bluuebunny/crossref_metadata_2025 huggingface dataset:
Anaerobic Ammonia-Oxidizing Bacteria and Related Activity in Baltimore Inner Harbor Sediment
Authors: Tal Yossi , Watts Joy E. M. , Schreier Harold J.
Published: April 2005

The discovery of bacteria capable of anaerobic ammonia oxidation (anammox) has generated interest in understanding the activity, diversity, and distribution of these bacteria in the environment. In this study anammox activity in sediment samples obtained from the Inner Harbor of Baltimore, Md., was detected by
15
N tracer assays. Anammox-specific oligonucleotide primer sets were used to screen a
Planctomycetales
-specific 16S rRNA gene library generated from sediment DNA preparations, and four new anammox bacterial sequences were identified. Three of these sequences form a cohesive new branch of the anammox group, and the fourth sequence branches separately from this group. Denaturing gradient gel electrophoresis analysis of sediment incubated with anammox-specific media confirmed the presence of the four anammox-related 16S rRNA gene sequences. Evidence for the presence of anammox bacteria in Inner Harbor sediment was also obtained by using an anammox-specific probe in fluorescence in situ hybridization studies. To our knowledge, this is the first report of anammox activity and related bacterial 16S rRNA gene sequences from the Chesapeake Bay basin area, and the results suggest that this pathway plays an important role in the nitrogen cycle of this estuarine environment. Furthermore, the presence of these bacteria and their activity in sediment strengthen the contention that anammox-related
Plactomycetales
are globally distributed.
Rapid Engineering of the Geldanamycin Biosynthesis Pathway by Red/ET Recombination and Gene Complementation
Authors: Vetcher Leandro , Tian Zong-Qiang , McDaniel Robert , Rascher Andreas , Revill W. Peter , Hutchinson C. Richard , Hu Zhihao
Published: April 2005

Genetic manipulation of antibiotic producers, such as
Streptomyces
species, is a rational approach to improve the properties of biologically active molecules. However, this can be a slow and sometimes problematic process. Red/ET recombination in an
Escherichia coli
host has permitted rapid and more versatile engineering of geldanamycin biosynthetic genes in a complementation plasmid, which can then be readily transferred into the
Streptomyces
host from which the corresponding wild type gene(s) has been removed. With this rapid Red/ET recombination and gene complementation approach, efficient gene disruptions and gene replacements in the geldanamycin biosynthetic gene cluster have been successfully achieved. As an example, we describe here the creation of a ketoreductase 6 null mutation in an
E. coli
high-copy-number plasmid carrying
gdmA2A3
from
Streptomyces hygroscopicus
NRRL3602 and the subsequent complementation of a
gdmA2A3
deletion host with this plasmid to generate a novel geldanamycin analog.
Biotinylation Facilitates the Uptake of Large Peptides by
Escherichia coli
and Other Gram-Negative Bacteria
Authors: Walker Jennifer R. , Altman Elliot
Published: April 2005

Gram-negative bacteria such as
Escherichia coli
can normally only take up small peptides less than 650 Da, or five to six amino acids, in size. We have found that biotinylated peptides up to 31 amino acids in length can be taken up by
E. coli
and that uptake is dependent on the biotin transporter. Uptake could be competitively inhibited by free biotin or avidin and blocked by the protonophore carbonyl
m
-chlorophenylhydrazone and was abolished in
E. coli
mutants that lacked the biotin transporter. Biotinylated peptides could be used to supplement the growth of a biotin auxotroph, and the transported peptides were shown to be localized to the cytoplasm in cell fractionation experiments. The uptake of biotinylated peptides was also demonstrated for two other gram-negative bacteria,
Salmonella enterica
serovar Typhimurium and
Pseudomonas aeruginosa
. This finding may make it possible to create new peptide antibiotics that can be used against gram-negative pathogens. Researchers have used various moieties to cause the illicit transport of compounds in bacteria, and this study demonstrates the illicit transport of the largest known compound to date.
Nutrient Effects on Biocontrol of
Penicillium roqueforti
by
Pichia anomala
J121 during Airtight Storage of Wheat
Authors: Druvefors Ulrika Ädel , Passoth Volkmar , Schnürer Johan
Published: April 2005

The biocontrol yeast
Pichia anomala
inhibits the growth of a variety of mold species. We examined the mechanism underlying the inhibition of the grain spoilage mold
Penicillium roqueforti
by the biocontrol yeast
P. anomala
J121 during airtight storage. The biocontrol effect in a model grain silo with moist wheat (water activity of 0.96) was enhanced when complex medium, maltose, or glucose was added. Supplementation with additional nitrogen or vitamin sources did not affect the biocontrol activity of the yeast. The addition of complex medium or glucose did not significantly influence the yeast cell numbers in the silos, whether in the presence or absence of
P. roqueforti
. Mold growth was not influenced by the addition of nutrients, if cultivated without yeast. The products of glucose metabolism, mainly ethanol and ethyl acetate, increased after glucose addition to
P. anomala
-inoculated treatments. Our results suggest that neither competition for nutrients nor production of a glucose-repressible cell wall lytic enzyme is the main mode of action of biocontrol by
P. anomala
in this grain system. Instead, the mold-inhibiting effect probably is due to the antifungal action of metabolites, most likely a combination of ethyl acetate and ethanol, derived from glycolysis. The discovery that sugar amendments enhance the biocontrol effect of
P. anomala
suggests novel ways of formulating biocontrol yeasts.
Folate Cross-Feeding Supports Symbiotic Homoacetogenic Spirochetes
Authors: Graber Joseph R. , Breznak John A.
Published: April 2005

