bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional analysis of the Pseudomonas syringae rulAB determinant in tolerance to ultraviolet B (290–320 nm) radiation and distribution of rulAB among P. syringae pathovars
Authors: Sundin George W. , Murillo Jesús
Published: March 2003

The effect of the plasmid‐encoded rulAB (resistance to ultraviolet radiation) determinant on responses of Pseudomonas syringae to ultraviolet‐B (UV‐B) radiation and the distribution of rulAB among pathovars of P. syringae were determined. The cloned rulAB determinant and the native rulAB+ plasmid pPSR1 both conferred approximately a 10‐fold increase in survival on P. syringae pv. syringae FF5 following increasing doses of UV‐B radiation. rulAB+P. syringae strains also maintained significantly larger epiphytic populations on leaf surfaces irradiated with UV‐B. rulAB‐insertional mutants, constructed in two native rulAB+ strains, were from 10‐ to 100‐fold more sensitive to UV‐B radiation. The UV tolerance phenotype and the rulAB genes were widely distributed among P. syringae pathovars isolated from varied plant hosts throughout the world and within a broad range of genotypic backgrounds of P. syringae pv. syringae. With one exception, the rulAB determinant was harboured on pPT23A‐like plasmids; these replicons are indigenous residents of the species P. syringae and also tend to encode determinants of importance in host–pathogen interactions.
Signalling by the fungus Pythium ultimum represses expression of two ribosomal RNA operons with key roles in the rhizosphere ecology of Pseudomonas fluorescens F113
Authors: Smith Loraine M. , Tola Elisabetta , DeBoer Paulo , O'gara Fergal
Published: March 2003

Pseudomonas fluorescens F113 produces antifungal metabolites that protect the roots of sugarbeet from the fungus Pythium ultimum. The phytopathogen, in turn, has the ability to downregulate the expression of genes fundamental to the rhizosphere competence of the bacterial strain. This paper describes the characterization of two of these genes, which were isolated by screening a mini‐Tn5 ::lacZ mutant bank for differential expression of β‐galactosidase in the presence of P. ultimum. In order to identify the genes affected in reporter mutants SF3 and SF5, the transposons and flanking regions were cloned. Sequence analysis of the regions flanking the transposons in SF3 revealed that mini‐Tn5 ::lacZ had inserted into a tRNAIle gene, which maps within a ribosomal RNA (rrn ) operon. In SF5, the transposon inserted between the promoter of a second rrn operon and a gene encoding a 16S rRNA. Southern blot analysis demonstrated that there are five rrn operons in P. fluorescens F113 and that the transposons in SF3 and SF5 had inserted into two different operons. Further characterization of these mutants suggests that their reduced rhizosphere competence is not the result of reduced viability in the short term but may be accounted for partly by reduced growth rates under conditions that support rapid growth. Analysis of lacZ expression in the reporter mutants indicate that the marked rrn operons are regulated differently, suggesting different physiological roles.
Cultivatable microbial biodiversity: gnawing at the Gordian knot
Authors: Tindall Brian J. , Brambilla Evelyne , Steffen Maike , Neumann Regine , Pukall Rüdiger , Kroppenstedt Reiner M. , Stackebrandt Erko
Published: March 2003

Rapid and inexpensive sorting of bacterial isolates may be achieved using Fourier transform infrared spectroscopy (FT‐IR), a method that has hitherto been applied to identification and classification. The comprehensive characterization of environmental samples requires the isolation of large numbers of isolates using different growth media and growth conditions. In such cases, sorting the isolates is critical before isolates are subjected to more detailed studies. Using FT‐IR, isolates are grown under standardized conditions, and 100 strains can be tested within less than 8 h. Chemotaxonomic and molecular characterization of members of clusters emerging from FT‐IR analysis either at a level of spectral distance values below 20–30 (analysis of region 600–800 cm−1, average linkage algorithm) or at spectral heterogeneity values below 75 (regions 1200–900, 3000–2798 and 901–698, scaling to first region, Ward's algorithm) reveals great similarities in fatty acids and 16S rDNA sequences. As judged from riboprinting analyses and fatty acid analyses, FT‐IR analysis is able to unravel intraspecific subclustering. The example used in this study of 100 isolates from a mat system, Lake Fryxell, Dry Valleys, Antarctica, selected from a larger number of isolates, picked mainly on the basis of colony pigmentation and form, reveals the utility of the method for identifying the number of putative species quickly. The method described is able to select strains rapidly that represent clusters at the specific and intraspecific level for subsequent characterization.
Characteristics of attachment and growth of Thiobacillus caldus on sulphide minerals: a chemotactic response to sulphur minerals?
Authors: Edwards Katrina J. , Bond Philip L. , Banfield Jillian F.
Published: March 2003

