bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic integration: studying the ecology and evolution of complex phenotypes
Authors: Pigliucci Massimo
Published: March 2003

Phenotypic integration refers to the study of complex patterns of covariation among functionally related traits in a given organism. It has been investigated throughout the 20th century, but has only recently risen to the forefront of evolutionary ecological research. In this essay, I identify the reasons for this late flourishing of studies on integration, and discuss some of the major areas of current endeavour: the interplay of adaptation and constraints, the genetic and molecular bases of integration, the role of phenotypic plasticity, macroevolutionary studies of integration, and statistical and conceptual issues in the study of the evolution of complex phenotypes. I then conclude with a brief discussion of what I see as the major future directions of research on phenotypic integration and how they relate to our more general quest for the understanding of phenotypic evolution within the neo‐Darwinian framework. I suggest that studying integration provides a particularly stimulating and truly interdisciplinary convergence of researchers from fields as disparate as molecular genetics, developmental biology, evolutionary ecology, palaeontology and even philosophy of science.
The generation of multiple co‐existing mal‐regulatory mutations through polygenic evolution in glucose‐limited populations of Escherichia coli
Authors: Notley‐McRobb Lucinda , Ferenci Thomas
Published: March 2003

The multicomponent glucose transport system of Escherichia coli was used to study the polygenic basis of increased fitness in prolonged nutrient‐limited, continuous cultures. After 280 generations of glucose‐limited growth, nearly all bacteria in four independent chemostat populations exhibited increased glucose transport and contained multiple, stable mutations. Fitter bacteria increased outer membrane permeability for glucose through overexpression of the LamB glycoporin. Three classes of mutation influenced LamB levels as well as regulation of other mal genes. Low‐level mal/lamB constitutivity resulted from mlc mutations acquired in all populations as well as changes at another uncharacterized locus. Larger increases in transporter content resulted from widespread acquisition of a regulatory malT–con mutation in fit isolates. The malT mutations sequenced from 67 adapted isolates were all single base substitutions resulting in amino acid replacements in the N‐terminal third of the MalT activator protein. Analysis of malT–con sequences revealed a mutational spectrum distinct from that found in plate‐selected malT mutants, suggesting that mutational pathways were affected by environmental factors. A second major finding was the remarkable allele diversity in malT within a population derived from a single clone, with at least 11 different alleles co‐existing in a population. The multiplicity of alleles (as well as those found in adaptive mgl changes in the accompanying study) suggest that the periodic selection events observed previously in such populations are not a major factor in reducing genetic diversity. A simple model is presented for the generation of genetic heterogeneity in bacterial populations undergoing polygenic selection.
Evidence for recent intergeneric transfer of a new tetracycline resistance gene, tet(W), isolated from Butyrivibrio fibrisolvens, and the occurrence of tet(O) in ruminal bacteria
Authors: Barbosa Teresa M. , Scott Karen P. , Flint Harry J.
Published: March 2003

We have previously reported high‐frequency transfer of tetracycline resistance between strains of the rumen anaerobic bacterium Butyrivibrio fibrisolvens. Donor strains were postulated to carry two TcR genes, one of which is transferred on a novel chromosomal element. It is shown here that coding sequences within the non‐transmissible gene in B. fibrisolvens 1.230 are identical to those of the Streptococcus pneumoniae tet(O) gene. This provides the first evidence for genetic exchange between facultatively anaerobic bacteria and rumen obligate anaerobes. In contrast, the product of the transmissible TcR gene shares only 68% amino acid sequence identity with the TetO and TetM proteins and represents a new class of ribosome protection tetracycline resistance determinant, designated Tet W. The tet(W) coding region shows a higher DNA G + C content (53%) than other B. fibrisolvens genes or other ribosome protection‐type tet genes, suggesting recent acquisition from a high G + C content genome. Tet(W) genes with almost identical sequences are also shown to be present in TcR strains of B. fibrisolvens from Australian sheep and in TcR strains of two other genera of rumen obligate anaerobes, Selenomonas ruminantium and Mitsuokella multiacidus. This provides compelling evidence for recent intergeneric transfer of resistance genes between ruminal bacteria. Tet(W) is not restricted to ruminal bacteria, as it was also present in a porcine strain of M. multiacidus.
Phylogeny and in situ identification of a morphologically conspicuous bacterium, Candidatus Magnospira bakii, present at very low frequency in activated sludge
Authors: Snaidr Jiri , Fuchs Bernhard , Wallner Günter , Wagner Michael , Schleifer Karl‐Heinz , Amann Rudolf
Published: March 2003