Treponema primitia
, an H
2
-consuming CO
2
-reducing homoacetogenic spirochete in termite hindguts, requires an exogenous source of folate for growth. Tetrahydrofolate (THF) acts as a C
1
carrier in CO
2
-reductive acetogenesis, a microbially mediated process important to the carbon and energy requirements of termites. To examine the hypothesis that other termite gut microbes probably supply some form of folate to
T. primitia
in situ, we used a bioassay to screen for and isolate folate-secreting bacteria from hindguts of
Zootermopsis angusticollis
, which is the host of
T. primitia
. Based on morphology, physiology, and 16S rRNA gene sequences, the major folate secretors were identified as strains of
Lactococcus lactis
and
Serratia grimesii
. During growth, these isolates secreted 5-formyl-THF at levels up to 146 ng/ml, and their cell-free culture fluids satisfied the folate requirement of
T. primitia
strains in vitro. Analysis of
Z. angusticollis
hindgut fluid revealed that 5-formyl-THF was the only detectable folate compound and occurred at an in situ concentration (1.3 μg/ml) which was more than sufficient to support the growth of
T. primitia
. These results imply that cross-feeding of 5-formyl-THF by other community members is important for growth of symbiotic hindgut spirochetes and thus termite nutrition and survival.
Cytotoxicity Potential and Genotypic Characterization of
Escherichia coli
Isolates from Environmental and Food Sources
Authors: Maldonado Yadilka , Fiser Jennifer C. , Nakatsu Cindy H. , Bhunia Arun K.
Published: April 2005

The presence of
Escherichia coli
isolates in the environment is a potential source of contamination of food and water supplies. Moreover, these isolates may harbor virulence genes that can be a source of new forms of pathogenic strains. Here, using multiplex PCR, we examined the presence of virulence gene markers (
stx
1
,
stx
2
,
eaeA
,
hlyA
) in 1,698 environmental isolates of
E. coli
and 81 isolates from food and clinical sources. The PCR analysis showed that ∼5% (79 of 1,698) of the total environmental isolates and 96% (79 of 81) of the food and clinical isolates were positive for at least one of the genes. Of the food and clinical isolates, 84% (68 of 81 isolates) were positive for all four genes. Of the subset of environmental isolates chosen for further analysis, 16% (13 of 79 isolates) were positive for
stx
2
and 84% (66 of 79 isolates) were positive for
eaeA
; 16 of the latter strains were also positive for
hlyA
. The pathogenic potentials of 174 isolates (81 isolates from food and clinical sources and 93 isolates from environmental sources) were tested by using a cytotoxicity assay based on lactate dehydrogenase release from Vero cells. In general, 97% (79 of 81) of the food and clinical isolates and 41% (39 of 93) of the environmental isolates exhibited positive cytotoxicity. High cytotoxicity values correlated to the presence of
stx
genes. The majority of
hly
-positive but
stx
-negative environmental isolates also exhibited a certain degree of cytotoxicity. Isolates were also tested for sorbitol utilization and were genotyped by ribotyping and by repetitive extragenic palindromic PCR (REP-PCR) as potential means of quickly identifying virulent strains from the environment, but none of these methods could be used to distinguish cytotoxic environmental isolates. Only 31% of the isolates were negative for sorbitol fermentation, and none of the isolates had common ribotypes or REP-PCR fingerprints. This study suggests that overall higher cytotoxicity values correlated with the production of
stx
genes, and the majority of
hly
-positive but
stx
-negative environmental isolates also exhibited a certain degree of cytotoxicity. This study demonstrated that there is widespread distribution of potentially virulent
E. coli
strains in the environment that may be a cause of concern for human health.
Genes Involved in SkfA Killing Factor Production Protect a
Bacillus subtilis
Lipase against Proteolysis
Authors: Westers Helga , Braun Peter G. , Westers Lidia , Antelmann Haike , Hecker Michael , Jongbloed Jan D. H. , Yoshikawa Hirofumi , Tanaka Teruo , van Dijl Jan Maarten , Quax Wim J.
Published: April 2005

Small lipases of
Bacillus
species, such as LipA from
Bacillus subtilis
, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the
B. subtilis
genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes,
skfABCD
, located in the prophage 1 region. Intact
skfABCD
genes are required not only for LipA production at wild-type levels by
B. subtilis
168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient
B. subtilis
strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor.
Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes fromMycobacteriumsp. Strain M156
Authors: Chan Kwo Chion Chan K. , Askew Sarah E. , Leak David J.
Published: April 2005

Propene monooxygenase has been cloned fromMycobacteriumsp. strain M156, based on hybridization with theamoABCDgenes ofRhodococcus corallinusB276. Sequencing indicated that the mycobacterial enzyme is a member of the binuclear nonheme iron monooxygenase family and, in gene order and sequence, is most similar to that fromR. corallinusB-276. Attempts were made to express thepmoABCDoperon inEscherichia coliandMycobacterium smegmatismc2155. In the former, there appeared to be a problem resolving overlapping reading frames betweenpmoAand -Band betweenpmoCand -D, while in the latter, problems were encountered with plasmid instability when thepmoABCDgenes were placed under the control of thehsp60heat shock promoter in the pNBV1 vector. Fortuitously, constructs with the opposite orientation were constitutively expressed at a level sufficient to allow preliminary mutational analysis. Two PMO active-site residues (A94 and V188) were targeted by site-directed mutagenesis to alter their stereoselectivity. The results suggest that changing the volume occupied by the side chain at V188 leads to a systematic alteration in the stereoselectivity of styrene oxidation, presumably by producing different orientations for substrate binding during catalysis. Changing the volume occupied by the side chain at A94 produced a nonsystematic change in stereoselectivity, which may be attributable to the role of this residue in expansion of the binding site during substrate binding. Neither set of mutations changed the enzyme's specificity for epoxidation.
Formation of
trans
Fatty Acids Is Not Involved in Growth-Linked Membrane Adaptation of
Pseudomonas putida
Authors: Härtig Claus , Loffhagen Norbert , Harms Hauke
Published: April 2005