To further our understanding of the ecological role of sulphur‐oxidizing microorganisms in the generation of acid mine drainage (AMD), growth and attachment of the chemoautotrophic sulphur‐oxidizing bacterium, Thiobacillus caldus, on the sulphide minerals pyrite, marcasite and arsenopyrite was studied. Growth curves were estimated based on total cells detected in the system (in suspension and attached to mineral surfaces). In general, higher cell numbers were detected on surfaces than in suspension. Fluorescent in situ hybridizations to cells on surfaces at mid‐log growth confirmed that cells on surfaces were metabolically active. Total cell (both surface and solution phase) generation times on pyrite and marcasite (both FeS2) were calculated to be ≈ 7 and 6 h respectively. When grown on pyrite (not marcasite), the number of T. caldus cells in the solution phase decreased, while the total number of cells (both surface and solution) increased. Additionally, marcasite supported about three times more total cells (≈ 3 × 109) than pyrite (≈ 8 × 108). This may be attributed to the dissolution rate of marcasite, which is twice that of pyrite. Epifluorescent and scanning electron microscopy (SEM) were used to analyse the cell orientation on surfaces. Results of Fourier transform analysis of fluorescent images confirmed that attachment to all three sulphides occurred in an oriented manner. Results from high‐resolution SEM imaging showed that cell orientation coincides with dissolution pit edges and secondary sulphur minerals that develop during dissolution. Preferential colonization of surfaces relative to solution and oriented cell attachment on these sulphide surfaces suggest that T. caldus may chemotactically select the optimal site for chemoautotrophic growth on sulphur (i.e. the mineral surface).
Impact of surface thermodynamics on bacterial transport
Authors: Chen Gang , Strevett Keith A.
Published: March 2003

Microbial surface thermodynamics correlated with bacterial transport in saturated porous media. The surface thermodynamics was characterized by contact‐angle measurement and the wicking method, which was related to surface free energies of Lifshitz–van der Waals interaction, Lewis acid–base interaction, and electrostatic interaction between the bacteria and the medium matrix. Transport of three different strains of bacteria present at three physiological states was measured in columns of silica gel and sand from the Canadian River Alluvium (Norman, OK, USA). Microorganisms in stationary state had the highest deposit on solid matrix, compared with logarithmic and decay states. The deposition correlated with the total surface free energy (ΔG132TOT) and the differences in ΔG132TOT were mainly controlled by the Lewis acid–base interaction. Infrared spectroscopy showed that the increased deposition correlated with an increase in the hydrogen‐bonding functional groups on the cell surfaces.
Sensitive characterization of microbial ubiquinones from biofilms by electrospray/mass spectrometry
Authors: Lytle Cory A. , Gan Ying Dong M. , Salone Kimberely , White David C.
Published: March 2003

Utilizing high‐performance liquid chromatography/electrospray/tandem mass spectrometric analysis of the neutral lipid extract of microbial cells and biofilm communities, respiratory ubiquinone (UQ) (1‐methyl‐2‐isoprenyl‐3,4‐dimethoxyparabenzoquinone) isoprenologues can be separated isocratically in minutes and assayed with a limit of quantification (LOQ) of 9 p.p.b. (11.1 fmol UQ9 µL−1). This corresponds to about 1.29 × 107 cells of Pseudomonas putida. Highest sensitivity is achieved using flow‐injection analysis with multiple reaction monitoring wherein ammoniated molecular ions of specific isoprenologues pass through quadrupole one, are collisionally dissociated in quadrupole two and identified from the product ion fragment at m/z 197.1 in quadrupole three. This assay has a repeatability of between 6% and 10% over three orders of magnitude (r2 = 0.996). Quinone profiling based on dominant isoprenologue patterns provides taxonomic insights. Detection of prominent UQ isoprenologues indicates presence of microeukaryotes and αProteobacteria with UQ10, obligatory aerobic Gram‐negative bacteria with UQ4‐14, facultative Gram‐negative (and some γProteobacteria growing in microniches with oxygen or to a much lesser extent nitrate as a terminal electron acceptor with UQ8, and other γProteobacteria with UQ9. High sensitivity is essential as the phospholipid fatty acid (PLFA) to UQ molar ratios are 130 or greater. Previous studies have established that recovery of sediment communities with high PLFA/UQ ratios corresponded to areas of aerobic metabolism, an important consideration in bioremediation or nuclide mobilization.
Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton
Authors: Suzuki Marcelino T. , Béjà Oded , Taylor Lance T. , DeLong Edward F.
Published: March 2003

Analyses of small subunit ribosomal RNA genes (SSU rDNAs) have significantly influenced our understanding of the composition of aquatic microbial assemblages. Unfortunately, SSU rDNA sequences often do not have sufficient resolving power to differentiate closely related species. To address this general problem for uncultivated bacterioplankton taxa, we analysed and compared sequences of polymerase chain reaction (PCR)‐generated and bacterial artificial chromosome (BAC)‐derived clones that contained most of the SSU rDNAs, the internal transcribed spacer (ITS) and the large subunit ribosomal RNA gene (LSU rDNA). The phylogenetic representation in the rRNA operon PCR library was similar to that reported previously in coastal bacterioplankton SSU rDNA libraries. We observed good concordance between the phylogenetic relationships among coastal bacterioplankton inferred from SSU or LSU rDNA sequences. ITS sequences confirmed the close intragroup relationships among members of the SAR11, SAR116 and SAR86 clades that were predicted by SSU and LSU rDNA sequence analyses. We also found strong support for homologous recombination between the ITS regions of operons from the SAR11 clade.
Development of specific oligonucleotide probes for the identification and in situ detection of hydrocarbon‐degrading Alcanivorax strains
Authors: Syutsubo Kazuaki , Kishira Hideo , Harayama Shigeaki
Published: March 2003

