bluuebunny/crossref_metadata_2025 huggingface dataset:
Algal Species and Light Microenvironment in a Low-pH, Geothermal Microbial Mat Community
Authors: Ferris M. J. , Sheehan K. B. , Kühl M. , Cooksey K. , Wigglesworth-Cooksey B. , Harvey R. , Henson J. M.
Published: November 2005

Unicellular algae are the predominant microbial mat-forming phototrophs in the extreme environments of acidic geothermal springs. The ecology of these algae is not well known because concepts of species composition are inferred from cultivated isolates and microscopic observations, methods known to provide incomplete and inaccurate assessments of species in situ. We used sequence analysis of 18S rRNA genes PCR amplified from mat samples from different seasons and different temperatures along a thermal gradient to identify algae in an often-studied acidic (pH 2.7) geothermal creek in Yellowstone National Park. Fiber-optic microprobes were used to show that light for algal photosynthesis is attenuated to <1% over the 1-mm surface interval of the mat. Three algal sequences were detected, and each was present year-round. ACyanidioschyzon merolaesequence was predominant at temperatures of ≥49°C. AChlorella protothecoidesvar.acidicolasequence and aParadoxia multisita-like sequence were predominant at temperatures of ≤39°C.
Effect of Oxidation Rate and Fe(II) State on Microbial Nitrate-Dependent Fe(III) Mineral Formation
Authors: Senko John M. , Dewers Thomas A. , Krumholz Lee R.
Published: November 2005

A nitrate-dependent Fe(II)-oxidizing bacterium was isolated and used to evaluate whether Fe(II) chemical form or oxidation rate had an effect on the mineralogy of biogenic Fe(III) (hydr)oxides resulting from nitrate-dependent Fe(II) oxidation. The isolate (designated FW33AN) had 99% 16S rRNA sequence similarity to
Klebsiella oxytoca.
FW33AN produced Fe(III) (hydr)oxides by oxidation of soluble Fe(II) [Fe(II)
sol
] or FeS under nitrate-reducing conditions. Based on X-ray diffraction (XRD) analysis, Fe(III) (hydr)oxide produced by oxidation of FeS was shown to be amorphous, while oxidation of Fe(II)
sol
yielded goethite. The rate of Fe(II) oxidation was then manipulated by incubating various cell concentrations of FW33AN with Fe(II)
sol
and nitrate. Characterization of products revealed that as Fe(II) oxidation rates slowed, a stronger goethite signal was observed by XRD and a larger proportion of Fe(III) was in the crystalline fraction. Since the mineralogy of Fe(III) (hydr)oxides may control the extent of subsequent Fe(III) reduction, the variables we identify here may have an effect on the biogeochemical cycling of Fe in anoxic ecosystems.
Generation and Molecular Characterization of New Temperature-Sensitive Plasmids Intended for Genetic Engineering of
Pasteurellaceae
Authors: Briggs Robert E. , Tatum Fred M.
Published: November 2005

Temperature-sensitive (TS) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin resistance parent plasmid pD70, isolated from
Mannheimia hemolytica
serotype 1. Three TS plasmids which failed to replicate at or above 42°C in
M. hemolytica
but which were fully functional below 31°C were selected for further analysis. Two of the TS plasmids were shown by sequencing to possess unique single-base-pair mutations. The third TS plasmid contained a unique base pair substitution and a second mutation that had been previously identified. These mutations were clustered within a 200-bp region of the presumed plasmid origin of replication. Site-directed single-nucleotide substitutions were introduced into the wild-type pD70 origin of replication to confirm that mutations identified by sequencing had conferred thermoregulated replication. Deletion analysis on the wild-type pD70 plasmid replicon revealed that approximately 720 bp are necessary for plasmid maintenance. Replication of the TS plasmids was thermoregulated in
Pasteurella multocida
and
Haemophilus somnus
as well. To consistently transform
H. somnus
with TS plasmid, in vitro DNA methylation with commercially available HhaI methyltransferase was necessary to protect against the organism's restriction enzyme HsoI (recognition sequence 5′-GCGC-3′) characterized herein.
Construction of In-Frame
aroA
Deletion Mutants of
Mannheimia haemolytica
,
Pasteurella multocida
, and
Haemophilus somnus
by Using a New Temperature-Sensitive Plasmid
Authors: Tatum Fred M. , Briggs Robert E.
Published: November 2005