Fatty acid compositions in growing and resting cells of several strains of
Pseudomonas putida
(P8, NCTC 10936, and KT 2440) were studied, with a focus on alterations of the saturation degree,
cis-trans
isomerization, and cyclopropane formation. The fatty acid compositions of the strains were very similar under comparable growth conditions, but surprisingly, and contrary to earlier reports,
trans
fatty acids were not found in either exponentially growing cells or stationary-phase cells. During the transition from growth to the starvation state, cyclopropane fatty acids were preferentially formed, an increase in the saturation degree of fatty acids was observed, and larger amounts of hydroxy fatty acids were detected. A lowered saturation degree and concomitant higher membrane fluidity seemed to be optimal for substrate uptake and growth. The incubation of cells under nongrowth conditions rapidly led to the formation of
trans
fatty acids. We show that harvesting and sample preparation for analysis could provoke the enzyme-catalyzed formation of
trans
fatty acids. Freeze-thawing of resting cells and increased temperatures accelerated the formation of
trans
fatty acids. We demonstrate that
cis-trans
isomerization only occurred in cells that were subjected to an abrupt disturbance without having the possibility of adapting to the changed conditions by the de novo synthesis of fatty acids. The
cis-trans
isomerization reaction was in competition with the
cis
-to-cyclopropane fatty acid conversion. The potential for the formation of
trans
fatty acids depended on the cyclopropane content that was already present.
Diversity and Phylogenetic Affiliations of Morphologically Conspicuous Large Filamentous Bacteria Occurring in the Pelagic Zones of a Broad Spectrum of Freshwater Habitats
Authors: Schauer Michael , Hahn Martin W.
Published: April 2005

Filamentous bacteria with a conspicuous morphology were found in the majority of the bacterioplankton samples from a variety of freshwater habitats that were studied. These heterotrophic filaments typically account for <1 to 11% of the total number of bacteria. The biovolume of this morphotype can exceed 40% of the biovolume for all bacteria. Surprisingly, we found hardly any data on these morphologically conspicuous filaments in the literature. Mixed cultures containing these filamentous bacteria were established by cultivation and isolation experiments with samples from different freshwater lakes. Nearly full-length 16S rRNA gene sequences were obtained from several mixed cultures and environmental samples from habitats in Europe, Africa, China, Australia, and New Zealand. Phylogenetic analysis of the sequences showed that three groups form a single monophyletic cluster, the SOL cluster, in the family
Saprospiraceae
. We developed a set of six nested probes for fluorescence in situ hybridization. Of the six probes, one probe was specific for
Haliscomenobacter hydrossis
, three probes were specific for the three subclusters (each probe was specific for one subcluster), one probe was specific for the entire SOL cluster, and another probe targeted almost the entire
Saprospiraceae
family. Specific hybridization of environmental samples and enrichments showed that the members of the three subclusters exhibited the same filamentous morphology. So far, using the subcluster-specific probes, we have not been able to detect any bacteria with a differing morphology. We conclude that the SOL cluster bacteria are an integral part of bacterioplankton in many freshwater habitats. They potentially account for a large fraction of the total bacterial biomass but have been underrepresented in molecular diversity studies so far.
Identification and Molecular Epidemiology of
Campylobacter coli
Isolates from Human Gastroenteritis, Food, and Animal Sources by Amplified Fragment Length Polymorphism Analysis and Penner Serotyping
Authors: Siemer B. L. , Nielsen E. M. , On S. L. W.
Published: April 2005

Campylobacter coli
is an infrequently studied but important food-borne pathogen with a wide natural distribution. We investigated its molecular epidemiology by use of amplified fragment length polymorphism (AFLP)-based genotyping and Penner serotyping. Serotype reference strains and 177 Danish isolates of diverse origin identified by routine phenotyping as
C. coli
were examined. Molecular tools identified some 12% of field isolates as
Campylobacter jejuni
, emphasizing the need for improved identification methods in routine laboratories. Cluster analysis of AFLP profiles of 174 confirmed
C. coli
isolates revealed a difference in the distribution of isolates from pig and poultry (chicken, duck, turkey, and ostrich) species and indicated the various poultry species, but not pigs, to be likely sources of human
C. coli
infection. A poor correlation was observed between serotyping and AFLP profiling, suggesting that the former method has limited value in epidemiological studies of this species.
Sec-Mediated Secretion of Bacteriocin Enterocin P by
Lactococcus lactis
Authors: Herranz Carmen , Driessen Arnold J. M.
Published: April 2005

Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in
Lactococcus lactis
. Signal peptide mutations and the SecA inhibitor azide blocked secretion. These observations demonstrate that EntP is secreted by the Sec translocase.
Culture-Dependent and -Independent Methods To Investigate the Microbial Ecology of Italian Fermented Sausages
Authors: Rantsiou Kalliopi , Urso Rosalinda , Iacumin Lucilla , Cantoni Carlo , Cattaneo Patrizia , Comi Giuseppe , Cocolin Luca
Published: April 2005