The genus Alcanivorax comprises diverse hydrocarbon‐degrading marine bacteria. Novel 16S rRNA‐targeted oligonucleotide DNA probes (ALV735 and ALV735‐b) were developed to quantify two subgroups of the Alcanivorax/Fundibacter group by fluorescence in situ hybridization (FISH), and the conditions for the single‐mismatch discrimination of the probes were optimized. The specificity of the probes was improved further using a singly mismatched oligonucleotide as a competitor. The growth of Alcanivorax cells in crude oil‐contaminated sea water under the biostimulation condition was investigated by FISH with the probe ALV735, which targeted the main cluster of the Alcanivorax/Fundibacter group. The size of the Alcanivorax population increased with increasing incubation time and accounted for 91% of the 4′,6‐diamidino‐2‐phenylindole (DAPI) count after incubation for 2 weeks. The probes developed in this study are useful for detecting Alcanivorax populations in petroleum hydrocarbon‐degrading microbial consortia.
Methyl tert‐butyl ether (MTBE) degradation by a microbial consortium
Authors: Fortin Nathalie Y. , Morales Marcia , Nakagawa Yuka , Focht Dennis D. , Deshusses Marc A.
Published: March 2003

The widespread use of methyl tert‐butyl ether (MTBE) as a gasoline additive has resulted in a large number of cases of groundwater contamination. Bioremediation is often proposed as the most promising alternative after treatment. However, MTBE biodegradation appears to be quite different from the biodegradation of usual gasoline contaminants such as benzene, toluene, ethyl benzene and xylene (BTEX). In the present paper, the characteristics of a consortium degrading MTBE in liquid cultures are presented and discussed. MTBE degradation rate was fast and followed zero order kinetics when added at 100 mg l−1. The residual MTBE concentration in batch degradation experiments ranged from below the detection limit (1 µg l−1) to 50 µg l−1.The specific activity of the consortium ranged from 7 to 52 mgMTBE gdw−1 h−1 (i.e. 19–141 mgCOD gdw−1 h−1). Radioisotope experiments showed that 79% of the carbon‐MTBE was converted to carbon‐carbon dioxide. The consortium was also capable of degrading a variety of hydrocarbons, including tert‐butyl alcohol (TBA), tert‐amyl methyl ether (TAME) and gasoline constituents such as benzene, toluene, ethylbenzene and xylene (BTEX). The consortium was also characterized by a very slow growth rate (0.1 d−1), a low overall biomass yield (0.11 gdw g−1MTBE; i.e. 0.040 gdw gCOD−1), a high affinity for MTBE and a low affinity for oxygen, which may be a reason for the slow or absence of MTBE biodegradation in situ. Still, the results presented here show promising perspectives for engineering the in situ bioremediation of MTBE.
16S‐23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium‐oxidizing bacteria: implications for phylogeny and in situ detection
Authors: Schmid Markus , Schmitz‐Esser Stephan , Jetten Mike , Wagner Michael
Published: March 2003

Recently, anaerobic ammonium‐oxidizing bacteria (AAOB) were identified by comparative 16S rDNA sequence analysis as a novel, deep‐branching lineage within the Planctomycetales. This lineage consists currently of only two, not yet culturable bacteria which have been provisionally described as Candidatus‘Brocadia anammoxidans’ and Candidatus‘Kuenenia stuttgartiensis’. In this study, a large fragment of the rDNA operon, including the 16S rDNA, the intergenic spacer region (ISR) and approximately 2 000 bases of the 23S rDNA, was polymerase chain reaction (PCR) amplified, cloned and sequenced from both AAOB. The retrieved 16S rDNA sequences of both species contain an insertion at helix 9 with a previously overlooked pronounced secondary structure (new subhelices 9a and 9b). This insertion, which is absent in all other known prokaryotes, is detectable by fluorescence in situ hybridization (FISH) and thus present in the mature 16S rRNA. In contrast with the genera Pirellula, Planctomyces and Gemmata that possess unlinked 16S and 23S rRNA genes, both AAOB have the respective genes linked together by an ISR of approximately 450 bp in length. Phylogenetic analysis of the obtained 23S rRNA‐genes confirmed the deep branching of the AAOB within the Planctomycetales and allowed the design of additional specific FISH probes. Remarkably, the ISR of the AAOB also could be successfully detected by FISH via simultaneous application of four monolabelled oligonucleotide probes. Quantitative FISH experiments with cells of Candidatus‘Brocadia anammoxidans’ that were inhibited by exposure to oxygen for different time periods demonstrated that the concentration of transcribed ISR reflected the activity of the cells more accurately than the 16S or 23S rRNA concentration. Thus the developed ISR probes might become useful tools for in situ monitoring of the activity of AAOB in their natural environment.
Optimization of an oligonucleotide microchip for microbial identification studies: a non‐equilibrium dissociation approach
Authors: Liu Wen‐Tso , Mirzabekov Andrei D. , Stahl David A.
Published: March 2003