A morphologically conspicuous bacterium that constituted a very small fraction (< 0.01%) of the total microbial community of activated sludge was enriched and analysed phylogenetically by a combination of cultivation‐independent molecular and physical methods. The large, corkscrew‐shaped, filamentous bacteria were first detected in municipal activated sludge by light microscopy owing to their unusual rotating gliding motility. Various attempts at microbiological enrichment and pure culture isolation with traditional techniques failed, as did attempts to retrieve the morphotype of interest by micromanipulation. In situ hybridization with the group‐specific, rRNA‐targeted oligonucleotide probe CF319a indicated a phylogenetic affiliation to the Cytophaga–Flexibacter group of the Cytophaga–Flavobacterium–Bacteroides phylum. Based on strong morphological resemblance to members of the genus Saprospira, additional 16S rRNA‐targeted oligonucleotides with more narrow specificity were designed and evaluated for in situ hybridization to the morphotype of interest. Flow cytometric cell sorting based on the fluorescence conferred by probe SGR1425 and forward scatter enabled a physical enrichment of the helical coiled cells. Subsequent polymerase chain reaction (PCR) amplification of 16S rDNA fragments from whole fixed sorted cells with a primer pair based on probes CF319a and SGR1425 resulted in the retrieval of 12 almost identical partial 16S rDNA fragments with sequence similarities among each other of more than 99.2%. In situ hybridizations proved that the sequences that showed the highest similarity (88.4%) to the 16S rRNA of Saprospira grandis were indeed retrieved from the corkscrew‐shaped filaments. The bacterium is likely to be a member of a genus of which no species has been cultured hitherto. It was consequently tentatively named ‘Magnospira bakii’ and has the taxonomic rank of Candidatus Magnospira bakii, as the ultimate taxonomic placement has to await its cultivation. In this study, it was demonstrated that even bacteria occurring at very low frequencies in highly complex environmental samples can be retrieved selectively without cultivation for further molecular analysis.
Application of nested polymerase chain reaction for the detection of as yet uncultured organisms of the class Actinobacteria in environmental samples
Authors: Rheims Holger , Stackebrandt Erko
Published: March 2003

Different groups of as yet uncultured, phylogenetically deeply rooting organisms of the class Actinobacteria have been shown previously by analysis of 16S rDNA clone libraries to be present in different soil and marine environments of worldwide distribution. In order to monitor specifically the occurrence of two of the actinobacterial groups among bacterial DNA extracted from different environments, a nested polymerase chain reaction (PCR) method, aiming at the sensitive detection of 16S rDNA, was used. Analysis of a clone library generated from one of these 16S rDNA‐derived, group‐specific PCR products of DNA from a forest soil broadened the spectrum of putative taxa within the two phylogenetic groups. Application of this PCR system demonstrated the presence of these organisms in natural environments and in a sample of dried and moistened peat. Fluctuations in the detectability of the target groups in different samples from the same site strongly suggest that the taxon diversity of a 16S rDNA clone library must be regarded as a temporal genomic snapshot taken from this environmental community.
Methanogenic archaea and CO2‐dependent methanogenesis on washed rice roots
Authors: Lehmann‐Richter  , Großkopf  , Liesack  , Frenzel  , Conrad
Published: March 2003

Washed excised roots of rice (Oryza sativa) immediately started to produce CH4 when they were incubated in phosphate buffer under anoxic conditions (N2 atmosphere), with initial rates varying between 2 and 70 nmol h−1 g−1 dry weight of root material (mean ± SE: 20.3 ± 5.9 nmol h−1 g−1 dry weight; n = 18). Production of CH4 continued for at least 500 h, with rates usually decreasing slowly. CH4 production was not significantly affected by methyl fluoride, an inhibitor of acetoclastic methanogenesis. Less than 0.5% of added [2‐14C]‐acetate was converted to 14CH4, and conversion of 14CO2 to 14CH4 indicated that CH4 was almost exclusively produced from CO2. Occasionally, however, especially when the roots were incubated without additional buffer, CH4 production started to accelerate after about 200 h reaching rates of > 100 nmol h−1 g−1 dry weight. Methyl fluoride inhibited methanogenesis by more than 20% only in these cases, and the conversion of 14CO2 to 14CH4 decreased. These results indicate that CO2‐dependent rather than acetoclastic methanogenesis was primarily responsible for CH4 production in anoxically incubated rice roots. Determination of most probable numbers of methanogens on washed roots showed highest numbers (106 g−1 dry roots) on H2 and ethanol, i.e. substrates that support CH4 production from CO2. Numbers on acetate (105 g−1 dry roots) and methanol (104 g−1 dry roots) were lower. Methanogenic consortia enriched on H2 and ethanol were characterized phylogenetically by comparative sequence analysis of archaeal small‐subunit (SSU) ribosomal RNA‐encoding genes (rDNA). These sequences showed a high similarity to SSU rDNA clones that had been obtained previously by direct extraction of total DNA from washed rice roots. The SSU rDNA sequences recovered from the H2/CO2‐using consortium either belonged to a novel lineage of methanogens that grouped within the phylogenetic radiation of the Methanosarcinales and Methanomicrobiales or were affiliated with Methanobacterium bryantii. SSU rDNA sequences retrieved from the ethanol‐using consortium either grouped within the genus Methanosarcina or belonged to another novel lineage within the phylogenetic radiation of the Methanosarcinales and Methanomicrobiales. Cultured organisms belonging to either of the two novel lineages have not been reported yet.
Phylogenetic diversity of termite gut spirochaetes
Authors: Lilburn  , Schmidt  , Breznak
Published: March 2003

