the widespread uncultured SAR86 bacterial group in oceanic surface waters. To survey proteorhodopsin diversity, new degenerate sets of proteorhodopsin primers were designed based on a genomic proteorhodopsin gene sequence originating from an Antarctic fosmid library. New proteorhodopsin variants were identified in Red Sea samples that were most similar to the original green‐light absorbing proteorhodopsins found in Monterey Bay California. Unlike green‐absorbing proteorhodopsins however, these new variants contained a glutamine residue at position 105, the same site recently shown to control spectral tuning in naturally occurring proteorhodopsins. Different proteorhodopsin variants were also found in the Mediterranean Sea. These proteorhodopsins formed new and distinctive proteorhodopsin groups. Phylogenetic analyses show that some of the new variants were very different from previously characterized proteorhodopsins, and formed the deepest branching groups found so far among marine proteorhodopsins. The existence of these varied proteorhodopsin sequences suggests that this class of proteins has undergone substantial evolution. These variants could represent functionally divergent paralogous genes, derived from the same or similar species, or orthologous proteorhodopsins that are distributed amongst divergent planktonic microbial taxa.
Microbially mediated sulphide production in a thermal, acidic algal mat community in Yellowstone National Park
Authors: Ferris Michael J. , Magnuson Timothy S. , Fagg Jennifer A. , Thar Roland , Kühl Michael , Sheehan Kathy B. , Henson Joan M.
Published: September 2003

Our objective in this study was to characterize prokaryotic sulphide production within the oxygenic, predominantly eukaryotic algal mat in an acidic stream, Nymph Creek, in Yellowstone National Park (YNP). We used microsensors to examine fluctuations in H2S and O2 concentrations over time through the vertical aspect of the ∼3 mm mat in a 46–48°C region of the creek. We also used analyses of PCR‐amplified 16S rRNA gene sequences obtained from denaturing gradient gels, and PCR‐amplified sequences of a functional gene associated with microbial sulphate respiration (dsrA) to characterize the bacterial community in the same region of the mat. During midday, photosynthesis rates were high within the first 500 µm interval of the mat and high oxygen concentrations (600% air saturation) penetrated deeply (>1800 µm) into the mat. During early evening and night, oxygen concentrations within the first 1100 µm of the mat decreased over time from 60% air saturation (a.s) to 12% a.s. A precipitous decline in oxygen concentration occurred at a depth of 1100 µm in all night measurements and anoxic conditions were present below 1200 µm. Within this anoxic region, sulphide concentrations increased from nearly 0 µM at 1200 µm depth to 100 µM at 2400 µm depth. Enrichment cultures inoculated with Nymph Creek mat organisms also produced H2S. Sequence analyses of 16S rRNA and dsrA genes indicated the presence of at least five bacterial genera including species involved in dissimilative sulphate or sulphur reduction.
Bacterial diversity in hydrothermal sediment and epsilonproteobacterial dominance in experimental microcolonizers at the Mid‐Atlantic Ridge
Authors: López‐García Purificación , Duperron Sébastien , Philippot Pascal , Foriel Julien , Susini Jean , Moreira David
Published: September 2003

We report here a molecular survey based on 16S rRNA genes of the bacterial diversity found in two deep‐sea vent niches at the Mid‐Atlantic Ridge: hydrothermal sediment (Rainbow site), and microcolonizers made of three different substrates (organic‐rich, iron‐rich and pumice) that were exposed for 15 days to a vent emission. Bacterial diversity in sediment samples was scattered through many bacterial divisions. The most abundant and diverse environmental sequences (phylotypes) in our libraries corresponded to the Gammaproteobacteria, followed by the Acidobacteria. We detected members of all the subdivisions within the Proteobacteria. Myxobacterial lineages were the most represented within the delta subdivision. Phylotypes ascribing to the Cytophaga‐Flavobacterium‐Bacteroides, Planctomycetales, high and low G + C Gram‐positives, Nitrospirae, and the candidate division TM7 were also identified. Compared to this broad taxonomic coverage, microcolonizers were almost exclusively colonized by epsilonproteobacteria, although these exhibited considerable morphological and phylogenetic in‐group diversity. No specificity for any of the substrates tested was seen. This observation further supports the idea of the ecological dominance of epsilonproteobacteria in the fluid–seawater interface environment. Because oxidation of reduced S species and/or sulphur‐reduction is thought to be essential for their energetic metabolism in these areas, we mapped different oxidation states of S in individual bacterial filaments from the iron‐rich microcolonizer. For this, we used high‐resolution, non‐destructive synchrotron micro‐X‐ray Absorption Near‐Edge Spectroscopy (micro‐XANES), which revealed the co‐existence of different S oxidation states, from sulphide to sulphate, at the level of individual cells. This suggests that these cells were metabolizing sulphur in situ.
Microbial characterization of the Mars Odyssey spacecraft and its encapsulation facility
Authors: La Duc Myron T. , Nicholson Wayne , Kern Roger , Venkateswaran Kasthuri
Published: September 2003