The utility of a high‐density oligonucleotide microarray (microchip) for identifying strains of five closely related bacilli (Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus medusa and Bacillus subtilis) was demonstrated using an approach that compares the non‐equilibrium dissociation rates (‘melting curves’) of all probe–target duplexes simultaneously. For this study, a hierarchical set of 30 oligonucleotide probes targeting the 16S ribosomal RNA of these bacilli at multiple levels of specificity (approximate taxonomic ranks of domain, kingdom, order, genus and species) was designed and immobilized in a high‐density matrix of gel pads on a glass slide. Reproducible melting curves for probes with different levels of specificity were obtained using an optimized salt concentration. Clear discrimination between perfect match (PM) and mismatch (MM) duplexes was achieved. By normalizing the signals to an internal standard (a universal probe), a more than twofold discrimination (> 2.4×) was achieved between PM and 1‐MM duplexes at the dissociation temperature at which 50% of the probe–target duplexes remained intact. This provided excellent differentiation among representatives of different Bacillus species, both individually and in mixtures of two or three. The overall pattern of hybridization derived from this hierarchical probe set also provided a clear ‘chip fingerprint’ for each of these closely related Bacillus species.
Community shifts in a seeded 3‐chlorobenzoate degrading membrane biofilm reactor: indications for involvement of in situ horizontal transfer of the clc‐element from inoculum to contaminant bacteria
Authors: Springael Dirk , Peys Kurt , Ryngaert Annemie , Roy Sandra Van , Hooyberghs Liliane , Ravatn Roald , Heyndrickx Marc , Meer Jan‐Roelof van der , Vandecasteele Carlo , Mergeay Max , Diels Ludo
Published: March 2003

Pseudomonas putida BN210, carrying the self‐ transferable clc‐element encoding degradation of 3‐chlorobenzoate on the chromosome, was used as inoculum in different membrane biofilm reactors treating 3‐chlorobenzoate‐contaminated model wastewater. Analysis of the bacterial population in the effluent and in the biofilm showed the loss of BN210 beyond detection from the reactors and the appearance of several novel 3‐chlorobenzoate mineralizing bacteria mainly belonging to the β‐proteobacteria. In contrast, in non‐inoculated reactors, no 3‐chlorobenzoate degradation was observed and no 3‐chlorobenzoate degraders could be recovered. Southern blots hybridization of genomic DNA using clc‐element‐specific probes and FIGE analysis indicated the presence of the complete clc‐element in one or more copies in the isolates. Moreover, the isolates could transfer the clc genes to Ralstonia metallidu‐rans recipients. Two representative reactor isolates, Ralstonia sp. strains KP3 and KP9 demonstrated a higher growth rate on 3‐chlorobenzoate than strain BN210 in batch cultures. When BN210, KP3 and KP9 were simultaneously inoculated in a membrane reactor supplied with 3‐chlorobenzoate, strain KP3 outcompeted the two other strains and remained the major 3‐chlorobenzoate degrading population in the reactor. Our data suggest that in situ horizontal transfer of the clc‐element from the inoculum to contaminant bacteria in the reactors was involved in the establishment of novel 3‐chlorobenzoate degrading populations that were more competitive under the defined reactor conditions than the inoculum strain.
Persistence of Escherichia coli O157:H7 in soil and on plant roots
Authors: Gagliardi Joel V. , Karns Jeffrey S.
Published: March 2003

Soil microcosms were inoculated with Escherichia coli O157:H7 to test persistence in fallow soil, on roots of cover crops and in presence of manure. In fallow soils, E. coli O157:H7 persisted for 25–41 days, on rye roots for 47–96 days and on alfalfa roots, in a silt loam soil, for 92 days whereas on other legumes persistence ranged from 25–40 days, similar to fallow soil. Manure did not seem to affect the persistence of E. coli O157:H7 in these soils. Indigenous and manure‐applied coliform populations often decreased faster when E. coli O157:H7 was applied, indicating possible competition between microflora. Coliform populations in microcosms not inoculated with E. coli O157:H7 decreased more slowly or increased. Microbial community analyses showed little effect for E. coli O157:H7 inoculation or addition of manure. Microbial community metabolic activity was enhanced from rye roots after 14 days and by 63 days from alfalfa roots. Microbial community lactose utilization increased over time on rye roots in all soils and on alfalfa roots in a silt loam soil when E. coli O157:H7 was inoculated. Lactose utilization also increased for uninoculated rye roots, soil around rye roots and in some fallow soils. Our data suggest that clay increases persistence and activity of E. coli O157:H7 and other coliforms. In frozen soil stored for over 500 days, E. coli O157:H7 was viable in 37% of tested samples. In summary, E. coli O157:H7 persisted longer and activity was enhanced with some cover crops in these soils due to plant roots, the presence of clay and freezing.
Seasonal dynamics of particle‐associated and free‐living marine Proteobacteria in a salt marsh tidal creek as determined using fluorescence in situ hybridization
Authors: Dang Hongyue , Lovell Charles R.
Published: March 2003

The seasonal distributions of salt marsh free‐living and particle‐associated bacteria belonging to three subdivisions of the Proteobacteria were determined by fluorescence
in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM).
More than 66% (median = 78%) of total bacterial cells that were stainable
with the fluorescent DNA stain Yo‐Pro‐1 were also detected using the bacterial probe
EUB338. The α‐Proteobacteria, especially those from the marine
Rhodobacter group, were abundant on suspended particles and as free‐living
cells all year round. The marine Rhodobacter group constituted more than 25%
of the particle‐associated bacteria and more than 18% of the free‐living
bacteria. Probes specific for three subgroups within the marine Rhodobacter
group detected more than 49% of the total marine Rhodobacter group
cells. These subgroups displayed different seasonal dynamics. The marine Rhodobacter group is clearly a widespread, diverse and important bacterial lineage in bacterioplankton and particle‐associated assemblages in south‐eastern United States salt marshes at all times of the year.
Stratification and seasonal stability of diverse bacterial communities in a Pinus merkusii (pine) forest soil in central Java, Indonesia
Authors: Krave Agna S. , Lin Bin , Braster Martin , Laverman Anniet M. , Straalen Nico M. van , Röling Wilfred F. M. , Verseveld Henk W. van
Published: March 2003

