bluuebunny/crossref_metadata_2025 huggingface dataset:
When leaves act like flowers: how dwarf palms attract their pollinators
Authors: Dufaÿ Mathilde , Hossaert‐McKey Martine , Anstett Marie Charlotte
Published: March 2003

In pollination mutualisms, floral odours are signals advertising the presence and location of rewards. However, in the case of the dwarf palm (Chamaerops humilis) and its species‐specific pollinating weevil (Derelomus chamaeropsis), rewards and advertisements are spatially separated. Flowers provide their specific pollinators with food and sites for both egg laying and larval development, but do not advertise them with floral odours or visually conspicuous petals. Insect behavioural bioassays revealed that pollinators are attracted by scents emitted by the leaves, which provide no rewards. These scents are released by large structures located at the sinuses of the palmate leaf. Such scent‐releasing structures have not been previously reported on palm leaves, and we suggest that they may represent an ‘exaptation’ (pre‐existing trait that acquired new functions). We also propose that such functional crossovers between vegetative and reproductive domains may be more frequent in plants than is currently documented.
Predation‐dependent oviposition habitat selection by the mosquitoCuliseta longiareolata: a test of competing hypotheses
Authors: Kiflawi Moshe , Blaustein Leon , Mangel Marc
Published: March 2003

We investigated the mechanism underlying oviposition habitat selection (OHS) in the mosquitoCuliseta longiareolata. The putative outcome of a trade‐off between the risk of predation and detrimental density dependence, OHS in this species presents an opportunity to test two competing alternatives: (1) a polymorphic scenario, in which a fixed proportion of females constantly avoid ‘predator pools’, while the remainder oviposits at random; and (2) a monomorphic scenario, in which all females oviposit in predator pools with a certain probability. We present a conceptual framework that demonstrates how a simple experimental design – whereby predator incidence in artificial pools is alternated between 0.25 and 0.75 – can distinguish between, or refute, the two scenarios. Given the proportional use, by ovipositing females, of predator‐free pools observed under each treatment, and a bootstrap estimate of the ratio of daily oviposition rates, we find the monomorphic scenario twice as likely as the polymorphic.
Evidence for collective medication in ants
Authors: Christe Philippe , Oppliger Anne , Bancalà Francesco , Castella Grégoire , Chapuisat Michel
Published: March 2003

Social organisms are exposed to many pathogens, and have evolved various defence mechanisms to limit the cost of parasitism. Here we report the first evidence that ants use plant compounds as a collective mean of defence against microorganisms. The wood ants Formica paralugubris often incorporate large quantities of solidified conifer resin into their nests. By creating resin‐free and resin‐rich experimental nests, we demonstrate that this resin inhibits the growth of microorganisms in a context mimicking natural conditions. Such a collective medication probably confers major ecological advantages, and may be an unrecognized yet common feature of large, complex and successful societies.
Temperature‐based population segregation in birch
Authors: Kelly Colleen K. , Chase Mark W. , De Bruijn Anette , Fay Michael F. , Woodward F. Ian
Published: March 2003

Mean temperature of establishment years for warm‐ and cold‐year subpopulations of a naturally occurring stand of Betula pendula (birch) shows a difference equivalent to that between current temperatures and temperatures projected for 35–55 years hence, given ‘business as usual.’ The existence of ‘pre‐adapted’ individuals in standing tree populations would reduce temperature‐based advantages for invading species and, if general, bring into question assumptions currently used in models of global climate change. Our results demonstrate a methodology useful for investigating the important ecological issue of adaptation vs. range shifts as a means of response to climate change.
Predicting biodegradation
Authors: Wackett Lawrence P. , Ellis Lynda B. M.
Published: March 2003

Biodegradation is important for natural and industrial cycling of environmental chemicals. Industries and government regulators increasingly seek to know the fate of chemicals in the environment and thus prevent potential negative impacts on human or ecosystem health. However, millions of organic compounds are known, and most will remain unstudied with respect to biodegradation. This necessitates the development of organized biodegradation information coupled with predictive methods. Biodegradation prediction methods are being developed using the information contained in the University of Minnesota Biocatalysis/Biodegradation database. Heuristic rules are derived from compiled biodegradation information. Additional rules are generated by deconstructing compounds into a set of the 40 most common organic functional groups. The rules consist of deriving biochemically plausible catabolic reactions for each of the functional groups. More complex compounds, containing multiple functional groups, are analysed using higher order rules requiring prioritizing enzymatic attack and reactions cleaving functional groups. While biodegradation prediction, like weather prediction, will never be perfect, it can be an important tool for guiding industry, regulators and experimentalists.
Environmental and clinical isolates of Pseudomonas aeruginosa show pathogenic and biodegradative properties irrespective of their origin
Authors: Alonso Ana , Rojo Fernando , Martínez José L.
Published: March 2003