Microbial characterization of the Mars Odyssey spacecraft and the Kennedy Space Center Spacecraft Assembly and Encapsulation Facility II (SAEF‐II) was carried out by both culture‐based and molecular methods. The most dominant cultivable microbes were species of Bacillus, with comamonads, microbacteria and actinomycetales also represented. Several spore‐forming isolates were resistant to γ‐radiation, UV, H2O2 and desiccation, and one Acinetobacter radioresistens isolate and several Aureobasidium, isolated directly from the spacecraft, survived various conditions. Sequences arising in clone libraries were fairly consistent between the spacecraft and facility; predominant genera included Variovorax, Ralstonia and Aquaspirillum. This study improves our understanding of the microbial community structure, diversity and survival capabilities of microbes in an encapsulation facility and physically associated with colocated spacecraft.
Involvement of rpoS in the survival of Escherichia coli in the viable but non‐culturable state
Authors: Boaretti Marzia , Del Mar Lleò Maria , Bonato Barbara , Signoretto Caterina , Canepari Pietro
Published: September 2003

When exposed to stress‐provoking environmental conditions such as those of ground waters, many medically important bacteria have been shown to be capable of activating a survival strategy known as the viable but non‐culturable (VBNC) state. In this state bacteria are no longer culturable on conventional growth media, but the cells maintain their viability and pathogenicity genes/factors and can start dividing again, in a part of the cell population, upon restoration of favourable environmental conditions. Little is known about the genetic mechanisms underlying the VBNC state. In this study we show evidence of involvement of the rpoS gene in persistence of Escherichia coli in the VBNC state. The kinetics of entry into the non‐culturable state and duration of cell viability were measured in two E. coli mutants carrying an inactivated rpoS gene and compared with those of the parents. For these experiments, laboratory microcosms consisting of an artificial oligotrophic medium incubated at 4°C were used. The E. coli parental strains reached the non‐culturable state in 33 days when the plate counts were evaluated on Luria–Bertani agar containing sodium pyruvate, whereas cells of the rpoS mutants lost their culturability in only 21 days. Upon reaching unculturability the parents yielded respiring cells and cells with intact membranes for at least the next three weeks and resuscitation was allowed during this time. In contrast, the RpoS‐ mutant cells demonstrated intact membranes for only two weeks and a very restricted (<7 days) resuscitation capability. Guanosine 3′,5′‐bispyrophosphate (ppGpp) acts as a positive regulator during the production and functioning of RpoS. A mutant deficient in ppGpp production behaved like the rpoS mutants, while overproducers of ppGpp displayed a vitality at least comparable to that of RpoS+ strains. These results suggest that the E. coli parental strains enter the VBNC state which lasts for, at least, three weeks, after which apparently all the cells die. The rpoS mutants do not activate this survival strategy and early die. This implies involvement of the rpoS gene in E. coli persistence in the VBNC state.
Persistence of adhesive properties in Vibrio cholerae after long‐term exposure to sea water
Authors: Pruzzo Carla , Tarsi Renato , Del Mar Lleò Maria , Signoretto Caterina , Zampini Massimiliano , Pane Luigi , Colwell Rita R. , Canepari Pietro
Published: September 2003