A molecular phylogenetic analysis was done of not‐yet‐cultured spirochaetes inhabiting the gut of the termite, Reticulitermes flavipes (Kollar). Ninety‐eight clones of near‐full‐length spirochaetal 16S rDNA genes were classified by ARDRA pattern and by partial sequencing. All clones grouped within the genus Treponema, and at least 21 new species of Treponema were recognized within R. flavipes alone. Analysis of 190 additional clones from guts of Coptotermes formosanus Shiraki and Zootermopsis angusticollis (Hagen), as well as published data on clones from Cryptotermes domesticus (Haviland), Mastotermes darwiniensis Froggatt, Nasutitermes lujae (Wasmann) and Reticulitermes speratus (Kolbe), revealed a similar level of novel treponemal phylogenetic diversity in these representatives of five of the seven termite families. None of the clones was closely related (i.e. all bore ≤ 91% sequence similarity) to any previously recognized treponeme. The data also revealed the existence of two major phylogenetic groups of treponemes: one containing all of the currently known isolates of Treponema and a large number of phylotypes from the human gingival crevice, but only a minority of the termite gut spirochaete clones; another containing the majority of termite spirochaete clones and two Spirochaeta (S. caldaria and S. stenostrepta ), which, although free living, group within the genus Treponema on the basis of 16S rRNA sequence. Signature nucleotides that almost perfectly distinguished the latter group, herein referred to as the ‘termite cluster’, occurred at the following (E. coli numbering) positions: 289‐G · C‐311; A at 812; and an inserted nucleotide at 1273. The emerging picture is that the long‐recognized and striking morphological diversity of termite gut spirochaetes is paralleled by their phylogenetic diversity and may reflect substantial physiological diversity as well.
Novel bacterial endosymbionts of Acanthamoeba spp. related to the Paramecium caudatum symbiont Caedibacter caryophilus
Authors: Horn Matthias , Fritsche Thomas R. , Gautom Romesh K. , Schleifer Karl‐Heinz , Wagner Michael
Published: March 2003

Acanthamoebae are increasingly being recognized as hosts for obligate bacterial endosymbionts, most of which are presently uncharacterized. In this study, the phylogeny of three Gram‐negative, rod‐shaped endosymbionts and their Acanthamoeba host cells was analysed by the rRNA approach. Comparative analyses of 16S rDNA sequences retrieved from amoebic cell lysates revealed that the endosymbionts of Acanthamoeba polyphaga HN‐3, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39 are related to the Paramecium caudatum endosymbionts Caedibacter caryophilus, Holospora elegans and Holospora obtusa. With overall 16S rRNA sequence similarities to their closest relative, C. caryophilus, of between 87% and 93%, these endosymbionts represent three distinct new species. In situ hybridization with fluorescently labelled endosymbiont‐specific 16S rRNA‐targeted probes demonstrated that the retrieved 16S rDNA sequences originated from the endosymbionts and confirmed their intracellular localization. We propose to classify provisionally the endosymbiont of Acanthamoeba polyphaga HN‐3 as ‘Candidatus Caedibacter acanthamoebae’, the endosymbiont of Acanthamoeba sp. strain UWC9 as ‘Candidatus Paracaedibacter acanthamoebae’ and the endosymbiont of Acanthamoeba sp. strain UWE39 as ‘Candidatus Paracaedibacter symbiosus’. The phylogeny of the Acanthamoeba host cells was analysed by comparative sequence analyses of their 18S rRNA. Although Acanthamoeba polyphaga HN‐3 clearly groups together with most of the known Acanthamoeba isolates (18S rRNA sequence type 4), Acanthamoeba sp. UWC9 and UWE39 exhibit < 92% 18S rRNA sequence similarity to each other and to other Acanthamoeba isolates. Therefore, we propose two new sequence types (T13 and T14) within the genus Acanthamoeba containing, respectively, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39.
Development and characterization of a lux‐modified 2,4‐dichlorophenol‐degrading Burkholderia sp. RASC
Authors: Shaw  , Beaton  , Glover  , Killham  , Meharg
Published: March 2003

lux‐marked biosensors for assessing the toxicity and bioremediation potential of polluted environments may complement traditional chemical techniques. luxCDABE genes were introduced into the chromosome of the 2,4‐dichlorophenol (2,4‐DCP)‐mineralizing bacterium, Burkholderia sp. RASC c2, by biparental mating using the Tn4431 system. Experiments revealed that light output was constitutive and related to cell biomass concentration during exponential growth. The transposon insertion was stable and did not interrupt 2,4‐DCP‐degradative genes, and expression of luxCDABE did not constitute a metabolic burden to the cell. A bioluminescence response was detectable at sublethal 2,4‐DCP concentrations: at < 10.26 μg ml−1, bioluminescence was stimulated (e.g. 218% of control), but at concentrations > 60 μg ml−1 it declined to < 1%. Investigating the effect of [14C]‐2,4‐DCP concentration on the evolution of 14CO2 revealed that, for initial concentrations of 2.5–25 μg ml−1, ≈55% of the added 14C was mineralized after 24 h compared with < 1% at 50 and 100 μg ml−1. Inhibition of 2,4‐DCP mineralization between 25 and 50 μg ml−1 corresponded well to the EC50 value (33.83 μg ml−1) obtained from bioluminescence inhibition studies. lux‐marked RASC c2 may therefore be used as a functionally (i.e. 2,4‐DCP degrader) and environmentally relevant biosensor of toxicity and biodegradation inhibition.
Cell envelope mutants of Pseudomonas putida: physiological characterization and analysis of their ability to survive in soil
Authors: Rodríguez‐Herva José‐Juan , Reniero Daniela , Galli Enrica , Ramos Juan L.
Published: March 2003