Virulence properties of pathogenic bacteria, as well as resistance to antibiotics, are thought to arise through a specialization process favoured by the strong selection pressure imposed in clinical treatments. Nevertheless, in the case of opportunistic pathogens, it is unclear whether strains can be classified into virulent and non‐virulent isolates. Clones of the opportunistic pathogen Pseudomonas aeruginosa do not seem to be associated to a particular biovar or pathovar, which suggests that virulence characteristics in opportunistic pathogens may already be present in environmental (non‐clinical) isolates. We have explored this possibility, studying environmental isolates (mainly from oil‐contaminated soils) and clinical isolates (from bacteraemia and cystic fibrosis patients) of P. aeruginosa. All environmental strains were found to actively efflux quinolones, which are synthetic antibiotics not expected to be present in the environment. These strains contained multidrug resistance determinants, were capable of invading epithelial cells and presented genes from the quorum‐sensing and type III secretion systems. Some of them expressed either haemolytic or proteolytic activities or both, characteristics considered to be typical of virulent strains. All the strains tested, of clinical or environmental origin, could use alkanes (oil hydrocarbons) as a carbon source. Our results suggest that clinical and non‐clinical P. aeruginosa strains might be functionally equivalent in several traits relevant for their virulence or environmental properties. Selection of clinically relevant traits, such as antibiotic resistance or cellular invasiveness, in opportunistic pathogens present in soil ecosystems is discussed.
Aerotaxis in Desulfovibrio
Authors: Eschemann Andrea , Kühl Michael , Cypionka Heribert
Published: March 2003

Aerotaxis of two sulphate‐reducing bacteria, the freshwater strain Desulfovibrio desulfuricans CSN (DSM 9104) and the marine strain Desulfovibrio oxyclinae N13 (DSM 11498), was studied using capillary microslides, microscopy and oxygen microsensors. The bacteria formed ring‐shaped bands in oxygen diffusion gradients surrounding O2 bubbles, which were placed into anoxic sulphate‐free cell suspensions in capillary microslides. The radial expansion of the oxic volume by diffusion was stopped by aerobic respiration. Bands were formed by cells avoiding high O2 levels near the O2 bubble, as well as by cells entering from the surrounding anoxic zone. At the inner edge of the bands, O2 levels of up to 20% air saturation (50 μM O2) were found, while the outer edge always coincided with the oxic–anoxic interface. Ring diameters and O2 concentrations at the inner edge of the band depended on the cell density and the strain used in the suspension. Band formation did not occur in the absence of an electron donor (5 mM lactate) or when N2 gas bubbles were used. Both strains were highly motile with velocities of ≈ 32 μm s−1 during forward runs, and 7 μm s−1 during backward runs respectively. Within the bands, cells moved in circles of about 20 μm diameter, while cells outside the band exhibited straighter or only slightly bent traces. It is concluded that the capacity of respiration at high rates and the positive and negative aerotactical responses of Desulfovibrio provide an efficient strategy for removing O2 from the habitat in situations where sufficient electron donors and high cell densities are present.
Fluorescence in situ hybridization analysis of the prokaryotic community inhabiting crystallizer ponds
Authors: Antón Josefa , Llobet‐Brossa Enric , Rodríguez‐Valera Francisco , Amann Rudolf
Published: March 2003

A fluorescence in situ hybridization (FISH) protocol suitable for the identification of prokaryotes inhabiting hypersaline environments was developed and applied to several crystallizer ponds with salinities above 36% from a multipond solar saltern in Alicante, Spain. Two morphotypes were abundant in these environments: rods and square or square‐like prokaryotes that could be affiliated to Bacteria and Archaea, respectively, by FISH with domain‐specific probes. FISH with a newly designed probe proved that the archaeal 16S rDNA sequence most frequently recovered from the crystallizers, SPhT, originated from the dominant square‐like prokaryotes. These uncultured prokaryotes have the morphology of Walsby's square bacteria. Additionally, FISH with a probe targeted to the genus Haloarcula, members of which are frequently isolated from this environment, indicated that this genus accounts for less than 0.1% of the total prokaryotic community.
Examination of two years of community dynamics in an anaerobic bioreactor using fluorescence polymerase chain reaction (PCR) single‐strand conformation polymorphism analysis
Authors: Zumstein Emmanuelle , Moletta René , Godon Jean‐Jacques
Published: March 2003

The structures of the bacterial and archaeal communities in an anaerobic digester were monitored over a 2 year period. The study was performed on a fluidized bed reactor fed with vinasse. The objective was to characterize the population dynamics over a long time period under constant environmental parameters. Total bacterial and archaeal populations were measured independently by fluorescence‐based polymerase chain reaction (PCR) single‐strand conformation polymorphism (SSCP) analysis using an automated DNA sequencer. With the current level of accuracy, the technique was able to monitor 45 bacterial and seven archaeal 16S rDNA molecules. The community dynamics were compared with molecular inventories of the microbial community based on 16S rDNA sequences done at the beginning of the study. The six archaeal and the 22 most frequent bacterial operational taxonomic units (OTUs) identified were associated with their SSCP peak counterparts. Overall, the data indicated that, throughout the period of the study, rapid significant shifts in the species composition of the bacterial community occurred, whereas the archaeal community remained relatively stable.
Ecological study of a bioaugmentation failure
Authors: Bouchez  , Patureau  , Dabert  , Juretschko  , Doré  , Delgenès  , Moletta  , Wagner
Published: March 2003