In Java, Indonesia, many nutrient‐poor soils are intensively reforested with Pinus
merkusii (pine). Information on nutrient cycles and microorganisms involved in
these cycles will benefit the management of these important forests. Here, seasonal
effects on the stratification of bacterial community structure in the soil profile
of a tropical pine forest are described, and differences in bacterial communities
are related to chemical and physical soil parameters. Culture‐independent community
profiles of litter, fragmented litter and mineral soil layers were made by denaturing
gradient gel electrophoresis (DGGE) of 16S rDNA‐specific polymerase chain reaction
(PCR) fragments. The community profiles of the different soil layers clustered separately,
correlating with significant differences in organic matter content between the three
layers. The bacterial communities appeared to be stable during the wet season of
1998. The drought in 1997, caused by the El Niño climatic effect,
did not influence the bacterial communities in fragmentation and mineral soil, although
moisture content and other soil parameters were markedly lower than in the wet season.
However, communities in litter were influenced by drought. In the litter layer, the
moisture content was significantly lower than in the fragmentation and mineral layers
during the dry season. A clone library was made from a litter sample taken during
the wet season. Partial sequencing of 74 clones and linking the DGGE banding positions
of these clones to bands in the DGGE profile of the sample from which the clone library
was derived showed considerable bacterial diversity. Alpha‐proteobacteria (40.5%
of the clones, of which 57% belonged to the Rhizobium–Agrobacterium group) and high‐G+C content, Gram‐positive bacteria (36.5%) dominated the clone library.
Picobenthic cyanobacterial populations revealed by 16S rRNA‐targeted in situ hybridization
Authors: Abed Raeid M. M. , Schönhuber Wilhelm , Amann Rudolf , Garcia‐Pichel Ferran
Published: March 2003

We report on the morphological identification of a population of benthic cyanobacteria from microbial mats, known previously only from molecular analyses of field samples, based on the retrieval of environmental 16S rRNA sequences. We used in situ hybridization with horseradish peroxidase‐labelled oligonucleotide probes designed to target the 16S rRNA of our unidentified population. Two probes were designed and checked for target binding ability and specificity using membrane hybridization against electroblotted bands from a denaturant gradient gel electrophoresis (DGGE) fingerprint of 16S rDNA gene fragments from the original cyanobacterial community. Under in situ hybridization, these probes bound specifically to extremely small, unicellular, colony‐forming cyanobacteria, 0.75–1 μm in diameter, which were embedded in abundant mucilaginous investments. We propose the term picobenthos, by analogy with picoplankton, to describe those unicellular benthic microbes around or less than 1 μm in diameter. Although picoplanktonic cyanobacteria are abundant in ocean and freshwaters, picobenthic (<1 μm) unicellular cyanobacteria are not typically recognized as a major component of microbial mats. The small size and low levels of photopigment autofluorescence from these cells probably rendered them cryptic or indistinguishable from heterotrophic bacteria in routine microscopic observations. It is not known how widespread picobenthic cyanobacteria may be in other environments.
nifH gene diversity in the bacterial community associated with the rhizosphere of Molinia coerulea, an oligonitrophilic perennial grass
Authors: Hamelin Jérôme , Fromin Nathalie , Tarnawski Sonia , Teyssier‐Cuvelle Sylvie , Aragno Michel
Published: March 2003

Rhizosphere associative dinitrogen fixation could be a valuable source of nitrogen in many nitrogen limited natural ecosystems, such as the rhizosphere of Molinia coerulea, a hemicryptophytic perennial grass naturally occurring in contrasted oligonitrophilic soils. The diversity of the dinitrogen‐fixing bacteria associated with this environment was assessed by a cloning–sequencing approach on the nifH gene directly amplified from environmental DNA extracts. Seventy‐seven randomly picked clones were analysed. One type of NifH sequence was dominant in both roots and surrounding soil, and represented 56% of all retrieved sequences. This cluster included previously described environmental clones and did not contain any NifH sequences similar to cultivated diazotrophs. The predominance of few NifH sequence types in the roots and the rhizosphere of Molinia coerulea indicate that the plant environment mediates a favourable niche for such dinitrogen‐fixing bacteria.
Diversity and distribution of a deeply branched novel proteobacterial group found in anaerobic–aerobic activated sludge processes
Authors: Kong Yunhong , Ong Say Leong , Ng Wun Jun , Liu Wen‐Tso
Published: March 2003

A novel coccobacilli group found previously in enhanced biological phosphorus removal (EBPR) systems was further revealed to have a high degree of diversity and distribution in various activated sludge systems. Phylogenetic analysis based on 14 existing and 18 newly retrieved 16S rRNA sequences revealed that these sequences formed a novel cohesive cluster with seven subgroups in the γ‐Proteobacteria. Fluorescence in situ hybridization with a set of probes designed specifically targeting the novel group at different hierarchical levels showed that the novel group with a coccoid (2–4 µm) to occasionally long‐rod (up to 20 µm) shape widely distributed and in some cases predominated in sludge samples taken from nine lab‐ and full‐scale EBPR systems (10–50% of total cells) and four conventional activated sludge systems (1–10%). Variation of predominance was also observed among those subgroups in systems showing deteriorated or effective EBPR activity.
A novel assay to monitor predator–prey interactions for Bdellovibrio bacteriovorus 109 J reveals a role for methyl‐accepting chemotaxis proteins in predation
Authors: Lambert Carey , Smith Margaret C. M. , Sockett R. Elizabeth
Published: March 2003