To generate mutants with altered lipopolysaccharides (LPS) of the wild‐type Pseudomonas putida KT2442, we used the mini‐Tn5luxAB‐Km transposon. A mutant was found among luminescent colonies and selected as a negative clone in enzyme‐linked immunosorbent assay (ELISA) with monoclonal antibody (mAb) 7.3B, which recognizes the O‐antigen of P. putida LPS. The DNA region of the LPS mutant interrupted by the minitransposon insertion was cloned and sequenced. Comparison of the deduced amino acid sequence with protein sequence databases showed similarity to the O‐antigen polymerase (Wzy) of Salmonella enterica (muenchen). The wild‐type gene was rescued by polymerase chain reaction (PCR), cloned into a broad‐host‐range plasmid and used to carry out complementation assays. The cloned gene was able to restore the wild‐type phenotype of the P. putida wzy mutant. We constructed an isogenic mutant of the luminescent wzy mutant to which an oprL mutation was transferred by homologous recombination with an oprL::xylE cassette. The wzy mutants of P. putida were more sensitive to SDS, deoxycholate and EDTA than the corresponding parental strains. We analysed the ability of wzy, oprL and wzy oprL mutants of P. putida to colonize soil. In comparison with the wild‐type strain, the ability of single mutants to colonize soil decreased; this characteristic was more evident for the double mutant, especially at high temperatures.
Desulfotomaculum genus‐ and subgenus‐specific 16S rRNA hybridization probes for environmental studies
Authors: Hristova Krassimira R. , Mau Margit , Zheng Dandan , Aminov Rustam I. , Mackie Roderick I. , Gaskins H. Rex , Raskin Lutgarde
Published: March 2003

Based on comparative analysis of 16S rRNA sequences and the recently established phylogeny of the genus Desulfotomaculum, a set of phylogenetically nested hybridization probes was developed and characterized. A genus‐specific probe targets all known Desulfotomaculum species (with the exception of Desulfotomaculum acetoxidans ), and five specific probes target subclusters within the Desulfotomaculum genus. The dissociation temperature of each probe was determined experimentally. Probe specificities were verified through hybridizations with pure culture rRNA isolated from a wide variety of target and non‐target organisms and through an evaluation of probe ‘nesting’ using samples obtained from four different environments. Fixation and hybridization conditions for fluorescence in situ hybridizations were also optimized. The probes were used in quantitative membrane hybridizations to determine the abundance of Desulfotomaculum species in thermophilic anaerobic digesters, in soil, in human faeces and in pig colon samples. Desulfotomaculum rRNA accounted for 0.3–2.1% of the total rRNA in the digesters, 2.6–6.6% in soil, 1.5–3.3% in human faeces and 2.5–6.2% in pig colon samples.
Regulation of urease gene expression by Streptococcus salivarius growing in biofilms
Authors: Li Yung‐Hua , Chen Yi‐Ywan M. , Burne Robert A.
Published: March 2003

The metabolism of urea by urease enzymes of oral bacteria profoundly influences oral biofilm pH homeostasis and oral microbial ecology. The purpose of this study was to gain insight into the regulation of expression of the low pH‐inducible urease genes in populations of Streptococcus salivarius growing in vitro in biofilms and to explore whether urease regulation or the levels of urease expression in biofilm cells differed significantly from planktonic cells. Two strains of S. salivarius harbouring urease promoter fusions to a chloramphenicol acetyltransferase (cat ) gene were used: PureICAT, containing a fusion to the full‐length, pH‐sensitive promoter; or PureIΔ100CAT, a constitutively derepressed deletion derivative of the urease gene promoter. The strains were grown in a Rototorque biofilm reactor in a tryptone–yeast extract–sucrose medium with or without pH control. Both CAT and urease activities in biofilms were measured at ‘quasi‐steady state’ and after a 25 mM glucose pulse. The results showed that CAT expression in PureICAT was repressed at relatively neutral pH values, and that expression could be induced by acidic pH after carbohydrate challenge. Biofilms of PureICAT grown at low pH, without buffering, had about 20‐fold higher CAT levels, and only a modest further induction could be elicited with carbohydrate pulsing. The levels of CAT in biofilms of PureICAT grown in buffered medium were slightly higher than those reported for planktonic cells cultured at pH 7.0, and the levels of CAT in PureICAT growing at low pH or after induction were similar to those reported for fully induced planktonic cells. CAT activity in PureIΔ100CAT was constitutively high, regardless of growth conditions. Interestingly, urease activity detected in biofilms of the parent strain, S. salivarius 57.I, could be as much as 130‐fold higher than that reported for fluid chemostat cultures grown under similar conditions. The higher level of urease activity in biofilms was probably caused by the accumulation of the stable urease enzyme within biofilm cells, low pH microenvironments and the growth phase of populations of cells in the biofilm. The ability of S. salivarius biofilm cells to upregulate urease expression in response to pH gradients and to accumulate greater quantities of urease enzyme when growing in bioiflms may have a significant impact on oral biofilm pH homeostasis and microbial ecology in vivo. Additionally, S. salivarius carrying the pH‐sensitive urease gene promoter fused to an appropriate reporter gene may be a useful biological probe for sensing biofilm pH in situ.
Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage
Authors: Béjà Oded , Suzuki Marcelino T. , Koonin Eugene V. , Aravind L. , Hadd Andrew , Nguyen Linh P. , Villacorta Rachel , Amjadi Mojgan , Garrigues Corey , Jovanovich Stevan B. , Feldman Robert A. , DeLong Edward F.
Published: March 2003