A nitrifying sequencing batch reactor was inoculated twice with the aerobic denitrifying bacterium Microvirgula aerodenitrificans and fed with acetate. No improvement was obtained on nitrogen removal. The second more massive inoculation was even followed by a nitrification breakdown, while at the same time, nitrification remained stable in a second reactor operated under the same conditions without bioaugmentation. Fluorescent in situ hybridization with rRNA‐targeted probes revealed that the added bacteria almost disappeared from the reactor within 2 days, and that digestive vacuoles of protozoa gave strong hybridization signals with the M. aerodenitrificans‐specific probe. An overgrowth of protozoa, coincident with the disappearance of free‐living bacteria, was monitored by radioactive dot‐blot hybridization only in the bioaugmented reactor. Population dynamics were analysed with a newly developed in situ quantification procedure of the probe‐targeted bacteria. The nitrifying groups of bacteria decreased in a similar way in the bioaugmented and non‐bioaugmented reactors. Other bacterial groups evolved differently. The involvement of different ecological parameters are discussed separately for each reactor. These results underline the importance of predator–prey interaction and illustrate the undesirable effects of massive bioaugmentation.
Identification of conserved traits in fluorescent pseudomonads with antifungal activity
Authors: Ellis Richard J. , Timms‐Wilson Tracey M. , Bailey Mark J.
Published: March 2003

A collection of 29 fluorescent pseudomonads, some with known biological control activity against a range of phytopathogenic fungi, were characterized phenotypically and genotypically by comparing carbon source utilization patterns, suppression ofPythium ultimumbothin plantaandin vitroand the potential to produce known secondary metabolites. Fatty acid profiling and restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA operon (ribotyping) were used to determine the diversity of isolates. A small group of genetically relatedPseudomonasspp. with similar properties was identified; each isolate produced a diffusible bioactive productin vitroand was active againstPythium ultimum in planta. However, other isolates that were able to suppress damping off disease but did not inhibit hyphal extensionin vitroclustered outside this group. Phenotypic analyses revealed that the accumulation of C17:0 cyclopropane fatty acid (17CFA) and the production of hydrogen cyanide correlated significantly with biological control activity and with the antagonism of fungal development. The potential of 17CFA as a marker for the selection of fluorescent pseudomonads with biocontrol agent (BCA) potential was demonstrated by the isolation of a novel active strain. This was selected after the screening of 13 clonal groups of fluorescent pseudomonads identified from 500 isolates from the phytosphere of sugar beet. Levels of 17CFA synthesis possibly reflect the efficacy of therpoSallele in particular strains.
From protozoa to mammalian cells: a new paradigm in the life cycle of intracellular bacterial pathogens
Authors: Harb Omar S. , Gao Lian‐Yong , Kwaik Yousef Abu
Published: March 2003

It is becoming apparent that several intracellular bacterial pathogens of humans can also survive within protozoa. This interaction with protozoa may protect these pathogens from harsh conditions in the extracellular environment and enhance their infectivity in mammals. This relationship has been clearly established in the case of the interaction between Legionella pneumophila and its protozoan hosts. In addition, the adaptation of bacterial pathogens to the intracellular life within the primitive eukaryotic protozoa may have provided them with the means to infect the more evolved mammalian cells. This is evident from the existence of several similarities, at both the phenotypic and the molecular levels, between the infection of mammalian and protozoan cells by L. pneumophila. Thus, protozoa appear to play a central role in the transition of bacteria from the environment to mammals. In essence, protozoa may be viewed as a ‘biological gym’, within which intracellular bacterial pathogens train for their encounters with the more evolved mammalian cells. Thus, intracellular bacterial pathogens have benefited from the structural and biochemical conservation of cellular processes in eukaryotes. The interaction of intracellular bacterial pathogens and protozoa highlights this conservation and may constitute a simplified model for the study of these pathogens and the evolution of cellular processes in eukaryotes. Furthermore, in addition to being environmental reservoirs for known intracellular pathogens of humans and animals, protozoa may be sources of emerging pathogenic bacteria. It is thus critical to re‐examine the relationship between bacteria and protozoa to further our understanding of current human bacterial pathogenesis and, possibly, to predict the appearance of emerging pathogens.
A Pseudomonas stutzeri gene cluster encoding the biosynthesis of the CCl4‐dechlorination agent pyridine‐2,6‐bis(thiocarboxylic acid)
Authors: Lewis Thomas A. , Cortese Marc S. , Sebat Jonathan L. , Green Tonia L. , Lee Chang‐Ho , Crawford Ronald L.
Published: March 2003