Bdellovibrio bacteriovorus are Gram‐negative bacteria that prey upon other Gram‐negative bacteria, including some pathogens, in a wide variety of habitats including soil, sewage, marine and estuarine environments. In order to facilitate studies on predation by this organism, we have developed a method that assays killing of luminescent Escherichia coli by B. bacteriovorus. Moreover, we have used this assay to compare predation of cells by derivatives of B. bacteriovorus containing targeted mutations in genes we have identified. Two genes are described; one, mcp2, encoding a methyl‐accepting chemotaxis protein (MCP) and the other, an mviN homologue. Bdellovibrio bacteriovorus mcp2::aphII were less efficient predators on luminescent E. coli than B. bacteriovorus containing a randomly inserted aphII gene via TnphoA transposition. These and other chemotaxis experiments implicated at least a minor role for chemotaxis in predation by B. bacteriovorus. They also open the way for further studies on Bdellovibrio ecology, genomics and predator–prey interactions. The results further confirm that Bdellovibrio uses a chemotaxis system in order to sense, and respond to, changes in its environment, including prey.
Intracellular symbionts of sharpshooters (Insecta: Hemiptera: Cicadellinae) form a distinct clade with a small genome
Authors: Moran Nancy A. , Dale Colin , Dunbar Helen , Smith Wendy A. , Ochman Howard
Published: March 2003

The leafhoppers (Insecta: Hemiptera: Cicadellidae) are the most species‐rich group of invertebrates in which intracellular symbionts are usual. Here we present the first molecular characterization of bacteriome‐associates in the leafhoppers, with focus on the subfamily Cicadellinae (sharpshooters). Phylogenetic analyses of 16S rDNA sequences from intracellular symbionts residing in the bacteriomes of five host species indicate that these symbionts form a well‐defined clade within the γ‐3 Proteobacteria, consistent with an ancient colonization and strict vertical transmission. More extensive gene sequence information is reported for the symbiont of Homalodisca coagulata (Say). The genome size, as determined by pulsed field gel electrophoresis, is approximately 680 kb. This finding, when combined with published results for symbionts of aphids, ants, psyllids and tsetse flies, adds to an emerging pattern which suggests that bacteriome associates often descend from ancient infections by γ Proteobacteria, and that these lineages have undergone pronounced genome reduction. A new genus and species name, ‘Candidatus Baumannia cicadellinicola’ (sp. nov.) is proposed for this newly characterized clade of symbiotic bacteria.
Diversity of microbial communities correlated to physiochemical parameters in a digestion basin of a zero‐discharge mariculture system
Authors: Cytryn Eddie , Gelfand Ilya , Barak Yoram , Van Rijn Jaap , Minz Dror
Published: March 2003

Bacterial community structure and physiochemical parameters were examined in a sedimentation basin of a zero‐discharge mariculture system. The system consisted of an intensively stocked fish basin from which water was recirculated through two separate treatment loops. Surface water from the basin was pumped over a trickling filter in one loop while bottom‐water was recirculated through a sedimentation basin followed by a fluidized bed reactor in the other. Ammonia oxidation to nitrate in the trickling filter and organic matter digestion together with nitrate reduction in the sedimentation basin and fluidized bed reactor, allowed zero‐discharge operation of the system. Relatively high concentrations of oxygen, nitrate, sulphate and organic matter detected simultaneously in the digestion basin suggested the potential for a wide range of microbially‐mediated transformation processes. In this study, physiochemical parameters were correlated to bacterial diversity and distribution in horizontal and vertical profiles within this basin in an effort to obtain a basic understanding of the chemical and microbial processes in this system. Chemical activity and microbial diversity, the latter measured by denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR) amplified 16S rDNA fragments, were higher in the sludge layer than in the overlying aqueous layer of the basin. Chemical parameters in sludge samples close to the basin inlet suggested enhanced microbial activity relative to other sampling areas with evidence of both nitrate and sulphate reduction. Four of the nine DGGE bands identified in this zone were affiliated with the Bacteroidetes phylum. Detected sequences closely related to sequences of organisms involved in the sulphur cycle included Desulfovibrio, Dethiosulfovibrio and apparent sulphur oxidizers from the gamma‐proteobacteria. In addition, a number of sequences from the beta and alpha‐proteobacteria were identified.
Long‐term results of colorectal resection for slow transit constipation
Authors: Hasegawa  , Radley  , Fatah  , Keighley
Published: March 2003

Objective

To determine the long‐term outcome for surgery for slow transit constipation.

Patients and methods

A long‐term retrospective review of 61 patients who had undergone surgery for slow transit constipation between 1977 and 1996. Patients with megabowel, Hirschsprung's disease or primary anorectal pathology were excluded from the study.