In this study, the microbial ecology of three naturally fermented sausages produced in northeast Italy was studied by culture-dependent and -independent methods. By plating analysis, the predominance of lactic acid bacteria populations was pointed out, as well as the importance of coagulase-negative cocci. Also in the case of one fermentation, the fecal enterocci reached significant counts, highlighting their contribution to the particular transformation process. Yeast counts were higher than the detection limit (>100 CFU/g) in only one fermented sausage. Analysis of the denaturing gradient gel electrophoresis (DGGE) patterns and sequencing of the bands allowed profiling of the microbial populations present in the sausages during fermentation. The bacterial ecology was mainly characterized by the stable presence of
Lactobacillus curvatus
and
Lactobacillus sakei
, but
Lactobacillus paracasei
was also repeatedly detected. An important piece of evidence was the presence of
Lactococcus garvieae
, which clearly contributed in two fermentations. Several species of
Staphylococcus
were also detected. Regarding other bacterial groups,
Bacillus
sp.,
Ruminococcus
sp., and
Macrococcus caseolyticus
were also identified at the beginning of the transformations. In addition, yeast species belonging to
Debaryomyces hansenii
, several
Candida
species, and
Willopsis saturnus
were observed in the DGGE gels. Finally, cluster analysis of the bacterial and yeast DGGE profiles highlighted the uniqueness of the fermentation processes studied.
Effects of Artificial Defoliation of Pines on the Structure and Physiology of the Soil Fungal Community of a Mixed Pine-Spruce Forest
Authors: Cullings Ken , Raleigh Christopher , New Michael H. , Henson Joan
Published: April 2005

Loss of photosynthetic area can affect soil microbial communities by altering the availability of fixed carbon. We used denaturing gradient gel electrophoresis (DGGE) and Biolog filamentous-fungus plates to determine the effects of artificial defoliation of pines in a mixed pine-spruce forest on the composition of the fungal community in a forest soil. As measured by DGGE, two fungal species were affected significantly by the defoliation of pines (
P
< 0.001); the frequency of members of the ectomycorrhizal fungus genus
Cenococcum
decreased significantly, while the frequency of organisms of an unidentified soil fungus increased. The decrease in the amount of
Cenococcum
organisms may have occurred because of the formation of extensive hyphal networks by species of this genus, which require more of the carbon fixed by their host, or because this fungus is dependent upon quantitative differences in spruce root exudates. The defoliation of pines did not affect the overall composition of the soil fungal community or fungal-species richness (number of species per core). Biolog filamentous-fungus plate assays indicated a significant increase (
P
< 0.001) in the number of carbon substrates utilized by the soil fungi and the rate at which these substrates were used, which could indicate an increase in fungal-species richness. Thus, either small changes in the soil fungal community give rise to significant increases in physiological capabilities or PCR bias limits the reliability of the DGGE results. These data indicate that combined genetic and physiological assessments of the soil fungal community are needed to accurately assess the effect of disturbance on indigenous microbial systems.
Diversity of Oxygenase Genes from Methane- and Ammonia-Oxidizing Bacteria in the Eastern Snake River Plain Aquifer
Authors: Erwin Daniel P. , Erickson Issac K. , Delwiche Mark E. , Colwell Frederick S. , Strap Janice L. , Crawford Ronald L.
Published: April 2005

PCR amplification, restriction fragment length polymorphism, and phylogenetic analysis of oxygenase genes were used for the characterization of in situ methane- and ammonia-oxidizing bacteria from free-living and attached communities in the Eastern Snake River Plain aquifer. The following three methane monooxygenase (MMO) PCR primer sets were used: A189-A682, which amplifies an internal region of both thepmoAgene of the MMO particulate form and theamoAgene of ammonia monooxygenase; A189-mb661, which specifically targets thepmoAgene; and mmoXA-mmoXB, which amplifies themmoXgene of the MMO soluble form (sMMO). Whole-genome amplification (WGA) was used to amplify metagenomic DNA from each community to assess its applicability for generating unbiased metagenomic template DNA. The majority of sequences in each archive were related to oxygenases of type II-like methanotrophs of the genusMethylocystis. A small subset of type I sequences found only in free-living communities possessed oxygenase genes that grouped nearest toMethylobacterandMethylomonasspp. Sequences similar to that of theamoAgene associated with ammonia-oxidizing bacteria (AOB) most closely matched a sequence from the uncultured bacterium BS870 but showed no substantial alignment to known cultured AOB. Based on these functional gene analyses, bacteria related to the type II methanotrophMethylocystissp. were found to dominate both free-living and attached communities. Metagenomic DNA amplified by WGA showed characteristics similar to those of unamplified samples. Overall, numerous sMMO-like gene sequences that have been previously associated with high rates of trichloroethylene cometabolism were observed in both free-living and attached communities in this basaltic aquifer.
Genetic Diversity of Benzoyl Coenzyme A Reductase Genes Detected in Denitrifying Isolates and Estuarine Sediment Communities
Authors: Song Bongkeun , Ward Bess B.
Published: April 2005