The effect of exposure to artificial sea water (ASW) on the ability of classical Vibrio cholerae O1 cells to interact with chitin‐containing substrates and human intestinal cells was studied. Incubation of vibrios in ASW at 5°C and 18°C resulted in two kinds of cell responses: the viable but non‐culturable (VBNC) state (i.e. <0.1 colony forming unit ml−1) at 5°C, and starvation (i.e. maintenance of culturability of the population) at 18°C. The latter remained rod shaped and, after 40 days’ incubation, presented a 47–58% reduction in the number of cells attached to chitin, a 48–53% reduction in the number of bacteria adhering to copepods, and a 48–54% reduction in the number of bacteria adhering to human cultured intestinal cells, compared to control cells not suspended in ASW. Bacteria suspended in ASW at 5°C became coccoid and, after 40 days, showed 34–42% fewer cells attached to chitin, 52–55% fewer adhering to copep‐ods, and 45–48% fewer cells adhering to intestinal cell monolayers, compared to controls. Sarkosyl‐insoluble membrane proteins that bind chitin particles were isolated and analysed by SDS‐PAGE. After 40 days incubation in ASW at both 5°C and 18°C vibrios expressed chitin‐binding ligands similar to bacteria harvested in the stationary growth phase. It is concluded that as vibrios do not lose adhesive properties after long‐term exposure to ASW, it is important to include methods for VBNC bacteria when testing environmental and clinical samples for purposes of public health safety.
Enduring toxicity of transgenic Anabaena PCC 7120 expressing mosquito larvicidal genes from Bacillus thuringiensis ssp. israelensis†
Authors: Manasherob Robert , Otieno‐Ayayo Zachariah Ngalo , Ben‐Dov Eitan , Miaskovsky Rina , Boussiba Sammy , Zaritsky Arieh
Published: September 2003

Persistence of biological control agents against mosquito larvae was tested under simulated field conditions. Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing cry4Aa, cry11Aa and p20 from Bacillus thuringiensis ssp. israelensis was greater than B. thuringiensis ssp. israelensis primary powder (fun 89C06D) or wettable powder (WP) (Bactimos products) when either mixed with silt or exposed to sunlight outdoors. Reduction of Bactimos primary powder toxicity was at least 10‐fold higher than Anabaena’s after mixing with silt. In outdoors experiments, Bactimos WP remained toxic (over 30% mortality of 3rd instar Aedes aegypti larvae) for 2–4 days only, while transgenic Anabaena’s toxicity endured 8–21 days.
The influence of antibiotic production and pre‐emptive colonization on the population dynamics of Pantoea agglomerans (Erwinia herbicola) Eh1087 and Erwinia amylovora in planta
Authors: Giddens Stephen R. , Houliston Gary J. , Mahanty H. Khris
Published: September 2003

Stigma colonization by Erwinia amylovora is the crucial first step in the development of most fire blight infections in apple and pear trees. Suppression at this point of the disease process by antagonists of E. amylovora, such as Pantoea agglomerans (Erwinia herbicola) strain Eh1087, is a rational approach to control fire blight. We tested the hypothesis that the ability of E. amylovora to compete with Eh1087 for colonization of a stigma is reduced by the potential for Eh1087 to produce the phenazine antibiotic, d‐alanylgriseoluteic acid (AGA). In competition experiments on the stigmas of apple flowers, E. amylovora was significantly less successful against Eh1087 (AGA+) than against EhΔAGA (AGA‐). Further experiments to test the importance of pre‐emptive colonization of the stigma by either the pathogen or the antagonist suggested that AGA production significantly enhanced the competitiveness of Eh1087 when it was applied at the same time or 24 h before the pathogen. We also found that pre‐emptive stigma colonization by either the pathogen or the antagonist resulted in a population that was resilient to subsequent invasion by a second species suggesting that niche exclusion has a dominant influence on the dynamics of bacterial populations on stigmas.
A strain isolated from gas oil‐contaminated soil displays chemotaxis towards gas oil and hexadecane
Authors: Lanfranconi Mariana P. , Alvarez Héctor M. , Studdert Claudia A.
Published: September 2003

In this report we describe the isolation of a strain from soil contaminated with gas oil by taking bacteria from a chemotactic ring on gas oil‐containing soft agar plates. Partial 16 S rDNA sequencing of the isolated strain showed 99.1% identity with Flavimonas oryzihabitans. It was not only able to degrade different aliphatic hydrocarbons but it was also chemotactic towards gas oil and hexadecane, as demonstrated by the use of three different chemotaxis methods, such as agarose plug and capillary assays and swarm plate analysis. In addition, the strain was chemotactic to a variety of carbon sources that serve as growth substrates, including glucose, arabinose, mannitol, glycerol, gluconate, acetate, succinate, citrate, malate, lactate and casaminoacids. This is the first report on chemotaxis of a hydrocarbon‐degrading bacterium towards a pure alkane, such as hexadecane.