Results

Subtotal colectomy was performed in 48 patients and segmental colectomy in 13. Twenty‐four patients (39%) required further surgery. Fourteen patients (23%) eventually had a permanent stoma. Twenty‐nine patients (48%) had normal bowel habit and improved symptoms (grade 1) following treatment. The permanent stoma rate was significantly higher in patients with proven psychological disturbance (70% vs 14%, P = 0.0005). Unsatisfactory outcomes (grades 3 and 4) were more common in patients with a psychological disorder (n = 10), 70% vs 18% (P = 0.002), and those with slow transit and impaired rectal evacuation, 39% vs 11% (P = 0.025). Satisfactory outcomes (grades 1 and 2) were achieved in 95% of the psychologically stable patients with slow transit constipation after subtotal colectomy.

Conclusion

The long‐term results of surgery for slow transit constipation may be poor. However, a good outcome can be predicted in patients with slow transit constipation without impaired evacuation and overt psychological or psychiatric disease.
Disappointing results of glyceryl trinitrate ointment in the treatment of chronic fissure‐in‐ano in a district general hospital
Authors: Pitt  , Dawas  , Dawson
Published: March 2003

Objective

 The recent introduction of glyceryl trinitrate ointment (GTN) has greatly changed the management of chronic fissure‐in‐ano. We have investigated the results obtained from this new treatment and the consequent alterations to surgical practice.

Patients and methods

 All patients who presented with chronic fissure‐in‐ano from March 1997 to March 1998 were treated with GTN ointment and prospectively evaluated until healing or lateral internal sphincterotomy. Long‐term follow up was obtained using a telephone questionnaire.

Results

 Forty‐five patients were treated with GTN ointment. Twenty‐two (49%) were healed initially, but 10/22 (45%) recurred within 36 weeks. Twelve (27%) patients were healed for the duration of this study. Twenty‐six (58%) patients complained of headache, of whom five (11%) were forced to abandon treatment. In comparison with the previous year, rates of lateral internal sphincterotomy reduced by 24%.

Conclusion

 GTN is a useful adjunct to the treatment of chronic anal fissure, but sphincterotomy remains the mainstay of treatment.
How accurate is the endoscopist’s assessment of visualization of the left colon seen at flexible sigmoidoscopy?
Authors: Adam  , Ali  , Shorthouse
Published: March 2003

Objective

There are concerns that the left colon may be incompletely examined by the standard 60‐cm flexible sigmoidoscope. Our objective was to compare the endoscopist’s assessment of the length of colon seen with the actual length visualized.

Patients and methods

During flexible sigmoidoscopy, endoscopists were asked to state how far they had reached. This was compared with the length of colon actually seen, assessed by a novel electromagnetic imaging (EMI) device that records the three‐dimensional position of the scope within a magnetic field pervading the patient’s abdomen. If EMI showed that the splenic flexure had not been reached, the endoscopist was asked to use the imager to see if it helped advance the scope further.

Results

In 94 patients, the endoscopist’s assessment of position reached was correct in only 47 cases (50%), with an overestimate of length of colon visualized in 24 (25.5%) and an underestimate in 23 (24.5%). EMI showed the splenic flexure had not been reached in 56 patients (59.6%). Using information from the EMI system, further progress was subsequently made in 45/56 (80.4%).

Conclusion

Caution is required in the diagnosis of rectal bleeding using flexible sigmoidoscopy alone. This has implications for the use of the 60‐cm flexible sigmoidoscope as a screening tool.
Endosonographic diagnosis of early colorectal tumour invasion using ultrasound miniature probe
Authors: Hizawa  , Kawasaki  , Hirakawa  , Aoyagi  , Yao  , Fujishima
Published: March 2003

Objective

We sought to determine the accuracy of differentiating between mucosal and submucosal colorectal tumour invasion using a high‐frequency ultrasound miniature probe.

Patients and methods

We analysed 43 lesions sonographically interpreted as T1 colorectal tumours, and evaluated the accuracy of detecting mucosal neoplasia (true‐positive and true‐negative mucosal neoplasias divided by all lesions).

Results

The overall accuracy of detecting mucosal neoplasia was 74%. Sonographic images were easily obtained under direct visualization, even for small and non‐polypoid lesions anywhere in the colon and rectum. However, diagnostic accuracy tended to be low in polypoid lesions >20 mm in width, and pure carcinoma with submucosal invasion. The submucosal layer in 16 lesions was visualized as a more detailed laminar structure, whereas the remaining 27 lesions showed a single hyperechoic submucosal layer. However, there were no significant differences in accuracy between the two.

Conclusion

The high‐frequency ultrasound miniature probe system may be useful for evaluating small non‐polypoid colorectal tumours because of easy manipulation rather than high resolution visualization.
Cigarette smoking and risk of death from colorectal cancer in women
Authors: Rohan  , Jain  , Rehm  , Ashley  , Bondy  , Ferrence  , Cohen  , Miller
Published: March 2003

Objective

Much of the epidemiological evidence concerning the relationship between cigarette smoking and colorectal cancer has come from studies in men, and less is known about the role of smoking in the aetiology and pathogenesis of colorectal cancer in women. Therefore, the purpose of the present study was to investigate the association between cigarette smoking and risk of death from colorectal cancer in a large cohort of women.

Subjects and methods

The study was conducted within the cohort of 56 837 women who were enrolled in the Canadian National Breast Screening Study (NBSS) and who completed self‐administered dietary questionnaires. (The NBSS is a randomized controlled trial of screening for breast cancer in women aged 40–59.) During follow up to December 31, 1993, 90 women died from colorectal cancer (79 from colon cancer, 11 from rectal cancer). Deaths were ascertained by means of record linkage to the Canadian Mortality Database. Cox proportional hazards models were used to estimate hazards ratios (HR) and 95% confidence intervals (CI) for the association between cigarette smoking and risk of death from colorectal cancer, after adjustment for age alone, or age, energy intake, alcohol consumption, and other potentially relevant confounders.