Benzoyl coenzyme A (benzoyl-CoA) reductase is a central enzyme in the anaerobic degradation of organic carbon, which utilizes a common intermediate (benzoyl-CoA) in the metabolism of many aromatic compounds. The diversity of benzoyl-CoA reductase genes in denitrifying bacterial isolates capable of degrading aromatic compounds and in river and estuarine sediment samples from the Arthur Kill in New Jersey and the Chesapeake Bay in Maryland was investigated. Degenerate primers were developed from the known benzoyl-CoA reductase genes from
Thauera aromatica
,
Rhodopseudomonas palustris
, and
Azoarcus evansii
. PCR amplification detected benzoyl-CoA reductase genes in the denitrifying isolates belonging to α-, β-, or γ-
Proteobacteria
as well as in the sediment samples. Phylogenetic analysis, sequence similarity comparison, and conserved indel determination grouped the new sequences into either the
bcr
type (found in
T. aromatica
and
R. palustris)
or the
bzd
type (found in
A. evansii
). All the
Thauera
strains and the isolates from the genera
Acidovorax
,
Bradyrhizobium
,
Paracoccus
,
Ensifer
, and
Pseudomonas
had
bcr
-type benzoyl-CoA reductases with amino acid sequence similarities of more than 97%. The genes detected from
Azarocus
strains were assigned to the
bzd
type. A total of 50 environmental clones were detected from denitrifying consortium and sediment samples, and 28 clones were assigned to either the
bcr
or the
bzd
type of benzoyl-CoA reductase genes. Thus, we could determine the genetic capabilities for anaerobic degradation of aromatic compounds in sediment communities of the Chesapeake Bay and the Arthur Kill on the basis of the detection of two types of benzoyl-CoA reductase genes. The detected genes have future applications as genetic markers to monitor aromatic compound degradation in natural and engineered ecosystems.
Vertical Distribution of the Free-Living Amoeba Population in Soil under Desert Shrubs in the Negev Desert, Israel
Authors: Rodriguez-Zaragoza Salvador , Mayzlish Einav , Steinberger Yosef
Published: April 2005

A field study was designed to examine the effect of desert shrubs on the dynamics of free-living amoebae in arid soil. Soil samples from 0- to 50-cm depths were collected at 10-cm intervals in each of the four seasons. The vertical distributions of the four main morphological types of amoebae, grouped according to their mobility, and of small flagellate populations were measured under the canopies of
Hammada scoparia
and
Atriplex halimus
, shrubs belonging to the chloride-absorbing xerohalophytes. The result obtained from the field study demonstrated that the total number of protozoa was significantly higher during the wet seasons (winter and spring) than during the dry seasons. The protozoan population was more diverse under the canopy of
H. scoparia
during the wet seasons, reaching 8,000 individuals per 1 g of dry soil, whereas during the dry seasons, the populations were higher under the canopy of
A. halimus
, with a mean of 250 individuals. The protozoan population in the deeper layers (40 to 50 cm) was found to be as active as that in the upper layers, demonstrating that, in the desert, soil columns below 20 cm are fertile and worth studying. The type 1 amoebae (e.g.,
Acanthamoeba
and
Filamoeba
spp.) were the most abundant throughout the study period, and their numbers were significantly higher than those of the other amoeba types.
Molecular Assays for Targeting Human and Bovine Enteric Viruses in Coastal Waters and Their Application for Library-Independent Source Tracking
Authors: Fong Theng-Theng , Griffin Dale W. , Lipp Erin K.
Published: April 2005

Rapid population growth and urban development along waterways and coastal areas have led to decreasing water quality. To examine the effects of upstream anthropogenic activities on microbiological water quality, methods for source-specific testing are required. In this study, molecular assays targeting human enteroviruses (HEV), bovine enteroviruses (BEV), and human adenoviruses (HAdV) were developed and used to identify major sources of fecal contamination in the lower Altamaha River, Georgia. Two-liter grab samples were collected monthly from five tidally influenced stations between July and December 2002. Samples were analyzed by reverse transcription- and nested-PCR. PCR results were confirmed by dot blot hybridization. Eleven and 17 of the 30 surface water samples tested positive for HAdV and HEV, respectively. Two-thirds of the samples tested positive for either HEV or HAdV, and the viruses occurred simultaneously in 26% of samples. BEV were detected in 11 of 30 surface water samples. Binary logistic regression analysis showed that the presence of both human and bovine enteric viruses was not significantly related to either fecal coliform or total coliform levels. The presence of these viruses was directly related to dissolved oxygen and streamflow but inversely related to water temperature, rainfall in the 30 days preceding sampling, and chlorophyll-
a
concentrations. The stringent host specificity of enteric viruses makes them good library-independent indicators for identification of water pollution sources. Viral pathogen detection by PCR is a highly sensitive and easy-to-use tool for rapid assessment of water quality and fecal contamination when public health risk characterization is not necessary.
Explorative Multifactor Approach for Investigating Global Survival Mechanisms of
Campylobacter jejuni
under Environmental Conditions
Authors: Moen Birgitte , Oust Astrid , Langsrud Øyvind , Dorrell Nick , Marsden Gemma L. , Hinds Jason , Kohler Achim , Wren Brendan W. , Rudi Knut
Published: April 2005