Cultivation‐independent surveys of ribosomal RNA genes have revealed the existence of novel microbial lineages, many with no known cultivated representatives. Ribosomal RNA‐based analyses, however, often do not provide significant information beyond phylogenetic affiliation. Analysis of large genome fragments recovered directly from microbial communities represents one promising approach for characterizing uncultivated microbial species better. To assess further the utility of this approach, we constructed large‐insert bacterial artificial chromosome (BAC) libraries from the genomic DNA of planktonic marine microbial assemblages. The BAC libraries we prepared had average insert sizes of ≈ 80 kb, with maximal insert sizes > 150 kb. A rapid screening method assessing the phylogenetic diversity and representation in the library was developed and applied. In general, representation in the libraries agreed well with previous culture‐independent surveys based on polymerase chain reaction (PCR)‐amplified rRNA fragments. A significant fraction of the genome fragments in the BAC libraries originated from as yet uncultivated microbial species, thought to be abundant and widely distributed in the marine environment. One entire BAC insert, derived from an uncultivated, surface‐dwelling euryarchaeote, was sequenced completely. The planktonic euryarchaeal genome fragment contained some typical archaeal genes, as well as unique open reading frames (ORFs) suggesting novel function. In total, our results verify the utility of BAC libraries for providing access to the genomes of as yet uncultivated microbial species. Further analysis of these BAC libraries has the potential to provide significant insight into the genomic potential and ecological roles of many indigenous microbial species, cultivated or not.
Effects of O2 and CH4 on presence and activity of the indigenous methanotrophic community in rice field soil
Authors: Henckel Thilo , Roslev Peter , Conrad Ralf
Published: March 2003

The activity and distribution of methanotrophs in soil depend on the availability of CH4 and O2. Therefore, we investigated the activity and structure of the methanotrophic community in rice field soil under four factorial combinations of high and low CH4 and O2 concentrations. The methanotrophic population structure was resolved by denaturant gradient gel electrophoresis (DGGE) with different PCR primer sets targeting the 16S rRNA gene, and two functional genes coding for key enzymes in methanotrophs, i.e. the particulate methane monooxygenase (pmoA) and the methanol dehydrogenase (mxaF). Changes in the biomass of type I and II methanotrophic bacteria in the rice soil were determined by analysis of phospholipid‐ester‐linked fatty acid (PLFA) biomarkers. The relative contribution of type I and II methanotrophs to the measured methane oxidation activity was determined by labelling of soil samples with 14CH4 followed by analysis of [14C]‐PLFAs. CH4 oxidation was repressed by high O2 (20.5%), and enhanced by low O2 (1%). Depending on the CH4 and O2 mixing ratios, different methanotrophic communities developed with a higher diversity at low than at high CH4 concentration as revealed by PCR‐DGGE. However, a prevalence of type I or II populations was not detected. The [14C]‐PLFA fingerprints, on the other hand, revealed that CH4 oxidation activity was dominated by type I methanotrophs in incubations with low CH4 mixing ratios (1000 p.p.m.v.) and during initiation of CH4 consumption regardless of O2 or CH4 mixing ratio. At high methane mixing ratios (10 000 p.p.m.v.), type I and II methanotrophs contributed equally to the measured CH4 metabolism. Collectively, type I methanotrophs responded fast and with pronounced shifts in population structure and dominated the activity under all four gas mixtures. Type II methanotrophs, on the other hand, although apparently more abundant, always present and showing a largely stable population structure, became active later and contributed to CH4 oxidation activity mainly under high CH4 mixing ratios.
Microbiological, molecular biological and stable isotopic evidence for nitrogen fixation in the open waters of Lake Michigan
Authors: MacGregor Barbara J. , Van Mooy Benjamin , Baker Brett J. , Mellon Mark , Moisander Pia H. , Paerl Hans W. , Zehr Jonathan , Hollander David , Stahl David A.
Published: March 2003

We have used a combination of microbiological, molecular biological and stable isotope methods to relate specific microbial populations to elemental cycling at an offshore site in Lake Michigan. Several lines of evidence suggest that atmospheric N2 may be a significant source of nitrogen to the lake. Particulate organic nitrogen (PON) at ≈ 10–15 m depth in July and October had a δ15N of 0.5–1.5‰. These values closely reflect the 15N composition of atmospheric N2, suggesting biological nitrogen fixation. Historical data show a developing late‐summer N:P minimum at ≈ 15 m; low abundance of inorganic nitrogen relative to phosphorus favours species able to acquire atmospheric nitrogen. Microscopic examination of October water samples revealed abundant heterocystous cyanobacteria, including Nodularia sp. Potentially nitrogen‐fixing Anabaena spp. have been found in Lake Michigan before but, to our knowledge, this is the first report of Nodularia. Finally, we have amplified both cyanobacterial and non‐cyanobacterial nifH sequences (encoding the nitrogenase iron protein) from lakewater samples, evidence for the presence of bacteria capable of nitrogen fixation. The surface waters of Lake Michigan are considered to be phosphate limited in the stratified season and, under these conditions, energetically expensive nitrogen fixation is expected to be uncompetitive with assimilation of combined nitrogen. Our results suggest that, from both microbiological and biogeochemical perspectives, this may be an oversimplification.
Characterization of (R/S)‐mecoprop [2‐(2‐methyl‐4‐chlorophenoxy) propionic acid]‐degrading Alcaligenes sp. CS1 and Ralstonia sp. CS2 isolated from agricultural soils
Authors: Smejkal Christopher W. , Vallaeys Tatiana , Seymour Fabian A. , Burton Sara K. , Lappin‐Scott Hilary M.
Published: March 2003