Results

Cigarette smoking was not associated with altered risk of death from colorectal cancer. The multivariate HR (95% CI) for the risk in ever smokers compared with that in never smokers was 1.37 (0.86–2.18). There was no evidence for trends in risk with cigarette‐years of consumption, and by amount and duration of consumption.

Conclusion

The results of this study suggest that cigarette smoking is not associated with altered risk of death from colorectal cancer in women. However, given the possibility that the association between cigarette smoking and colorectal cancer might not become evident until at least 30–40 years after the commencement of smoking, it might still be too early to detect an association, since uptake of smoking by women occurred later than uptake by men.
Screening of patients at high risk of colorectal cancer
Authors: Hill J. , Walsh S. , Evans D. G.
Published: March 2003

Objective

To determine the present state of colonoscopic screening practice for patients at a high risk of developing colorectal cancer.

Methods

We assessed colonoscopic screening practice in nine colorectal surgery or gastroenterology units referring patients to the regional genetics centre in the North‐west of England. The validity of the pedigree was confirmed by inspection of Hospital records, use of cancer registries and records of general practitioners across the United Kingdom. The screening activity at the participating centres was investigated by means of a questionnaire, site visit and study of case notes.

Results

One hundred and eight high risk families were identified. Outside the genetics unit, family history was not taken in a structured manner and family pedigree was not confirmed. In four units screening was offered directly by the consultant surgeon. Differing protocols for screening were in place in six units, but were frequently not adhered to. Colonoscopy completion rates were known in only four units (range 83–97%). Frequency of screening varied from 2 to 5 years. Recording of information was minimal outside the patients’ records and there were no dedicated databases. Facilities for families to be screened together existed in three units and the psychological effect of screening was not determined outside the genetics centre.

Conclusions

Screening practice should be standardized and specific criteria met before regular screening is initiated. A dedicated database should be established to record information from units screening patients at high risk of colorectal cancer.
Effect of age on the functional outcome of total abdominal colectomy for colonic inertia
Authors: Pikarsky A. J. , Efron J. , Hamel C. T. , Weiss E. G. , Nogueras J. J. , Wexner S. D.
Published: March 2003

Objective

Colonic inertia (CI) usually presents in women in the third decade of life; however, elderly patients are at times diagnosed with the disease. Total abdominal colectomy (TAC) is considered the preferred surgical treatment for patients with well established CI refractory to conservative and medical management. Surgeons are reluctant to proceed with colectomy in aged patients because of anticipated high morbidity and poor functional outcome.

Materials and methods

We retrospectively reviewed the outcome in 14 patients over the age of 65 years (range 65–80) (Group I) who underwent TAC for CI between 1988 and 1996. The functional and clinical outcome was compared with that of 41 patients under the age of 65 (range 21–61) (Group II) operated upon during the same time period. Functional outcome was assessed by postal and telephone questionnaires at least 12 months following surgery.

Results

There was no major postoperative morbidity in either group. Three (21%) patients in Group I developed small bowel obstruction postoperatively and, of them, 2 required surgical treatment. In Group II the rate of obstruction was 7% (3 patients), with one patient requiring surgery. One patient in Group I subsequently underwent completion proctectomy and creation of an end ileostomy due to continued panenteric hypomotility. Three patients in Group I died during follow‐up from causes unrelated to surgery. The mean frequency of spontaneous bowel movements following surgery was 3.8 (range 1–10)/day in Group I and 2.9 (range 1–8)/day in Group II (P=NS). ‘Excellent’ outcome was reported by 7 patients (64%) in Group I and 39 patients (95%) in Group II (P=0.01).

Conclusion

TAC can be performed in elderly patients with established CI with acceptable functional results and no increase in morbidity, resulting in lifestyle improvement. Complete physiological evaluation with increased emphasis on small bowel and gastric motility studies is required in this patient population.
Curative resection for rectal carcinoma: definition influences outcome in terms of local recurrence
Authors: Anwar R. M. , Britton A. , Stevens W.
Published: March 2003

Objective

There are wide variations in local recurrence rate following curative surgery for rectal cancer and there are substantial inconsistencies among surgeons regarding the method of defining curative resection. This paper seeks to explore whether defining criteria is one of the important factors driving the variations in outcome.

Method

A literature review was undertaken to find all UK‐based studies that had data on curative resection and local recurrence rates. The studies were divided into groups with distinct definitions of curative resection for rectal cancer. Meta‐analyses were performed to pool the risks of local recurrence by group definition. Statistical tests were used to explore the variation in local recurrence by group. Confounding relationships of age, sex, Dukes stage, length of follow‐up and year of study were explored as far as possible given the limitations of the available data.

Results

For rectal cancers significant differences were found between the pooled local recurrence risks by group definition (P < 0.01). Meta‐regression tests including all the studies indicate that the definition of curative resection is an important predictor of local recurrence.

Conclusion

It is suggested that a standardized approach towards defining curative resection and local recurrence may have a significant effect on outcomes in colorectal cancer surgery and would enable comparisons to be made between different series.