A spontaneous mutant of Pseudomonas stutzeri strain KC lacked the carbon tetrachloride (CCl4) transformation ability of wild‐type KC. Analysis of restriction digests separated by pulsed‐field gel electrophoresis (PFGE) indicated that the mutant strain CTN1 differed from strain KC by deletion of approximately 170 kb of chromosomal DNA. CTN1 did not produce pyridine‐2,6‐bis(thiocarboxylic acid) (PDTC), the agent determined to be responsible for CCl4 dechlorination in cultures of strain KC. Cosmids from a genomic library of strain KC containing DNA from within the deleted region were identified by hybridization with a 148 kb genomic SpeI fragment absent in strain CTN1. Several cosmids identified in this manner were further screened for complementation of the PDTC biosynthesis‐negative (Pdt−) phenotype. One cosmid (pT31) complemented the Pdt− phenotype of CTN1 and conferred CCl4 transformation activity and PDTC production upon other pseudomonads. Southern analysis showed that none of three other P. stutzeri strains representing three genomovars contained DNA that would hybridize with the 25 746 bp insert of pT31. Transposon mutagenesis of pT31 identified open reading frames (ORFs) whose disruption affected the ability to make PDTC in the strain CTN1 background. These data describe the pdt locus of strain KC as residing in a non‐essential region of the chromosome subject to spontaneous deletion. The pdt locus is necessary for PDTC biosynthesis in strain KC and is sufficient for PDTC biosynthesis by other pseudomonads but is not a common feature of P. stutzeri strains.
New perspectives on anaerobic methane oxidation
Authors: Valentine David L. , Reeburgh William S.
Published: March 2003

Anaerobic methane oxidation is a globally important but poorly understood process. Four lines of evidence have recently improved our understanding of this process. First, studies of recent marine sediments indicate that a consortium of methanogens and sulphate‐reducing bacteria are responsible for anaerobic methane oxidation; a mechanism of ‘reverse methanogenesis’ was proposed, based on the principle of interspecies hydrogen transfer. Second, studies of known methanogens under low hydrogen and high methane conditions were unable to induce methane oxidation, indicating that ‘reverse methanogenesis’ is not a widespread process in methanogens. Third, lipid biomarker studies detected isotopically depleted archaeal and bacterial biomarkers from marine methane vents, and indicate that Archaea are the primary consumers of methane. Finally, phylogenetic studies indicate that only specific groups of Archaea and SRB are involved in methane oxidation. This review integrates results from these recent studies to constrain the responsible mechanisms.
Characterization of depth‐related population variation in microbial communities of a coastal marine sediment using 16S rDNA‐based approaches and quinone profiling
Authors: Urakawa Hidetoshi , Yoshida Tsutomu , Nishimura Masahiko , Ohwada Kouichi
Published: March 2003

Depth‐related changes in whole‐community structure were evaluated in a coastal marine sediment using a molecular fingerprinting method, terminal restriction fragment length polymorphism (T‐RFLP) analysis, and a chemotaxonomic technique (quinone profiling). Dendrograms derived from both T‐RFLP analysis and quinone profiling indicated a significant variation in microbial community structure between the 0–2 cm layer and deeper layers. This corresponded to the dramatic change in the redox potential, acid‐volatile sulphide‐sulphur and bacterial numbers observed at 0–2 cm and 2–4 cm depths. A significant change in the number of terminal restriction fragments (T‐RFs) was also detected at this transition depth. However, the change in major T‐RFs with depth was not seen in electropherograms. The population changes were primarily variations in minor ribotypes. Most quinone homologues were detected at all depths, although the quinone composition changed with depth. Therefore, quinone profiling also suggested that the depth‐related variation was primarily attributable to minor bacterial groups rather than change in the major population structure. 16S rDNA clone library analysis revealed that clones belonging to the generaVibrioandSerratiapredominated as major bacterial groups at all depths. Our data suggested that the sediment community might result from sedimentation effects of sinking particles. Overall, our results demonstrated that the combined methods of T‐RFLP analysis and quinone profiling were effective for assessing depth‐related microbial populations
A gene containment strategy based on a restriction–modification system
Authors: Torres Begoña , Jaenecke Susanne , Timmis Kenneth N. , García José Luis , Díaz Eduardo
Published: March 2003

Engineering barriers to the spread of specific genes are of great interest both to increase the predictability of recombinant microorganisms used for environmental applications and to study the role of gene transfer in the adaptation of microbial communities to changing environments. We report here a new gene containment circuit based on a toxin–antidote pair that targets the cell DNA, i.e. the type II EcoRI restriction–modification system. The set‐up involved linkage of the ecoRIR lethal gene encoding the EcoRI endonuclease (toxin) to the contained character in a plasmid and chromosomal insertion of the ecoRIM gene encoding the cognate EcoRI methylase (antidote) that protects the target DNA from restriction. Transfer of the contained character to a recipient cell lacking the antidote caused EcoRI‐mediated chromosomal breaks, leading to cell death, thereby preventing gene spread. Using transformation and conjugation as mechanisms of DNA transfer and different environmentally relevant bacteria as recipients, we have shown that the potentially universal EcoRI‐based containment system decreases gene transfer frequencies by more than four orders of magnitude. Analyses of the survivors escaping killing revealed a number of possible inactivation mechanisms.
Population genetics of Escherichia coli in a natural population of native Australian rats
Authors: Pupo Gulietta M. , Lan Ruiting , Reeves Peter R. , Baverstock Peter R.
Published: March 2003