The fact that environmental isolates show chemotaxis towards contaminant/s present in the site of isolation suggests that chemotaxis might enhance biodegradation by favouring contact between the degrading microorganism and its substrate.
Leitbakteria of microbial biofilm communities causing occlusion of biliary stents
Authors: Wenderoth Dirk F. , Ferslev Björn , Macarri Giampiero , Molinari Gabriella , Lünsdorf Heinrich , Timmis Kenneth N.
Published: September 2003

Biliary stents inserted to relieve obstructive jaundice caused by biliary or pancreatic malignancies inevitably become occluded by microbial growth in the form of diverse microbial community biofilms. The scarce information available on these communities is based on cultivation methods, but such methods usually provide distorted overviews of community composition, so commonalities and differences in biliary stent communities are uncertain. We extracted DNA and RNA from the microbial communities of 11 biliary stents explanted from nine patients in hospitals from two different countries, amplified 16S rRNA and rDNA sequences, analysed the amplicons by the single‐strand conformation polymorphism (SSCP) method, and sequenced and deduced phylogenetic assignments of the major amplicons representing the major biofilm community members. We used a Modified Robbins Device (MRD) to study de novo development of a stent biofilm from a patient stent microbial community. Single‐strand conformation polymorphism fingerprinting revealed the same six abundant bacterial species, here designated Leitbakteria, namely Klebsiella pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Enterobacter aerogenes, and two unculturable bacteria distantly related to E. coli and Shigella sonnei, in all of the stent biofilm communities. In the experimental biliary stent system, a sequential colonization of the stent surface was observed, with P. aeruginosa being the pioneer colonizer, followed by K. pneumoniae and one of the unculturable Leitbakteria, followed by the remainder of the community. The overview of microbial biofilm communities of biliary stents gained by the use of culture‐independent methods revealed new unculturable bacteria as major members of biliary stent biofilms, and the diversity of the abundant members of the stent biofilms is considerably lower than suggested from earlier studies based on cultivation methods, and that communities from different stents from different patients in different countries are remarkably similar and have similar major members, the stent Leitbakteria.
Inhibitors of swelling‐activated chloride channels increase infarct size and apoptosis in rabbit myocardium
Authors: Souktani Rachid , Ghaleh Bijan , Tissier Renaud , De Tassigny Alexandra d'Anglemont , Aouam Karim , Bedossa Pierre , Charlemagne Danièle , Samuel Janelyse , Henry Patrick , Berdeaux Alain
Published: September 2003

Apoptosis is a significant contributor to myocardial cell death during ischemia‐reperfusion and swelling‐activated chloride channels (ICl,swell) contribute to apoptosis. However, the relationship between ICl,swell, ischemia‐reperfusion and apoptosis remains unknown. To further investigate this, New Zealand rabbits underwent a 20‐min coronary artery occlusion (CAO) followed by 72 h of coronary artery reperfusion (CAR). Two ICl,swell blockers, 5‐nitro‐2‐[3‐phenylpropylamino]benzoic acid (NPPB) and indanyloxyacetic acid 94 (IAA‐94) (both 1 mg/kg), were administered prior to CAO and throughout the 72 h CAR. Infarct size (IS) was increased with NPPB and IAA‐94 compared with control (vehicle) rabbits (51 ± 2% and 48 ± 3% and vs. 35 ± 2%, respectively, P < 0.05). Similar results were found when NPPB was administered only during the reperfusion period. The percentage of TUNEL‐positive nuclei in the border zone of the infarct was increased with NPPB compared with control (37 ± 2% vs. 25 ± 3%, P < 0.05) as well as the number of cytoplasmic histone‐associated DNA fragments (0.45 ± 0.06 vs. 0.33 ± 0.04 absorbance units, P < 0.05). These findings support the concept that ICl,swell channels play an important role in the determination of myocardial infarct size and apoptosis during ischemia‐reperfusion.
Assessing immunotoxicity: guidelines
Authors: Putman E. , van der Laan J.W. , van Loveren H.
Published: September 2003

Over the last couple of years the assessment of immunotoxic potential of human pharmaceuticals has drawn considerable attention worldwide. Regulatory agencies entrusted with the registration of pharmaceuticals (or other compounds) found an increased need for guidance on this issue. This has resulted in the release of guidance documents on immunotoxicity in Europe, USA and Japan in close succession. In Europe the CPMP has released their immunotoxicity guidance documents that are now in force. The FDA and the Japanese Authorities are in the process of doing so, and will shortly enforce them.