A temperature-sensitive (TS) plasmid was generated from the endogenous streptomycin resistance plasmid of
Mannheimia hemolytica
and used to engineer in-frame
aroA
deletion mutants of
Mannheimia hemolytica
,
Pasteurella multocida
, and
Haemophilus somnus
. TS replacement plasmids carrying in-frame
aroA
deletions were constructed for each target species and introduced into host cells by electroporation. After recovery in broth, cells were spread onto plates containing antibiotic and incubated at 30°C, the permissive temperature for autonomous plasmid replication. Transfer of transformants to selective plates cultured at a nonpermissive temperature for plasmid replication selected for single-crossover mutants consisting of replacement plasmids that had integrated into host chromosomes by homologous recombination. Transfer of the single-crossover mutants back to a permissive temperature without antibiotic selection drove plasmid resolution, and, depending on where plasmid excision occurred, either deletion mutants or wild-type cells were generated. The system used here represents a broadly applicable means for generating unmarked mutants of
Pasteurellaceae
species.
In Silico Reconstruction of the Metabolic Pathways of
Lactobacillus plantarum
: Comparing Predictions of Nutrient Requirements with Those from Growth Experiments
Authors: Teusink Bas , van Enckevort Frank H. J. , Francke Christof , Wiersma Anne , Wegkamp Arno , Smid Eddy J. , Siezen Roland J.
Published: November 2005

On the basis of the annotated genome we reconstructed the metabolic pathways of the lactic acid bacterium
Lactobacillus plantarum
WCFS1. After automatic reconstruction by the Pathologic tool of Pathway Tools (
http://bioinformatics.ai.sri.com/ptools/
), the resulting pathway-genome database, LacplantCyc, was manually curated extensively. The current database contains refinements to existing routes and new gram-positive bacterium-specific reactions that were not present in the MetaCyc database. These reactions include, for example, reactions related to cell wall biosynthesis, molybdopterin biosynthesis, and transport. At present, LacplantCyc includes 129 pathways and 704 predicted reactions involving some 670 chemical species and 710 enzymes. We tested vitamin and amino acid requirements of
L. plantarum
experimentally and compared the results with the pathways present in LacplantCyc. In the majority of cases (32 of 37 cases) the experimental results agreed with the final reconstruction. LacplantCyc is the most extensively curated pathway-genome database for gram-positive bacteria and is open to the microbiology community via the World Wide Web (
www.lacplantcyc.nl
). It can be used as a reference pathway-genome database for gram-positive microbes in general and lactic acid bacteria in particular.
Ecology, Inhibitory Activity, and Morphogenesis of a Marine Antagonistic Bacterium Belonging to the
Roseobacter
Clade
Authors: Bruhn Jesper Bartholin , Nielsen Kristian Fog , Hjelm Mette , Hansen Michael , Bresciani José , Schulz Stefan , Gram Lone
Published: November 2005