Escherichia coli, a normal inhabitant of the intestinal tract of mammals and birds, is a diverse species. Most studies on E. coli populations involve organisms from humans or human‐associated animals. In this study, we undertook a survey of E. coli from native Australian mammals, predominantly Rattus tunneyi, living in a relatively pristine environment in the Bundjalung National Park. The genetic diversity was assessed and compared by multilocus enzyme electrophoresis (MLEE), sequence analysis of the mdh (malate dehydrogenase) gene and biotyping using seven sugars. Ninety‐nine electrophoretic types were identified from the 242 isolates analysed by MLEE and 15 sequences from the mdh genes sequenced from 21 representative strains. The Bundjalung isolates extend the diversity represented by the E. coli reference (ECOR) set, with new MLEE alleles found in six out of 10 loci. Many of the Bundjalung isolates fell into a discrete group in MLEE. Other Bundjalung strains fell into the recognized E. coli ECOR set groups, but tended to be at the base of both the MLEE and mdh gene trees, implying that these strains are derived independently from ancestral forms of the ECOR groups and that ECOR strains represent only a subset of E. coli adapted to humans and human‐associated animals. Linkage disequilibrium analysis showed that the Bundjalung population has an ‘epidemic’ population structure. The Bundjalung isolates were able to utilize more sugars than the ECOR strains, suggesting that diet plays a prominent role in adaptation of E. coli.
Nitrate‐dependent iron(II) oxidation in paddy soil
Authors: Ratering Stefan , Schnell Sylvia
Published: March 2003

Iron(III) profiles of flooded paddy soil incubated in the greenhouse indicated oxidation of iron(II) in the upper 6 mm soil layer. Measurement of oxygen with a Clark‐type microelectrode showed that oxygen was only responsible for the oxidation of iron(II) in the upper 3 mm. In the soil beneath, nitrate could be used as electron acceptor instead of oxygen for the oxidation of the iron(II). Nitrate was still available 3 mm below the soil surface, and denitrifying activity was indicated by higher concentrations of nitrite between 3 and 6 mm soil depth. Nitrate was generated by nitrification from ammonium. Ammonium concentrations increased beneath 6 mm soil depth, indicating ammonium release and diffusion from deeper soil layers. High concentrations of ammonium were also found at the surface, probably resulting from N2 fixation by cyanobacteria. Experimental adjustment of the nitrate concentration in the flooding water to 200 µM stimulated nitrate‐dependent iron(II) oxidation, which was indicated by significantly lower iron(II) concentrations in soil layers in which nitrate‐dependent iron(II) oxidation was proposed. Soil incubated in the dark showed high iron(III) concentrations only in the layer where oxygen was still available. In this soil, the nitrogen pool was depleted because of the lack of N2 fixation by cyanobacteria. In contrast, soil incubated in the dark with 500 µM nitrate in the flooding water showed significantly higher iron(III) and significantly lower iron(II) concentrations in the anoxic soil layers, indicating nitrate‐dependent iron(II) oxidation. Anoxic incubations of soil with nitrate in the flooding water also showed high concentrations of iron(III) and low concentrations of iron(II) in the upper 3 mm. As oxygen was excluded in anoxic incubations, the high iron(III) concentrations are a sign of the activity of nitrate‐dependent iron(II) oxidizers. The presence of these bacteria in non‐amended soil was also indicated by the most probable number (MPN) counts of nitrate‐dependent iron(II) oxidizers in the layer of 3–4 mm soil depth, which revealed 1.6 × 106 bacteria g−1 dry weight.
Molecular analyses of methyl‐coenzyme M reductase α‐subunit (mcrA) genes in rice field soil and enrichment cultures reveal the methanogenic phenotype of a novel archaeal lineage
Authors: Lueders Tillmann , Chin Kuk‐Jeong , Conrad Ralf , Friedrich Michael
Published: March 2003

The diversity of methanogen‐specific methyl‐coenzyme M reductase α‐subunit (mcrA/mrtA) genes in Italian rice field soil was analysed using a combination of molecular techniques and enrichment cultures. From 75 mcrA/mrtA clones retrieved from rice field soil, 52 were related to members of the Methanosarcinaceae, Methanosaetaceae and Methanobacteriaceae. However, 19 and four clones formed two novel clusters of deeply branching mcrA sequences, respectively, which could not be affiliated to known methanogens. A new methanogen‐specific fingerprinting assay based on terminal restriction fragment length polymorphism (T‐RFLP) analysis of fluorescently labelled polymerase chain reaction (PCR) products allowed us to distinguish all environmental mcrA/mrtA sequences via group‐specific Sau96I restriction sites. Even genes for the isoenzyme methyl‐coenzyme M reductase two (mrtA) of Methanobacteriaceae present in rice field soil were represented by a unique 470 bp terminal restriction fragment (T‐RF). Both cloning and T‐RFLP analysis indicated a significant representation of novel environmental mcrA sequences in rice field soil (238 bp T‐RF). To identify these mcrA sequences, methanogenic enrichment cultures with rice field soil as inoculum were established with H2/CO2 as substrates at a temperature of 50°C, and these were monitored using molecular tools. In subsequent transfers of these enrichment cultures, cloning and T‐RFLP analysis detected predominantly SSU rRNA genes of rice cluster I (RC‐I), an uncultivated euryarchaeotal lineage discovered previously in anoxic rice field soil. In parallel, both mcrA cloning and T‐RFLP analyses of the enrichment culture identified the more frequent cluster of novel environmental mcrA sequences as belonging to members of RC‐I. Thus, we could demonstrate the genotype and phenotype of RC‐I Archaea by the presence of a catabolic gene in a methanogenic enrichment culture before the isolation of pure cultures.
Molecular detection of marine bacterial populations on beaches contaminated by the Nakhodka tanker oil‐spill accident
Authors: Kasai Yuki , Kishira Hideo , Syutsubo Kazuaki , Harayama Shigeaki
Published: March 2003