Immune suppression and stimulation, hypersensitivity, photosensitivity, drug‐induced autoimmunity and developmental immunotoxicity are the focus of regulatory testing. This review discusses these kinds of immunotoxicity and their clinical implications. The three regional guidelines and screening tools for detection are discussed. Additionally, the scientific background on which these guidelines are based is briefly highlighted.
Activation of DNA biosynthesis in human hepatoblastoma HEPG2 cells by the nitric oxide donor, sodium nitroprusside
Authors: Sokołowska Maria , Rokita Hanna , Włodek Lidia
Published: September 2003

The role of nitric oxide (NO) in carcinogenesis is controversial as it has been shown to both stimulate and inhibit tumour growth. Also, there are contradictory opinions regarding the effects of NO on the proliferation of normal and tumour cells. The aim of our study was to use an in vitro model to determine the influence of exogenous NO donors on DNA biosynthesis by measuring [3H] thymidine incorporation in human hepatoblastoma cells (HepG2). The studies were conducted with the following NO precursors: sodium nitroprusside (SNP), S‐nitrosoglutathione, and nitroglycerine (NTG). Out of all three NO donors, SNP increased NO levels and strongly stimulated DNA biosynthesis. A SNP concentration of 150 μm induced optimal NO levels necessary for the activation of DNA biosynthesis. Lower levels of DNA biosynthesis (118% increase over the control) were observed in the presence of NTG, whereas S‐nitrosoglutathione had no effect. Antioxidants such as thiol‐containing drugs, N‐acetylcysteine and tocopherol, proved to be the most efficient co‐activators of SNP‐induced DNA synthesis. On the other hand, supplementing the SNP‐containing medium with compounds that induce oxidative stress and lower the level of –SH groups such as hydrogen peroxide, doxorubicin, and N‐ethylmaleimide, led to the inhibition of DNA synthesis. Therefore, our results firmly confirm the hypothesis that biological effects of exogenous NO donors depends on the redox status of the cell.
Neuroprotection assessment by topographic electroencephalographic analysis: effects of a sodium channel blocker to reduce polymorphic delta activity following ischaemic brain injury in rats
Authors: Williams Anthony J. , Lu Xi‐Chun M. , Hartings Jed A. , Tortella Frank C.
Published: September 2003

The spatiotemporal electroencephalogram (EEG) pathology associated with brain injury was studied using high‐resolution, 10‐electrode cortical EEG mapping in a rat model of middle cerebral artery occlusion (MCAo). Using this model we evaluated the ability of the novel sodium channel blocker and neuroprotective agent RS100642 to resolve injury‐induced EEG abnormalities as a measure of neurophysiological recovery from brain injury. The middle cerebral artery (MCA) was occluded for 1 h during which a dramatic loss of EEG power was measured over the injured cortex with near complete recovery upon reperfusion of blood to the MCA region in all rats. The resultant progression of the MCAo/reperfusion injury (6–72 h) included the appearance of diffuse polymorphic delta activity (PDA), as visually indicated by the presence of high‐amplitude slow‐waves recorded from both brain hemispheres. PDA was associated with large increases in EEG power, particularly evident in outer ‘peri‐infarct’ regions of the ipsilateral parietal cortex as visualized using topographic EEG mapping. Post‐injury treatment with RS100642 (1.0 mg/kg, i.v.) significantly reduced the PDA activity and attenuated the increase in EEG power throughout the course of the injury. These effects were associated with a reduction in brain infarct volume and improved neurological function. These methods of EEG analysis may be helpful tools to evaluate the physiological recovery of the brain from injury in humans following treatment with an experimental neuroprotective compound.
Inhibition of l‐arginine transport by reactive oxygen species in rat anococcygeus muscle
Authors: Tugba Durlu N. , Burcin Ismailoglu U. , Sahin‐Erdemli Inci
Published: September 2003

The effect of l‐arginine on nitrergic transmission and its alteration with reactive oxygen species (ROS) were investigated. l‐arginine potentiated the relaxation response induced by electrical field stimulation in rat anococygeus muscle. This effect was inhibited by l‐lysine, a cationic amino acid using y+L and y+ transport systems in a similar way with l‐arginine. The neutral amino acid l‐leucine, which uses only y+L system as a transport mechanism, inhibited this potentiation at only low frequency stimulation. Electrolysis of the physiological solution did not change the responses to electrical field stimulation, but inhibited the potentiation elicited by l‐arginine that was prevented in the presence of mannitol and N‐acetyl‐l‐cysteine.

In conclusion, l‐arginine is transported via y+ system predominantly to potentiate the relaxation response to nitrergic nerve stimulation in rat anococcygeus muscle. ROS, primarily hydroxyl radicals inhibited l‐arginine‐induced potentiation probably by interacting with the y+ amino acid transport system.