Roseobacter
strain 27-4 has been isolated from a turbot larval rearing unit and is capable of reducing mortality in turbot egg yolk sac larvae. Here, we demonstrate that the supernatant of
Roseobacter
27-4 is lethal to the larval pathogens
Vibrio anguillarum
and
Vibrio splendidus
in a buffer system and inhibited their growth in marine broth. Liquid chromatography (LC) with both UV spectral detection and high-resolution mass spectrometry (HR-MS) identified the known antibacterial compound thiotropocin or its closely related precursor tropodithietic acid in the bioactive fractions. Antibacterial activity correlated with the appearance of a brownish pigment and was only formed in marine broth under static growth conditions. A thick biofilm of multicellular star-shaped aggregated cells formed at the air-liquid interface under static growth conditions. Here, the bioactive compound was the base peak in the LC-UV chromatograms of the extracts where it constituted 15% of the total peak area. Aerated conditions results in 10-fold-higher cell yield, however, cultures were nonpigmented, did not produce antibacterial activity, and grew as single cells. Production of antibacterial compounds may be quorum regulated, and we identified the acylated homoserine lactone (3-hydroxy-decanoyl homoserine lactone) from cultures of
Roseobacter
27-4 using LC-HR-MS. The signal molecule was primarily detected in stagnant cultures.
Roseobacter
27-4 grew between 10 and 30°C but died rapidly at 37°C. Also, the antibacterial compounds was sensitive to heat and was inactivated at 37°C in less than 2 days and at 25°C in 8 days. Using
Roseobacter
27-4 as a probiotic culture will require that is be established in stagnant or adhered conditions and, due to the temperature sensitivity of the active compound, constant production must be ensured.
Ascending Migration of Endophytic Rhizobia, from Roots to Leaves, inside Rice Plants and Assessment of Benefits to Rice Growth Physiology
Authors: Chi Feng , Shen Shi-Hua , Cheng Hai-Ping , Jing Yu-Xiang , Yanni Youssef G. , Dazzo Frank B.
Published: November 2005

Rhizobia, the root-nodule endosymbionts of leguminous plants, also form natural endophytic associations with roots of important cereal plants. Despite its widespread occurrence, much remains unknown about colonization of cereals by rhizobia. We examined the infection, dissemination, and colonization of healthy rice plant tissues by four species of
gfp-
tagged rhizobia and their influence on the growth physiology of rice. The results indicated a dynamic infection process beginning with surface colonization of the rhizoplane (especially at lateral root emergence), followed by endophytic colonization within roots, and then ascending endophytic migration into the stem base, leaf sheath, and leaves where they developed high populations. In situ CMEIAS image analysis indicated local endophytic population densities reaching as high as 9 × 10
10
rhizobia per cm
3
of infected host tissues, whereas plating experiments indicated rapid, transient or persistent growth depending on the rhizobial strain and rice tissue examined. Rice plants inoculated with certain test strains of
gfp
-tagged rhizobia produced significantly higher root and shoot biomass; increased their photosynthetic rate, stomatal conductance, transpiration velocity, water utilization efficiency, and flag leaf area (considered to possess the highest photosynthetic activity); and accumulated higher levels of indoleacetic acid and gibberellin growth-regulating phytohormones. Considered collectively, the results indicate that this endophytic plant-bacterium association is far more inclusive, invasive, and dynamic than previously thought, including dissemination in both below-ground and above-ground tissues and enhancement of growth physiology by several rhizobial species, therefore heightening its interest and potential value as a biofertilizer strategy for sustainable agriculture to produce the world's most important cereal crops.
Copper-Adapted
Suillus luteus
, a Symbiotic Solution for Pines Colonizing Cu Mine Spoils
Authors: Adriaensen K. , Vrålstad T. , Noben J.-P. , Vangronsveld J. , Colpaert J. V.
Published: November 2005