In January 1997, the tanker Nakhodka sank in the Japan Sea, and more than 5000 tons of heavy oil leaked. The released oil contaminated more than 500 km of the coastline, and some still remained even by June 1999. To investigate the long‐term influence of the Nakhodka oil spill on marine bacterial populations, sea water and residual oil were sampled from the oil‐contaminated zones 10, 18, 22 and 29 months after the accident, and the bacterial populations in these samples were analysed by denaturing gradient gel electrophoresis (DGGE) of PCR‐amplified 16S rDNA fragments. The dominant DGGE bands were sequenced, and the sequences were compared with those in DNA sequence libraries. Most of the bacteria in the sea water samples were classified as the Cytophaga–Flavobacterium–Bacteroides phylum, α‐Proteobacteria or cyanobacteria. The bacteria detected in the oil paste samples were different from those detected in the sea water samples; they were types related to hydrocarbon degraders, exemplified by strains closely related to Sphingomonas subarctica and Alcanivorax borkumensis. The sizes of the major bacterial populations in the oil paste samples ranged from 3.4 × 105 to 1.6 × 106 bacteria per gram of oil paste, these low numbers explaining the slow rate of natural attenuation.
Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes
Authors: Allaway David , Schofield Neil A. , Leonard Mary E. , Gilardoni Liliana , Finan Turlough M. , Poole Philip S.
Published: March 2003

The techniques of differential fluorescence induction (DFI) and optical trapping (OT) have been combined to allow the identification of environmentally induced genes in single bacterial cells. Designated DFI‐OT, this technique allows the in situ isolation of genes driving the expression of green fluorescent protein (Gfp) using temporal and spatial criteria. A series of plasmid‐based promoter probe vectors (pOT) was developed for the construction of random genomic libraries that are linked to gfpUV or egfp. Bacteria that do not express Gfp on laboratory medium (i.e. non‐fluorescent) were inoculated into the environment, and induced genes were detected with a combined fluorescence/optical trapping microscope. Using this selection strategy, rhizosphere‐induced genes with homology to thiamine pyrophosphorylase (thiE) and cyclic glucan synthase (ndvB) were isolated. Other genes were expressed late in the stationary phase or as a consequence of surface‐dependent growth, including fixND and metX, and a putative ABC transporter of putrescine. This strategy provides a unique ability to combine spatial, temporal and physical information to identify environmental regulation of bacterial gene expression.
Short‐ and long‐term changes in proteome composition and kinetic properties in a culture of Escherichia coli during transition from glucose‐excess to glucose‐limited growth conditions in continuous culture and vice versa
Authors: Wick Lukas M. , Quadroni Manfredo , Egli Thomas
Published: March 2003

To investigate the ability of Escherichia coli K12 MG1655 to cope with excess and limitation of a carbon and energy source, we studied the changes in kinetic properties and two‐dimensional (2D) gel protein patterns of an E. coli culture. The population was transferred from glucose‐excess batch to glucose‐limited continuous culture (D = 0.3 h−1), in which it was cultivated for 500 h (217 generations) and then transferred back to glucose‐excess batch culture. Two different stages to glucose‐limitation were recognized: a short‐term physiological adaptation characterized by a general effort in enhancing the cell's substrate scavenging ability and mutations resulting in a population exhibiting increased glucose affinity. Physiological short‐term adaptation to glucose‐limitation was achieved by upregulation of 12 proteins, namely MglB, MalE, ArgT, DppA, RbsB, YdcS, LivJ (precursor), UgpB (precursor), AceA, AldA, AtpA and GatY. Eight of these proteins are periplasmic binding proteins of ABC transporters. Most of them are not involved in glucose transport regulons, but rather in chemotaxis and transport of other substrates, whereas MalE and MglB have previously been shown to belong to transport systems important in glucose transport under glucose‐limited conditions. Evolution under low glucose concentration led to an up to 10‐fold increase in glucose affinity (from a Ks of 366 ± 36 µg l−1 at the beginning to 44 ± 7 µg l−1). The protein pattern of a ‘500‐h‐old’ continuous culture showed a highly increased expression of MglB and MalE as well as of the regulator protein MalI. When adapted cells taken from the ‘500‐h‐old’ continuous culture were transferred to batch culture, an increased expression of MalE was observed, compared with cells from un‐adapted batch‐grown cells. Otherwise, no significant changes were observed in the protein pattern of batch‐grown populations before and after 500 h of evolution in the glucose‐limited continuous culture.
Characterization of nickel‐resistant bacteria isolated from serpentine soil
Authors: Mengoni A. , Barzanti R. , Gonnelli C. , Gabbrielli R. , Bazzicalupo M.
Published: March 2003