The herbicide mecoprop [2‐(2‐methyl‐4‐chlorophenoxy) propionic acid] is widely applied to corn fields in order to control broad‐leaved weeds. However, it is often detected in groundwater where it can be a persistent contaminant. Two mecoprop‐degrading bacterial strains were isolated from agricultural soils through their capability to degrade (R/S)‐mecoprop rapidly. 16S rDNA sequencing of the isolates demonstrated that one was closely related to the genera Alcaligenes sp. (designated CS1) and the other to Ralstonia sp. (designated CS2). Additionally, these isolates demonstrated ability to grow on other related herbicides, including 2,4‐D (2,4‐dichlorophenoxyacetic acid), MCPA [4‐chloro‐2‐methyl phenoxy acetic acid] and (R/S)‐2,4‐DP [2‐(2,4‐dichlorophenoxy)propionic acid] as sole carbon sources. tfdABC gene‐specific probes derived from the 2,4‐D‐degrading Variovorax paradoxus TV1 were used in hybridization analyses to establish whether tfd‐like genes are present in mecoprop‐degrading bacteria. Hybridization analysis demonstrated that both Alcaligenes sp. CS1 and Ralstonia sp. CS2 harboured tfdA, tfdB and tfdC genes on plasmids that have approximately > 60% sequence similarity to the tfdA, tfdB and tfdC genes of V. paradoxus. It is therefore likely that tfd‐like genes may be involved in the degradation of mecoprop, and we are currently investigating this further.
Oxygen tension and nutrient starvation are major signals that regulate agfD promoter activity and expression of the multicellular morphotype in Salmonella typhimurium
Authors: Gerstel Ulrich , Römling Ute
Published: March 2003

Expression of multicellular behaviour (rdar morphotype) is a characteristic of wild‐type Salmonella typhimurium strains. The key target for the regulation of rdar morphotype expression is the agfD promoter. The regulation of two rdar morphotypes, regulated and semi‐constitutive (the latter differs from the former by the insertion of A after position −17), by various environmental conditions was studied using transcriptional fusions to the regulated and semi‐constitutive agfD promoters by Western blot analysis and phenotypic analysis of the rdar morphotype. AgfD promoter activities were strongly dependent on oxygen tension. Expression maxima were observed in rich medium under microaerophilic conditions and in minimal medium under aerobic conditions. The regulated rdar morphotype was only expressed under conditions of maximal promoter activity. Glucose did not influence rdar morphotype expression, and the two promoters showed no consistent response to pH. In the stationary phase of growth, nitrogen and phosphate depletion were found to be signals that switch on the agfD promoters. In the logarithmic phase of growth, ethanol was the stress signal that enhanced rdar morphotype expression. The results indicate that, although the regulated and semi‐constitutive agfD promoters are key factors in the grade of expression of the multicellular behaviour, common signals such as oxygen tension, depletion of nutrients and ethanol vary their levels of expression significantly.
Bioaugmentation of soils by increasing microbial richness: missing links
Authors: Dejonghe Winnie , Boon Nico , Seghers Dave , Top Eva M. , Verstraete Willy
Published: March 2003

It is generally assumed that increased microbial diversity corresponds to increased catabolic potential and, hence, to better removal of metabolites and pollutants. Yet, microbial diversity, more specifically richness of species in environmental samples and sites, is difficult to assess. It is proposed to interpret this diversity more in the framework of Pareto's law, i.e. 20% of the species govern 80% of the energy flux of the ecosystem. Ecological studies should attempt to delineate the main energy fluxes and that group of species playing quantitative key roles in the system. Consequently, bioaugmentation should aim at the rearrangement of the group of organisms dominantly involved in the overall energy flux, so that specific catabolic traits necessary for the clean up of pollutants are part of that active group. For soil ecosystems, the capacity of plant roots as creators of physical and chemical discontinuity should be used more strategically to bring about such rearrangements. Overall, this paper identifies a number of ecological concepts, such as the Pareto law, the Gompertz model and plant community‐induced microbial competence, which may, given careful underpinning, open new perspectives for microbial ecology and biodegradation.
Biomonitoring of pJP4‐carrying Pseudomonas chlororaphis with Trb protein‐specific antisera
Authors: Schmidt‐Eisenlohr Heike , Rittig Michael , Preithner Susanne , Baron Christian
Published: March 2003

The transfer of catabolic genes on conjugative plasmids to indigenous organisms from which they may spread further into the community allows the introduction of new biodegradative pathways for metabolic conversion of pollutants to the community. Biomonitoring of IncP plasmid pJP4‐carrying Pseudomonas chlororaphis from the rhizosphere of Arabidopsis thaliana was achieved using antisera specific for proteins from the plasmid transfer machinery. Antisera were generated that recognized TrbC and TrbF, the putative major and minor components of pJP4‐determined pili, respectively, and the putative lipoprotein TrbH. Cell fractionation studies showed association of TrbC, TrbF and TrbH with the cells and suggested that TrbC and TrbF are part of extracellular pJP4‐determined pili. TrbF and TrbH antisera allowed specific detection of IncP compared with IncN or IncW plasmid‐carrying cells and even permitted differentiation between bacteria carrying IncPα plasmid RP4 and IncPβ plasmid pJP4. Immunofluorescence microscopy was applied to detect TrbF and TrbH signal at the cell periphery, allowing distinction from autofluorescing cells and soil debris. In situ experiments showed specific recognition of pJP4‐carrying cells from laboratory cultures, as well as from the rhizosphere of A. thaliana grown in natural soil. After co‐inoculation of donor P. chlororaphis pJP4 and recipient Ralstonia eutropha, a combination of immunofluorescence and oligonucleotide hybridization techniques permitted the detection of plasmid transfer between both organisms in the A. thaliana rhizosphere. This strategy may be generally applicable for the analysis of plasmid transfer in natural ecosystems.
Toxicity and synergism in transgenic Escherichia coli expressing four genes from Bacillus thuringiensis subsp. israelensis
Authors: Khasdan Vadim , Ben‐Dov Eitan , Manasherob Robert , Boussiba Sammy , Zaritsky Arieh
Published: March 2003