Natural populations thriving in heavy-metal-contaminated ecosystems are often subjected to selective pressures for increased resistance to toxic metals. In the present study we describe a population of the ectomycorrhizal fungus
Suillus luteus
that colonized a toxic Cu mine spoil in Norway. We hypothesized that this population had developed adaptive Cu tolerance and was able to protect pine trees against Cu toxicity. We also tested for the existence of cotolerance to Cu and Zn in
S. luteus
. Isolates from Cu-polluted, Zn-polluted, and nonpolluted sites were grown in vitro on Cu- or Zn-supplemented medium. The Cu mine isolates exhibited high Cu tolerance, whereas the Zn-tolerant isolates were shown to be Cu sensitive, and vice versa. This indicates the evolution of metal-specific tolerance mechanisms is strongly triggered by the pollution in the local environment. Cotolerance does not occur in the
S. luteus
isolates studied. In a dose-response experiment, the Cu sensitivity of nonmycorrhizal
Pinus sylvestris
seedlings was compared to the sensitivity of mycorrhizal seedlings colonized either by a Cu-sensitive or Cu-tolerant
S. luteus
isolate. In nonmycorrhizal plants and plants colonized by the Cu-sensitive isolate, root growth and nutrient uptake were strongly inhibited under Cu stress conditions. In contrast, plants colonized by the Cu-tolerant isolate were hardly affected. The Cu-adapted
S. luteus
isolate provided excellent insurance against Cu toxicity in pine seedlings exposed to elevated Cu levels. Such a metal-adapted
Suillus
-
Pinus
combination might be suitable for large-scale land reclamation at phytotoxic metalliferous and industrial sites.
Detection of Carp Interstitial Nephritis and Gill Necrosis Virus in Fish Droppings
Authors: Dishon Arnon , Perelberg Ayana , Bishara-Shieban Janette , Ilouze Maya , Davidovich Maya , Werker Shlomit , Kotler Moshe
Published: November 2005

Carp interstitial nephritis and gill necrosis virus (CNGV) is an unclassified large DNA virus that morphologically resembles members of the
Herpesviridae
but contains a large (ca. ∼280-kbp) linear double-stranded DNA. This virus has also been named koi herpesvirus, koi herpes-like virus, and cyprinid herpesvirus 3. CNGV is the cause of a lethal disease that afflicts common carp and koi. By using immunohistochemistry, molecular analysis, and electron microscopy we previously demonstrated that this virus is present mainly in the intestine and kidney of infected fish. Based on these observations, we postulated that viruses and/or viral components may appear in droppings of infected carp. Here we report that (i) by using PCR we demonstrated that fish droppings contain viral DNA, (ii) fish droppings contain viral antigens which are useful for CNGV diagnosis, and (iii) fish droppings contain active virus which can infect cultured common carp brain cells and induce the disease in naïve fish following inoculation. Thus, our findings show that CNGV can be identified by using droppings without taking biopsies or killing fish and that infectious CNGV is present in the stools of sick fish. The possibility that fish droppings preserve viable CNGV during the nonpermissive seasons is discussed.
Crucial Role of Extracellular Polysaccharides in Desiccation and Freezing Tolerance in the Terrestrial Cyanobacterium
Nostoc commune
Authors: Tamaru Yoshiyuki , Takani Yayoi , Yoshida Takayuki , Sakamoto Toshio
Published: November 2005

The cyanobacterium
Nostoc commune
is adapted to the terrestrial environment and has a cosmopolitan distribution. In this study, the role of extracellular polysaccharides (EPS) in the desiccation tolerance of photosynthesis in
N. commune
was examined. Although photosynthetic O
2
evolution was not detected in desiccated colonies, the ability of the cells to evolve O
2
rapidly recovered after rehydration. The air-dried colonies contained approximately 10% (wt/wt) water, and field-isolated, natural colonies with EPS were highly water absorbent and were rapidly hydrated by atmospheric moisture. The cells embedded in EPS in
Nostoc
colonies were highly desiccation tolerant, and O
2
evolution was not damaged by air drying. Although
N. commune
was determined to be a mesophilic cyanobacterium, the cells with EPS were heat tolerant in a desiccated state. EPS could be removed from cells by homogenizing colonies with a blender and filtering with coarse filter paper. This treatment to remove EPS did not damage
Nostoc
cells or their ability to evolve O
2
, but O
2
evolution was significantly damaged by desiccation treatment of the EPS-depleted cells. Similar to the EPS-depleted cells, the laboratory culture strain KU002 had only small amount of EPS and was highly sensitive to desiccation. In the EPS-depleted cells, O
2
evolution was also sensitive to freeze-thaw treatment. These results strongly suggest that EPS of
N. commune
is crucial for the stress tolerance of photosynthesis during desiccation and during freezing and thawing.
Antagonists of Hsp16.3, a Low-Molecular-Weight Mycobacterial Chaperone and Virulence Factor, Derived from Phage-Displayed Peptide Libraries
Authors: Saha Abhik , Sharma Archna , Dhar Amlanjyoti , Bhattacharyya Bhabatarak , Roy Siddhartha , Das Gupta Sujoy K.
Published: November 2005