In the present study, heterotrophic nickel‐resistant bacteria were isolated and characterized from three different serpentine outcrops in central Italy populated by the nickel‐hyperaccumulating plant Alyssum bertolonii. Bacteria were isolated from the rhizosphere of the plant and from soil portions at various distances from the plant. The proportion of nickel‐resistant cfu was higher in proximity to the plant than in free soil. A total of 138 isolates was collected and grouped into 47 different operational taxonomic units (OTUs) by means of amplified ribosomal DNA restriction analysis (ARDRA) and into 25 heavy‐metal resistant phenotypes. The phylogenetic position of strains belonging to 20 OTUs, representing more than the 70% of the total isolates, was determined by 16S rDNA sequencing. These analyses showed that the most represented genera in all three different outcrops were Pseudomonas and Streptomyces. Pseudomonas strains were found to be predominant in the plant rhizosphere, whereas Streptomyces strains were mainly present in the soil.
Comparative analysis of polychlorinated biphenyl‐dechlorinating communities in enrichment cultures using three different molecular screening techniques
Authors: Watts Joy E. M. , Wu Qingzhong , Schreier Susan B. , May Harold D. , Sowers Kevin R.
Published: March 2003

The catalysts for many microbially mediated environmental processes such as the dechlorination of polychlorinated biphenyls (PCBs) have been difficult to identify by traditional isolation techniques. Numerous, as yet unsuccessful, attempts have been made to isolate and culture the dechlorinating species. To overcome this limitation, amplified rDNA restriction analysis (ARDRA) of a clone library, denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (TRFLP) were used concurrently to compare their effectiveness for characterizing an enriched microbial community. These methods were applied to enrichment cultures that selectively dechlorinated double‐flanked chlorines in the PCB congener 2,3,4,5 chlorinated biphenyl. The methods have different biases, which were apparent from discrepancies in the relative clone frequencies (ARDRA), band intensities (DGGE) or peak heights (TRFLP) from the same enrichment culture. However, each method was effectively qualitative and identified the same organisms: a low G + C Gram‐positive eubacterium, an organism most similar to the green non‐sulphur bacteria, an Aminobacterium sp. and a Desulfovibrio sp. Overall, in community fingerprinting and preliminary identification, DGGE proved to be the most rapid and effective tool for the monitoring of microorganisms within a highly enriched culture. TRFLP results corroborated DGGE fingerprint analysis; however, identification required the additional step of creating a clone library. ARDRA provided an in‐depth analysis of the community and this technique detected slight intraspecies sequence variation in 16S rDNA. These molecular methods are common in environmental microbiology, but rarely are they compared with the same sample site or culture. In general, all three methods detected similar community profiles, but inherent biases resulted in different detection limits for individual OTUs (operational taxonomic units).
Reduction of humic substances by halorespiring, sulphate‐reducing and methanogenic microorganisms
Authors: Cervantes Francisco J. , Bok Frank A. M. de , Duong‐Dac Tuan , Stams Alfons J. M. , Lettinga Gatze , Field Jim A.
Published: March 2003

Physiologically distinct anaerobic microorganisms were explored for their ability to oxidize different substrates with humic acids or the humic analogue, anthraquinone‐2,6‐disulphonate (AQDS), as a terminal electron acceptor. Most of the microorganisms evaluated including, for example, the halorespiring bacterium, Desulfitobacterium PCE1, the sulphate‐reducing bacterium, Desulfovibrio G11 and the methanogenic archaeon, Methanospirillum hungatei JF1, could oxidize hydrogen linked to the reduction of humic acids or AQDS. Desulfitobacterium dehalogenans and Desulfitobacterium PCE1 could also convert lactate to acetate linked to the reduction of humic substances. Humus served as a terminal electron acceptor supporting growth of Desulfitobacterium species, which may explain the recovery of these microorganisms from organic rich environments in which the presence of chlorinated pollutants or sulphite is not expected. The results suggest that the ubiquity of humus reduction found in many different environments may be as a result of the increasing number of anaerobic microorganisms, which are known to be able to reduce humic substances.
Effect of oil pollution on euendolithic cyanobacteria of the Arabian Gulf
Authors: Al‐Thukair Assad A.
Published: March 2003

Microbial euendoliths (true borer) cyanobacteria are carbonate‐boring microorganisms found in modern and ancient marine environments. Modern euendoliths include a wide range of prokaryotes as well as eukaryotes, which have been reported world‐wide. The importance of euendolithic cyanobacteria concerns their role in bio‐erosion of calcium carbonate substrates and as ecological indicators of shallow, tropical and subtropical marine environments. Arabian Gulf ooids from four sites along the east coast of Saudi Arabia have been bored and inhabited by several species of euendolithic cyanobacteria. This assemblage of different species exists simultaneously within the same ooid grain. Comparisons of 1989 and 1992 data reveal a drastic reduction in active euendoliths, and the average numbers of colonies in these ooids. This study reveals the harmful effect of the 1991 oil spill on these unique microorganisms residing in these ooids.
Correlation between pigmentation and antifouling compounds produced by Pseudoalteromonas tunicata
Authors: Egan Suhelen , James Sally , Holmström Carola , Kjelleberg Staffan
Published: March 2003