Explorative approaches such as DNA microarray experiments are becoming increasingly important in microbial research. Despite these major technical advancements, approaches to study multifactor experiments are still lacking. We have addressed this problem by using rotation testing and a novel multivariate analysis of variance (MANOVA) approach (50-50 MANOVA) to investigate interacting experimental factors in a complex experimental design. Furthermore, a new rotation testing based method was introduced to calculate false-discovery rates for each response. This novel analytical concept was used to investigate global survival mechanisms in the environment of the major food-borne pathogen
C. jejuni.
We simulated nongrowth environmental conditions by investigating combinations of the factors temperature (5 and 25°C) and oxygen tension (anaerobic, microaerobic, and aerobic). Data were generated with DNA microarrays for information about gene expression patterns and Fourier transform infrared (FT-IR) spectroscopy to study global macromolecular changes in the cell. Microarray analyses showed that most genes were either unchanged or down regulated compared to the reference (day 0) for the conditions tested and that the 25°C anaerobic condition gave the most distinct expression pattern with the fewest genes expressed. The few up-regulated genes were generally stress related and/or related to the cell envelope. We found, using FT-IR spectroscopy, that the amount of polysaccharides and oligosaccharides increased under the nongrowth survival conditions. Potential mechanisms for survival could be to down regulate most functions to save energy and to produce polysaccharides and oligosaccharides for protection against harsh environments. Basic knowledge about the survival mechanisms is of fundamental importance in preventing transmission of this bacterium through the food chain.
Analysis of Fluorescent Protein Expression in Transformants ofRickettsia monacensis, an Obligate Intracellular Tick Symbiont
Authors: Baldridge Gerald D. , Burkhardt Nicole , Herron Michael J. , Kurtti Timothy J. , Munderloh Ulrike G.
Published: April 2005

We developed and applied transposon-based transformation vectors for molecular manipulation and analysis of spotted fever group rickettsiae, which are obligate intracellular bacteria that infect ticks and, in some cases, mammals. Using the Epicentre EZ::TN transposon system, we designed transposons for simultaneous expression of a reporter gene and a chloramphenicol acetyltransferase (CAT) resistance marker. Transposomes (transposon-transposase complexes) were electroporated intoRickettsia monacensis, a rickettsial symbiont isolated from the tickIxodes ricinus. Each transposon contained an expression cassette consisting of the rickettsialompApromoter and a green fluorescent protein (GFP) reporter gene (GFPuv) or theompBpromoter and a red fluorescent protein reporter gene (DsRed2), followed by theompAtranscription terminator and a secondompApromoter CAT gene cassette. Selection with chloramphenicol gave rise to rickettsial populations with chromosomally integrated single-copy transposons as determined by PCR, Southern blotting, and sequence analysis. Reverse transcription-PCR and Northern blots demonstrated transcription of all three genes. GFPuv transformant rickettsiae exhibited strong fluorescence in individual cells, but DsRed2 transformants did not. Western blots confirmed expression of GFPuv inR. monacensisand inEscherichia coli, but DsRed2 was expressed only inE. coli. The DsRed2 gene, but not the GFPuv gene, contains many GC-rich amino acid codons that are rare in the preferred codon suite of rickettsiae, possibly explaining the failure to express DsRed2 protein inR. monacensis. We demonstrated that our vectors provide a means to study rickettsia-host cell interactions by visualizing GFPuv-fluorescentR. monacensisassociated with actin tails in tick host cells.
Lipid Biomarkers and Carbon Isotope Signatures of a Microbial (
Beggiatoa
) Mat Associated with Gas Hydrates in the Gulf of Mexico
Authors: Zhang Chuanlun L. , Huang Zhiyong , Cantu James , Pancost Richard D. , Brigmon Robin L. , Lyons Timothy W. , Sassen Roger
Published: April 2005

White and orange mats are ubiquitous on surface sediments associated with gas hydrates and cold seeps in the Gulf of Mexico. The goal of this study was to determine the predominant pathways for carbon cycling within an orange mat in Green Canyon (GC) block GC 234 in the Gulf of Mexico. Our approach incorporated laser-scanning confocal microscopy, lipid biomarkers, stable carbon isotopes, and 16S rRNA gene sequencing. Confocal microscopy showed the predominance of filamentous microorganisms (4 to 5 μm in diameter) in the mat sample, which are characteristic of
Beggiatoa
. The phospholipid fatty acids extracted from the mat sample were dominated by 16:1ω7c/t (67%), 18:1ω7c (17%), and 16:0 (8%), which are consistent with lipid profiles of known sulfur-oxidizing bacteria, including
Beggiatoa
. These results are supported by the 16S rRNA gene analysis of the mat material, which yielded sequences that are all related to the vacuolated sulfur-oxidizing bacteria, including
Beggiatoa
,
Thioploca
, and
Thiomargarita
. The δ
13
C value of total biomass was −28.6‰; those of individual fatty acids were −29.4 to −33.7‰. These values suggested heterotrophic growth of
Beggiatoa
on organic substrates that may have δ
13
C values characteristic of crude oil or on their by-products from microbial degradation. This study demonstrated that integrating lipid biomarkers, stable isotopes, and molecular DNA could enhance our understanding of the metabolic functions of
Beggiatoa
mats in sulfide-rich marine sediments associated with gas hydrates in the Gulf of Mexico and other locations.
Mycobacterium aviumsubsp.paratuberculosisin the Catchment Area and Water of the River Taff in South Wales, United Kingdom, and Its Potential Relationship to Clustering of Crohn's Disease Cases in the City of Cardiff
Authors: Pickup R. W. , Rhodes G. , Arnott S. , Sidi-Boumedine K. , Bull T. J. , Weightman A. , Hurley M. , Hermon-Taylor J.
Published: April 2005