The persistence ofMycobacterium tuberculosisis a major cause of concern in tuberculosis (TB) therapy. In the persistent mode the pathogen can resist drug therapy, allowing the possibility of reactivation of the disease. Several protein factors have been identified that contribute to persistence, one of them being the 16-kDa low-molecular-weight mycobacterial heat shock protein Hsp16.3, a homologue of the mammalian eye lens protein alpha-crystallin. It is believed that Hsp16.3 plays a key role in the persistence phase by protecting essential proteins from being irreversibly denatured. Because of the close association of Hsp16.3 with persistence, an attempt has been made to develop inhibitors against it. Random peptide libraries displayed on bacteriophage M13 were screened for Hsp16.3 binding. Two phage clones were identified that bind to the Hsp16.3 protein. The corresponding synthetic peptides, an 11-mer and a 16-mer, were able to bind Hsp16.3 and inhibit its chaperone activity in vitro in a dose-dependent manner. Little or no effect of these peptides was observed on alphaB-crystallin, a homologous protein that is a key component of human eye lens, indicating that there is an element of specificity in the observed inhibition. Two histidine residues appear to be common to the selected peptides. Nuclear magnetic resonance studies performed with the 11-mer peptide indicate that in this case these two histidines may be the crucial binding determinants. The peptide inhibitors of Hsp16.3 thus obtained could serve as the basis for developing potent drugs against persistent TB.
Capsular Polysaccharide Surrounds Smooth and Rugose Types ofSalmonella entericaserovar Typhimurium DT104
Authors: de Rezende C. Eriksson , Anriany Y. , Carr L. E. , Joseph S. W. , Weiner R. M.
Published: November 2005

The biofilms and rugose colony morphology ofSalmonella entericaserovar Typhimurium strains are usually associated with at least two different exopolymeric substances (EPS), curli and cellulose. In this study, another EPS, a capsular polysaccharide (CP) synthesized constitutively inS. entericaserovar Typhimurium strain DT104 at 25 and 37°C, has been recognized as a biofilm matrix component as well. Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis indicated that the CP is comprised principally of glucose and mannose, with galactose as a minor constituent. The composition differs from that of known colanic acid-containing CP that is isolated from cells ofEscherichia coliand other enteric bacteria grown at 37°C. The reactivity of carbohydrate-specific lectins conjugated to fluorescein isothiocyanate or gold particles with cellular carbohydrates demonstrated the cell surface localization of CP. Further, lectin binding also correlated with the FACE analysis of CP. Immunoelectron microscopy, using specific antibodies against CP, confirmed that CP surrounds the cells. Confocal microscopy of antibody-labeled cells showed greater biofilm formation at 25°C than at 37°C. Since the CP was shown to be produced at both 37°C and 25°C, it does not appear to be significantly involved in attachment during the early formation of the biofilm matrix. Although the attachment ofS. entericaserovar Typhimurium DT104 does not appear to be mediated by its CP, the capsule does contribute to the biofilm matrix and may have a role in other features of this organism, such as virulence, as has been shown previously for the capsules of other gram-negative and gram-positive bacteria.
Community Composition of a Hypersaline Endoevaporitic Microbial Mat
Authors: Sørensen Ketil Bernt , Canfield Donald E. , Teske Andreas P. , Oren Aharon
Published: November 2005