Pseudoalteromonas tunicata is a marine bacterium with the ability to prevent biofouling by the production of at least four target‐specific compounds. In addition to these antifouling compounds, P. tunicata produces at least two pigments. These include a yellow and a purple pigment which, when combined, give the bacterium a dark green appearance. Transposon mutagenesis was used in this study to investigate the correlation between pigment production and the expression of specific antifouling phenotypes in P. tunicata. Four different categories of pigmentation mutants were isolated including yellow, dark‐purple, light‐purple and white mutants. The mutants were tested for their ability to inhibit the settlement of invertebrate larvae, algal spore germination, fungal growth and bacterial growth. The results showed that the yellow‐pigmented mutants retained full antifouling activity, whereas the purple and white mutant strains had lost some, or all, of their ability to inhibit target organisms. This demonstrates that the loss of antifouling capabilities correlates with the loss of yellow pigment and not purple pigment. Sequencing and analysis of the genes disrupted by the transposons in these mutants identified a number of potential biosynthetic enzymes and transport systems involved in the synthesis and regulation of pigmentation and fouling inhibitors in this organism.
Identification of complex composition, strong strain diversity and directional selection in local Pseudomonas stutzeri populations from marine sediment and soils
Authors: Sikorski Johannes , Möhle Martin , Wackernagel Wilfried
Published: March 2003

Members of Pseudomonas stutzeri have been isolated world‐wide from various habitats including aquatic and terrestrial ecosystems. The global population has a clonal structure, is of exceptionally high genetic diversity and has been grouped into eight genomovars. We have analysed four local populations (n = 89–125) from three geographically separated habitats (two from a marine sediment and two from different soils) by random amplified polymorphic DNA–polymerase chain reaction (RAPD‐PCR), restriction fragment length polymorphism (RFLP) of the rpoB gene and 16S rDNA sequences in order to quantify the influence of evolutionary forces on closely related groups of proliferating cells in situ. All populations consisted of a complex structure of genomic subgroups with variable numbers of members. The analyses revealed that the two populations from marine sediment were rather similar. At least three of the populations were influenced by migrational input as concluded from the presence of members from different genomovars. All populations showed very high strain diversity suggesting strong influence of mutability. Neutrality tests indicated that two or possibly three of the populations were shaped by directional selection. Thus, the local populations of P. stutzeri reflect already the high genetic diversity of the global population and are influenced, to different extents, by migration, mutation and directional selection.
Compatible solutes of organisms that live in hot saline environments
Authors: Santos Helena , Da Costa Milton S.
Published: March 2003

The accumulation of organic solutes is a prerequisite for osmotic adjustment of all microorganisms. Thermophilic and hyperthermophilic organisms generally accumulate very unusual compatible solutes namely, di‐myo‐inositol‐phosphate, di‐mannosyl‐di‐myo‐­inositol‐phosphate, di‐glycerol‐phosphate, mannosylglycerate and mannosylglyceramide, which have not been identified in bacteria or archaea that grow at low and moderate temperatures. There is also a growing awareness that some of these compatible solutes may have a role in the protection of cell components against thermal denaturation. Mannosylglycerate and di‐glycerol‐phosphate have been shown to protect enzymes and proteins from thermal denaturation in vitro as well, or better, than compatible solutes from mesophiles. The pathways leading to the synthesis of some of these compatible solutes from thermophiles and hyperthermophiles have been elucidated. However, large numbers of questions remain unanswered. Fundamental and applied interest in compatible ­solutes and osmotic adjustment in these organisms, drives research that, will, in the near future, allow us to understand the role of compatible solutes in osmotic protection and thermoprotection of some of the most fascinating organisms known on Earth.
Influence of the growth substrate on the mycolic acid profiles of mycobacteria
Authors: Wick Lukas Y. , Wattiau Pierre , Harms Hauke
Published: March 2003

The influence of growth substrates on mycolic acid profiles of PAH‐degrading Mycobacterium spp. LB501T, LB307T and VM552 was examined by high‐performance liquid chromatography (HPLC) using glucose, alkanes, polycyclic aromatic hydrocarbons (PAH) or Luria–Bertani medium (LB) as the sole carbon source. The substrates gave rise to varying mycolic acid profiles, as bacteria growing on poorly water‐soluble substrates exhibited more hydrophobic mycolic acids than cells grown on glucose. Our results indicate that mycobacteria respond to the growth substrate by changing the mycolic acid composition of their cell wall, pointing at the importance of the growth substrate for mycolic acid profiling as an identification method of actinomycetes.
Characterization of the surface hydrophobicity of filamentous fungi
Authors: Smits Theo H. M. , Wick Lukas Y. , Harms Hauke , Keel Christoph
Published: March 2003

A method for the quantitative analysis of the hydrophobicity of the mycelial mat of filamentous fungi based on contact angle measurements is presented. It was tested for a range of fungi belonging to the classes of basidiomycetes, ascomycetes and deuteromycetes. The measured contact angles of the mycelial mats ranged between hydrophilic (<30°) for the deuteromycetes Fusarium oxysporum Fo47 GUS1 and Trichoderma harzianum P1[pZEGA1] and hydrophobic (>60°) for the ascomycete Cladosporium sp. DSE48.1b and the basidiomycetes Paxillus involutus WSL 37.7, Hebeloma crustiliniforme WSL 6.2, Suillus bovinus WSL 48.1 and Laccaria bicolor WSL 73.1. For some fungi, variations in the hydrophobicity of the mycelium depending on the growth medium, the physiological state and the exposure to water were distinguished.