In South Wales, United Kingdom, a populated coastal region lies beneath hill pastures grazed by livestock in whichMycobacterium aviumsubsp.paratuberculosisis endemic. The Taff is a spate river running off the hills and through the principal city of Cardiff. We sampled Taff water above Cardiff twice weekly from November 2001 to November 2002.M. aviumsubsp.paratuberculosiswas detected by IS900PCR and culture. Thirty-one of 96 daily samples (32.3%) were IS900PCR positive, and 12 grewM. aviumsubsp.paratuberculosisbovine strains. Amplicon sequences from colonies were identical to the sequence with GenBank accession no.X16293, whereas 16 of 19 sequences from river water DNA extracts had a single-nucleotide polymorphism at position 214. This is consistent with a different strain ofM. aviumsubsp.paratuberculosisin the river, which is unculturable by the methods we used. Parallel studies showed thatM. aviumsubsp.paratuberculosisremained culturable in lake water microcosms for 632 days and persisted to 841 days. Of four reservoirs controlling the catchment area of the Taff,M. aviumsubsp.paratuberculosiswas present in surface sediments from three and in sediment cores from two, consistent with deposition over at least 50 years. Previous epidemiological research in Cardiff demonstrated a highly significant increase of Crohn's disease in 11 districts. These bordered the river except for a gap on the windward side. A topographical relief map shows that this gap is directly opposite a valley open to the prevailing southwesterly winds. This would influence the distribution of aerosols carryingM. aviumsubsp.paratuberculosisfrom the river.
New Tool for Metabolic Pathway Engineering in
Escherichia coli
: One-Step Method To Modulate Expression of Chromosomal Genes
Authors: Meynial-Salles Isabelle , Cervin Marguerite A. , Soucaille Philippe
Published: April 2005

A simple and highly efficient method was developed to produce a library of
Escherichia coli
clones that express a particular chromosomal gene at a wide range of expression levels. The basic strategy was to replace all or part of the upstream region of a coding sequence containing the elements involved in its expression (promoter, operator, gene coding for a regulator, ribosome binding site, and start codon) with a PCR-generated library of expression cassettes.
Novel Major Bacterial Candidate Division within a Municipal Anaerobic Sludge Digester
Authors: Chouari Rakia , Le Paslier Denis , Dauga Catherine , Daegelen Patrick , Weissenbach Jean , Sghir Abdelghani
Published: April 2005

In a previous study, we analyzed the molecular diversity ofPlanctomycetalesby PCR amplification and sequencing of 16S rRNA clone libraries generated from a municipal wastewater plant, using planctomycete-specific and universal primer sets (R. Chouari, D. Le Paslier, P. Daegelen, P. Ginestet, J. Weissenbach, and A. Sghir, Appl. Environ. Microbiol.69:7354-7363, 2003). Only a small fraction (4%) of the 16S rRNA gene sequences of the digester clone library corresponded to thePlanctomycetalesdivision. Importantly, 85.9% of the digester clone sequences are grouped into two different clusters named WWE1 (81.4% of the sequences) and WWE2 (4.5%) and are distantly affiliated with unidentified bacterial sequences retrieved from a methanogenic reactor community and from a termite gut, respectively. In phylogenetic analysis using 16S rRNA gene sequence representatives of the main phylogenetic bacterial divisions, the two clusters are monophyletic, branch apart from each other, and are distantly related toPlanctomycetalesand other bacterial divisions. A novel candidate division is proposed for WWE1, while the WWE2 cluster strongly affiliates with the recently proposedLentisphearaephylum. We designed and validated a 16S rRNA probe targeting WWE1 16S rRNA sequences by both fluorescent in situ hybridization (FISH) and dot blot hybridization (DBH). Results of FISH analysis show that WWE1 representative microorganisms are rods or filamentous shaped, while DBH shows that WWE1 accounts for 12% of the total bacterial rRNA within the anaerobic digester. The remaining 16S rRNA gene sequences are affiliated withVerrucomicrobiaor recently described candidate divisions with no known pure culture representatives, such as OD1, BRC1, or NBL-UPA2, making up less than 3.5% of the clone library, respectively. This inventory expands the known diversity of the latter bacterial division-level lineages.
Detection ofSalmonellaStrains andEscherichia coliO157:H7 in Feces of Small Ruminants and Their Isolation with Various Media
Authors: Pao Steven , Patel Dhartika , Kalantari Aref , Tritschler Joseph P. , Wildeus Stephan , Sayre Brian L.
Published: April 2005

Salmonellastrains andEscherichia coliO157:H7 were detected in 17 and 5 small ruminants in Virginia, respectively, of 287 tested. Background microflora interfered with the fecal analysis. The combination ofSalmonellaenzyme immunoassay (EIA) detection and xylose-lysine-deoxycholate agar isolation was satisfactory. Modifying enrichment to a 1:100 dilution enabled effectiveE. coliO157:H7 detection by EIA and isolation by sorbitol-MacConkey agar with cefixime-tellurite.
Comparative Analysis of Bacterial Diversity in Freshwater Sediment of a Shallow Eutrophic Lake by Molecular and Improved Cultivation-Based Techniques
Authors: Tamaki Hideyuki , Sekiguchi Yuji , Hanada Satoshi , Nakamura Kazunori , Nomura Nakao , Matsumura Masatoshi , Kamagata Yoichi
Published: April 2005

Comparative analysis of bacterial diversity in freshwater sediment collected from a shallow eutrophic lake was performed by using 16S rRNA gene clone library and improved cultivation-based techniques. Our study demonstrated that the use of gellan gum as a gelling reagent instead of agar was more effective at increasing culturability, cultivating a diverse array of novel microbes, and reducing the gaps of the results between molecular and cultivation-based analyses.
Viscoelasticity of
Staphylococcus aureus
Biofilms in Response to Fluid Shear Allows Resistance to Detachment and Facilitates Rolling Migration
Authors: Rupp Cory J. , Fux Christoph A. , Stoodley Paul
Published: April 2005