A hypersaline, endoevaporitic microbial community in Eilat, Israel, was studied by microscopy and by PCR amplification of genes for 16S rRNA from different layers. In terms of biomass, the oxygenic layers of the community were dominated by
Cyanobacteria
of the
Halothece
,
Spirulina
, and
Phormidium
types, but cell counts (based on 4′,6′-diamidino-2-phenylindole staining) and molecular surveys (clone libraries of PCR-amplified genes for 16S rRNA) showed that oxygenic phototrophs were outnumbered by the other constituents of the community, including chemotrophs and anoxygenic phototrophs. Bacterial clone libraries were dominated by phylotypes affiliated with the
Bacteroidetes
group and both photo- and chemotrophic groups of α-proteobacteria. Green filaments related to the
Chloroflexi
were less abundant than reported from hypersaline microbial mats growing at lower salinities and were only detected in the deepest part of the anoxygenic phototrophic zone. Also detected were nonphototrophic γ- and δ-proteobacteria,
Planctomycetes
, the TM6 group,
Firmicutes
, and
Spirochetes
. Several of the phylotypes showed a distinct vertical distribution in the crust, suggesting specific adaptations to the presence or absence of oxygen and light.
Archaea
were less abundant than
Bacteria
, their diversity was lower, and the community was less stratified. Detected archaeal groups included organisms affiliated with the
Methanosarcinales
, the
Halobacteriales
, and uncultured groups of
Euryarchaeota
.
Detection of AirborneStachybotrys chartarumMacrocyclic Trichothecene Mycotoxins in the Indoor Environment
Authors: Brasel T. L. , Martin J. M. , Carriker C. G. , Wilson S. C. , Straus D. C.
Published: November 2005

The existence of airborne mycotoxins in mold-contaminated buildings has long been hypothesized to be a potential occupant health risk. However, little work has been done to demonstrate the presence of these compounds in such environments. The presence of airborne macrocyclic trichothecene mycotoxins in indoor environments with knownStachybotrys chartarumcontamination was therefore investigated. In seven buildings, air was collected using a high-volume liquid impaction bioaerosol sampler (SpinCon PAS 450-10) under static or disturbed conditions. An additional building was sampled using an Andersen GPS-1 PUF sampler modified to separate and collect particulates smaller than conidia. Four control buildings (i.e., no detectableS. chartarumgrowth or history of water damage) and outdoor air were also tested. Samples were analyzed using a macrocyclic trichothecene-specific enzyme-linked immunosorbent assay (ELISA). ELISA specificity was tested using phosphate-buffered saline extracts of the fungal generaAspergillus,Chaetomium,Cladosporium,Fusarium,Memnoniella,Penicillium,Rhizopus, andTrichoderma, fiveStachybotrysstrains, and the indoor air allergens Can f 1, Der p 1, and Fel d 1. For test buildings, the results showed that detectable toxin concentrations increased with the sampling time and short periods of air disturbance. Trichothecene values ranged from <10 to >1,300 pg/m3of sampled air. The control environments demonstrated statistically significantly (P< 0.001) lower levels of airborne trichothecenes. ELISA specificity experiments demonstrated a high specificity for the trichothecene-producing strain ofS. chartarum. Our data indicate that airborne macrocyclic trichothecenes can exist inStachybotrys-contaminated buildings, and this should be taken into consideration in future indoor air quality investigations.
Distribution and Diversity of Natural Product Genes in Marine and Freshwater Cyanobacterial Cultures and Genomes
Authors: Ehrenreich Ian M. , Waterbury John B. , Webb Eric A.
Published: November 2005