The genes cyt1Aa and p20, encoding, respectively, cytolytic and accessory proteins of Bacillus thuringiensis subsp. israelensis, were introduced into previously constructed clones expressing cry4Aa and cry11Aa in Escherichia coli (Ben‐Dov et al., 1995). Fifteen clones with all possible combinations of the four genes were obtained and found to express the genes included. Two new combinations, pVE4‐ADRC and pVE4‐ARC, expressing cyt1Aa, p20 and cry4Aa, with or without cry11Aa, respectively, were more toxic than their counterparts without cyt1Aa. They displayed the highest toxicity against Aedes aegypti larvae ever reached in transgenic bacteria. Five out of the six clones (except pVE4‐DC) containing cry4Aa or cry11Aa (with or without p20) displayed varying levels of synergism with cyt1Aa: they are 1.5‐to 34‐fold more toxic than the respective clones without cyt1Aa against exposed larvae. Their lethal times also decreased (they kill larvae quicker), more so at higher cell concentrations. These clones are anticipated to dramatically reduce the likelihood of resistant development in the target organisms (Wirth et al., 1997).
Rhizobial position as a main determinant in the problem of competition for nodulation in soybean
Authors: López‐García Silvina L. , Vázquez Tirso E. E. , Favelukes Gabriel , Lodeiro Aníbal R.
Published: March 2003

Selected Bradyrhizobium japonicum strains inoculated on soybean seeds often fail to occupy a significant proportion of nodules when a competitor rhizobial population is established in the soil. This competition problem could result from a genetic/ physiological advantage of the adapted soil population over the introduced inoculant or from a positional advantage, as the soil population already occupies the soil profile where the roots will penetrate, whereas the inoculant remains concentrated around the seeds. Here, we have assessed the contribution of these factors with a laboratory model in which a rhizobial population is established in sterile vermiculite. We observed that the wild‐type strain B. japonicum LP 3004 was able to grow in pots with N‐free plant nutrient solution‐watered vermiculite for six or seven generations with a duplication rate of at least 0.7 day−1. In addition, the rhizobial population persisted for 3 months with 106–107 colony‐forming units ml−1 of the vermiculite‐retained solution. N‐starved, young rhizobial cultures are more efficient in performing several steps along their early association with soybean roots. However, N starvation during growth of rhizobia used for seed inoculation did not enhance their competitiveness against a 1 month vermiculite‐established rhizobial population, which occupied more than 72% of the nodules. When a similarly established rhizobial population was recovered from the vermiculite and homogeneously suspended in plant nutrient solution, these cells were significantly less competitive (29% of nodules occupied) than rhizobia obtained from a fresh, logarithmic culture in a N‐poor minimal medium, thus indicating that cell position rather than intrinsic competitiveness was the determinant for nodule occupation.
Genetic variability of Tuber uncinatum and its relatedness to other black truffles
Authors: Mello Antonietta , Cantisani Annamaria , Vizzini Alfredo , Bonfante Paola
Published: March 2003

Genetic variability is one of the major survival strategies developed by symbiotic fungi. We focused on the ectomycorrhizal fungus Tuber uncinatum Chatin that produces edible ascomata. In order to understand the degree of its variability and its relatedness to another morphologically‐similar truffle, T. aestivum Vittad., ascomata of T. uncinatum were collected from a single natural truffle‐ground located in the north of Italy and compared with samples from other Italian sites, as well as with T. aestivum ascomata from other European regions.

We used multi‐locus approaches, such as microsatellite‐primed PCR (polymerase chain reaction), and single locus markers, such as mitochondrial and nuclear ribosomal DNA on 30 samples. The results demonstrate that the level of genetic polymorphism among isolates of T. uncinatum was higher than in other Tuber species, like T. melanosporum. Neighbour‐joining analyses were carried out on a binary data matrix on 12 ascomata of T. uncinatum and T. aestivum, and on 15 internal transcribed spacer (ITS) sequences of these species and 5 from other Tuber species. Taken together, they clustered T. uncinatum and T. aestivum in two separate groups. The mitochondrial rDNA primers, NMS1 and NMS2, were not able to differentiate morphologically related and unrelated truffles. Moreover, a pair of primers, intentionally designed to differentiate isolates of T. aestivum and T. uncinatum from other Tuber species, successfully amplified DNA from all the samples of T. aestivum and T. uncinatum considered in our analysis. In conclusion, different molecular approaches separate T. aestivum and T. uncinatum according to their spore reticulum and their taste and smell.
Approaches to prokaryotic biodiversity: a population genetics perspective
Authors: Rodríguez‐Valera Francisco
Published: March 2003

The study of prokaryotic diversity has blossomed during the last 10–15 years as a result of the introduction of molecular identification, mostly based on direct 16S rRNA gene polymerase chain reaction (PCR) amplification and sequencing from natural samples. A large amount of information exists about the diversity of this specific gene. However, data from the field of bacterial population genetics and genomics make questionable the value of information regarding just one gene. Even if we accept 16S rRNA genes as useful for species identification, intraspecific variation in bacteria is so high that species catalogues are often of little value. The gene pools represented by an operational species are yet impossible to predict. On the other hand, adaptive features in prokaryotes are often coded in gene clusters (genomic islands) that can be cloned directly from the environment, sequenced and even expressed in a surrogate host. Thus, the study of the environmental genome or metagenome appears as an alternative that could eventually lead to a more realistic understanding of prokaryotic biodiversity, provide biotechnology with new tools and maybe even contribute to develop a model of prokaryotic evolution.
Adaptation to nickel spiking of bacterial communities in neocaledonian soils
Authors: Héry Marina , Nazaret Sylvie , Jaffré Tanguy , Normand Philippe , Navarro Elisabeth
Published: March 2003