Staphylococcus aureus
is a leading cause of catheter-related bloodstream infections and endocarditis. Both involve (i) biofilm formation, (ii) exposure to fluid shear, and (iii) high rates of dissemination. We found that viscoelasticity allowed
S. aureus
biofilms to resist detachment due to increased fluid shear by deformation, while remaining attached to a surface. Further, we report that
S. aureus
microcolonies moved downstream by rolling along the lumen walls of a glass flow cell, driven by the flow of the overlying fluid. The rolling appeared to be controlled by viscoelastic tethers. This tethered rolling may be important for the surface colonization of medical devices by nonmotile bacteria.
Characterization of the Export of Bulk Poly(A)
+
mRNA in
Saccharomyces cerevisiae
during the Wine-Making Process
Authors: Izawa Shingo , Takemura Reiko , Miki Takeo , Inoue Yoshiharu
Published: April 2005

Ethanol stress affects the nuclear export of mRNA similarly to heat shock in
Saccharomyces cerevisiae
. However, we have little information about mRNA transport in actual alcoholic fermentation. Here we characterized the transport of mRNA during wine making and found that bulk poly(A)
+
mRNA accumulated in the nucleus as fermentation progressed.
Degradation of β-Hexachlorocyclohexane by Haloalkane Dehalogenase LinB from
Sphingomonas paucimobilis
UT26
Authors: Nagata Yuji , Prokop Zbyněk , Sato Yukari , Jerabek Petr , Kumar Ashwani , Ohtsubo Yoshiyuki , Tsuda Masataka , Damborský Jiří
Published: April 2005

β-Hexachlorocyclohexane (β-HCH) is the most recalcitrant among the α-, β-, γ-, and δ-isomers of HCH and causes serious environmental pollution problems. We demonstrate here that the haloalkane dehalogenase LinB, reported earlier to mediate the second step in the degradation of γ-HCH in
Sphingomonas paucimobilis
UT26, metabolizes β-HCH to produce 2,3,4,5,6-pentachlorocyclohexanol.
Evidence for Involvement of an Electron Shuttle in Electricity Generation byGeothrix fermentans
Authors: Bond Daniel R. , Lovley Derek R.
Published: April 2005

In experiments performed using graphite electrodes poised by a potentiostat (+200 mV versus Ag/AgCl) or in a microbial fuel cell (with oxygen as the electron acceptor), the Fe(III)-reducing organismGeothrix fermentansconserved energy to support growth by coupling the complete oxidation of acetate to reduction of a graphite electrode. Other organic compounds, such as lactate, malate, propionate, and succinate as well as components of peptone and yeast extract, were utilized for electricity production. However, electrical characteristics and the results of shuttling assays indicated that unlike previously described electrode-reducing microorganisms,G. fermentansproduced a compound that promoted electrode reduction. This is the first report of complete oxidation of organic compounds linked to electrode reduction by an isolate outside of theProteobacteria.
Quantitative Detection of
Listeria monocytogenes
in Biofilms by Real-Time PCR
Authors: Guilbaud Morgan , de Coppet Pierre , Bourion Fabrice , Rachman Cinta , Prévost Hervé , Dousset Xavier
Published: April 2005

A quantitative method based on a real-time PCR assay to enumerate
Listeria monocytogenes
in biofilms was developed. The specificity for
L. monocytogenes
of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of
L. monocytogenes
detected growing in biofilms was 6 × 10
2
CFU/cm
2
.
Pathways for Methanogenesis and Diversity of Methanogenic Archaea in Three Boreal Peatland Ecosystems
Authors: Galand P. E. , Fritze H. , Conrad R. , Yrjälä K.
Published: April 2005

The main objectives of this study were to uncover the pathways used for methanogenesis in three different boreal peatland ecosystems and to describe the methanogenic populations involved. The mesotrophic fen had the lowest proportion of CH
4
produced from H
2
-CO
2
. The oligotrophic fen was the most hydrogenotrophic, followed by the ombrotrophic bog. Each site was characterized by a specific group of methanogenic sequences belonging to
Methanosaeta
spp. (mesotrophic fen), rice cluster-I (oligotrophic fen), and fen cluster (ombrotrophic bog).
por
Variable-Region Typing by DNA Probe Hybridization Is Broadly Applicable to Epidemiologic Studies of
Neisseria gonorrhoeae
Authors: Bash Margaret C. , Zhu Peixuan , Gulati Sunita , McKnew Durrie , Rice Peter A. , Lynn Freyja
Published: April 2005

The porin gene (
porB
) of
Neisseria gonorrhoeae
encodes the major outer membrane protein identified as PI or Por. To examine the utility of
por
variable-region (VR) typing,
porB
from 206 isolates was characterized by using oligonucleotide probes in a checkerboard hybridization assay that identifies the sequence types of five VRs of both PIA and PIB
porB
alleles. The strains represented temporally and geographically distinct isolates, isolates from a large cluster, epidemiologically linked partner isolates, and a collection of strains from disseminated gonococcal infections. By using rigorous epidemiologic criteria for transmission of infection between sex partners,
por
VR typing was more discriminatory than serovar typing in classifying isolates from both members of 43 epidemiologically linked pairs: 39 of 43 pairs were classified as coinciding by
por
VR typing compared to 43 of 43 by serovar determination (
P
= 0.058).
porB
sequence data confirmed the accuracy of the
por
VR method. Relationships between VR type and serovar typing monoclonal antibodies were observed for all six PIB and three of six PIA antibodies.
por
VR typing is a molecular tool that appears to have broad applicability. This method can be adapted to a wide range of technologies from simple hybridization to microarray and may allow for typing from noncultured clinical specimens.