Natural products are a functionally diverse class of biochemically synthesized compounds, which include antibiotics, toxins, and siderophores. In this paper, we describe both the detection of natural product activities and the sequence identification of gene fragments from two molecular systems that have previously been implicated in natural product production, i.e., nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), in diverse marine and freshwater cyanobacterial cultures. Using degenerate PCR and the sequencing of cloned products, we show that NRPSs and PKSs are common among the cyanobacteria tested. Our molecular data, when combined with genomic searches of finished and progressing cyanobacterial genomes, demonstrate that not all cyanobacteria contain NRPS and PKS genes and that the filamentous and heterocystous cyanobacteria are the richest sources of these genes and the most likely sources of novel natural products within the phylum. In addition to validating the use of degenerate primers for the identification of PKS and NRPS genes in cyanobacteria, this study also defines numerous gene fragments that will be useful as probes for future studies of the synthesis of natural products in cyanobacteria. Phylogenetic analyses of the cyanobacterial NRPS and PKS fragments sequenced in this study, as well as those from the cyanobacterial genome projects, demonstrate that there is remarkable diversity and likely novelty of these genes within the cyanobacteria. These results underscore the potential variety of novel products being produced by these ubiquitous organisms.
Identification and Molecular Characterization of the Chromosomal Exopolysaccharide Biosynthesis Gene Cluster from
Lactococcus lactis
subsp.
cremoris
SMQ-461
Authors: Dabour N. , LaPointe G.
Published: November 2005

The exopolysaccharide (EPS) capsule-forming strain SMQ-461 of
Lactococcus lactis
subsp.
cremoris
, isolated from raw milk, produces EPS with an apparent molecular mass of >1.6 × 10
6
Da. The EPS biosynthetic genes are located on the chromosome in a 13.2-kb region consisting of 15 open reading frames. This region is flanked by three IS
1077
-related
tnp
genes (
L. lactis
) at the 5′ end and
orfY
, along with an IS
981
-related
tnp
gene, at the 3′ end. The
eps
genes are organized in specific regions involved in regulation, chain length determination, biosynthesis of the repeat unit, polymerization, and export. Three (
epsGIK
) of the six predicted glycosyltransferase gene products showed low amino acid similarity with known glycosyltransferases. The structure of the repeat unit could thus be different from those known to date for
Lactococcus
. Reverse transcription-PCR analysis revealed that the
eps
locus is transcribed as a single mRNA. The function of the
eps
gene cluster was confirmed by disrupting the priming glycosyltransferase gene (
epsD
) in
Lactococcus cremoris
SMQ-461, generating non-EPS-producing reversible mutants. This is the first report of a chromosomal location for EPS genetic elements in
Lactococcus cremoris
, with novel glycosyltransferases not encountered before in lactic acid bacteria.
Real-Time PCR Quantification of Human Adenoviruses in Urban Rivers Indicates Genome Prevalence but Low Infectivity
Authors: Choi Samuel , Jiang Sunny C.
Published: November 2005

Real-time PCR was applied to quantify the abundance of human adenoviruses in two southern California urban rivers, the San Gabriel and Los Angeles. A total of 114 river samples from five different locations were collected over a 1-year period and analyzed for human adenoviruses, along with fecal indicator bacteria and coliphages. Adenoviruses were detected by real-time PCR in ∼16% of the samples, with concentrations ranging from 10
2
to 10
4
genomes per liter. However, a plaque assay using two human tissue culture cell lines, HEK-293A and A549, yielded negative results, suggesting that adenoviruses detected by real-time PCR are likely noninfectious. Enterovirus genome was detected in ∼7% of the samples by reverse transcription-PCR. Analysis by Spearman's rho rank order correlation showed significant correlations between fecal indicator bacteria and indicator virus (total coliform, fecal coliform, enterococcus, and coliphage values). However, no significant correlations were found between human adenoviruses quantified by real-time PCR and culturable coliphages or fecal indicator bacteria. Kruskal-Wallis chi-square analysis showed significant seasonal variability of all fecal indicator bacteria and coliphages, while no significant variability was observed for adenoviruses or enteroviruses. This study presents the first quantitative measurement of human adenovirus genomes in urban rivers and their statistical relationship to fecal indicator bacteria and coliphages. The uncoupling between high-number genome copies of adenoviruses detected by real-time PCR and the absence of infectivity detected by tissue culture suggests that genome-based detection methods are inadequate for direct assessment of human health risk.
Molecular Characterization of the Nonphotosynthetic Partner Bacterium in the Consortium “
Chlorochromatium aggregatum
”
Authors: Kanzler Birgit E. M. , Pfannes Kristina R. , Vogl Kajetan , Overmann Jörg
Published: November 2005