Adaptation to nickel of bacterial communities of two extreme neocaledonian soils (an ultramafic soil and an acidic soil) was investigated by nickel spiking and compared with adaptation in a non‐neocaledonian soil used as reference. Soil microcosms were amended with nickel chloride (NiCl2), and bacterial community structure was analysed with the ribosomal intergenic spacer analysis (RISA) technique. Then, bacterial populations that respond to nickel stress were identified by cloning and sequencing. In the ultramafic soil, a shift occurred on day zero on the assay profiles and consisted of the emergence of a bacterial group closely related to the Ralstonia/Oxalobacter/Burkholderia group. It is hypothesized that NiCl2 had a physico‐chemical impact on soil structure. Fourteen days after nickel spiking, another shift occurred in the two soils that concerned a bacterial group belonging to the Actinomycete group. Only a few changes occurred in the bacterial community structure of the neocaledonian soils compared with those of the reference soil, which is more affected by nickel spiking. These results suggest that neocaledonian soil bacteria are particularly well adapted to nickel.
Isolation and phylogenetic characterization of bacteria capable of inducing differentiation in the green alga Monostroma oxyspermum
Authors: Matsuo Yoshihide , Suzuki Makoto , Kasai Hiroaki , Shizuri Yoshikazu , Harayama Shigeaki
Published: March 2003

Many green algae cannot develop normally when they are grown under axenic conditions. Monostroma oxyspermum, for example, proliferates unicellularly in an aseptic culture, but develops into a normal foliaceous gametophyte in the presence of some marine bacteria. More than 1000 bacterial strains were isolated from marine algae and sponges and assayed for their ability to induce the morphogenesis of unicellular M. oxyspermum. Fifty bacterial strains exhibiting morphogenesis‐inducing activity against unicellular M. oxyspermum were isolated. The partial gyrB (≈ 1.2 kbp) and 16S rDNA (≈ 1.4 kbp) sequences of about 40 active strains were determined, and their phylogenetic relationships were analysed. All these strains were located within the Cytophaga–Flavobacterium–Bacteroides (CFB) complex, and most of these strains were clustered in a clade comprising Zobellia uliginosa. On the other hand, these bacteria also exhibited morphogenetic activity against germ‐free spores of Ulva pertusa, Ulva conglobata and Enteromorpha intestinalis. Moreover, these bacteria induced the release of spores from the leafy young gametophyte of M. oxyspermum. These results indicate that strains belonging to several groups in the CFB complex play an important role in the normal development of green algae in the marine coastal environment.
Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil
Authors: Anderson Ian C. , Campbell Colin D. , Prosser James I.
Published: March 2003

Four fungal 18S rDNA and internal transcribed spacer (ITS) polymerase chain reaction (PCR) primer pairs were tested for their specificity towards target fungal DNA in soil DNA extracts, and their ability to assess the diversity of fungal communities in a natural grassland soil was compared. Amplified PCR products were cloned, and ≈ 50 clones from each library were sequenced. Phylogenetic analysis and database searches indicated that each of the sequenced cloned DNA fragments was of fungal origin for each primer pair, with the exception of the sequences generated using the 18S rDNA primers nu‐SSU‐0817 and nu‐SSU‐1196, where 35 of the 50 sequenced clones represented soil invertebrates. Although some of the primers have previously been suggested to be biased towards certain fungal taxonomic groups, the ratio of sequences representing each of the four main fungal phyla, Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, was similar for each of the primer pairs, suggesting that primer bias may be less significant than previously thought. Collector's curves were plotted to estimate the coverage obtained for each of the clone libraries after clustering the sequences into operational taxonomic units at a level of 99% sequence similarity. The curves indicated that good coverage of diversity was achieved, with the exception of the clone library constructed using primers nu‐SSU‐0817 and nu‐SSU‐1196, on account of the high number of non‐fungal sequences obtained. The work demonstrates the usefulness of 18S rDNA and ITS PCR primers for assessing fungal diversity in environmental samples, and it also highlights some potential limitations of the approach with respect to PCR primer specificity and bias.
Attenuating CV risk factors in patients with diabetes: clinical evidence to clinical practice
Authors: Garber Alan J.
Published: March 2003

Individuals with diabetes are at high risk of cardiovascular (CV) disease, a risk that is significantly greater in the presence of traditional CV risk factors (hyperlipidaemia, hypertension, prothrombotic state). Glucose control and management of these risk factors decreases but does not eliminate CV events, reflecting the complexity of atherosclerosis. Novel risk factors (C‐reactive protein, lipoprotein a, homocysteine, and endothelial dysfunction) have been proposed and are potentially modifiable. However, clinical trials data are not yet available to guide therapy. At this time, no single agent can achieve adequate risk reduction in patients with diabetes. Even with the use of multiple agents and classes of agents to manage CV risk, 75% of patients with diabetes are expected to die from CV causes. Despite the recent advances in primary and secondary prevention of CV events, new approaches are needed. Data from the Heart Outcomes Prevention Evaluation (HOPE) trial demonstrated that CV risk can be further reduced by the addition of the ACE inhibitor ramipril to the existing treatment regimen of high‐risk patients with diabetes.