Phototrophic consortia represent valuable model systems for the study of signal transduction and coevolution between different bacteria. The phototrophic consortium “
Chlorochromatium aggregatum
” consists of a colorless central rod-shaped bacterium surrounded by about 20 green-pigmented epibionts. Although the epibiont was identified as a member of the green sulfur bacteria, and recently isolated and characterized in pure culture, the central colorless bacterium has been identified as a member of the β-
Proteobacteria
but so far could not be characterized further. In the present study, “
C. aggregatum
” was enriched chemotactically, and the 16S rRNA gene sequence of the central bacterium was elucidated. Based on the sequence information, fluorescence in situ hybridization probes targeting four different regions of the 16S rRNA were designed and shown to hybridize exclusively to cells of the central bacterium. Phylogenetic analyses of the 1,437-bp-long sequence revealed that the central bacterium of “
C. aggregatum
” represents a so far isolated phylogenetic lineage related to
Rhodoferax
spp.,
Polaromonas vacuolata
, and
Variovorax paradoxus
within the family
Comamonadaceae
. The majority of relatives of this lineage are not yet cultured and were found in low-temperature aquatic environments or aquatic environments containing xenobiotica or hydrocarbons. In CsCl-bisbenzimidazole equilibrium density gradients, genomic DNA of the central bacterium of “
Chlorochromatium aggregatum
” formed a distinct band which could be detected by quantitative PCR using specific primers. Using this method, the G+C content of the central bacterium was determined to be 55.6 mol%.
Rhodopseudomonas palustris
Regulons Detected by Cross-Species Analysis of Alphaproteobacterial Genomes
Authors: Conlan Sean , Lawrence Charles , McCue Lee Ann
Published: November 2005

Rhodopseudomonas palustris
, an α-proteobacterium, carries out three of the chemical reactions that support life on this planet: the conversion of sunlight to chemical-potential energy; the absorption of carbon dioxide, which it converts to cellular material; and the fixation of atmospheric nitrogen into ammonia. Insight into the transcription-regulatory network that coordinates these processes is fundamental to understanding the biology of this versatile bacterium. With this goal in mind, we predicted regulatory signals genomewide, using a two-step phylogenetic-footprinting and clustering process that we had developed previously. In the first step, 4,963 putative transcription factor binding sites, upstream of 2,044 genes and operons, were identified using cross-species Gibbs sampling. Bayesian motif clustering was then employed to group the cross-species motifs into regulons. We have identified 101 putative regulons in
R. palustris
, including 8 that are of particular interest: a photosynthetic regulon, a flagellar regulon, an organic hydroperoxide resistance regulon, the LexA regulon, and four regulons related to nitrogen metabolism (FixK
2
, NnrR, NtrC, and σ
54
). In some cases, clustering allowed us to assign functions to proteins that previously had been annotated with only putative functions; we have identified RPA0828 as the organic hydroperoxide resistance regulator and RPA1026 as a cell cycle methylase. In addition to predicting regulons, we identified a novel inverted repeat that likely forms a highly conserved stem-loop and that occurs downstream of over 100 genes.
Global Transcriptional Profiling of
Shewanella oneidensis
MR-1 during Cr(VI) and U(VI) Reduction
Authors: Bencheikh-Latmani Rizlan , Williams Sarah Middleton , Haucke Lisa , Criddle Craig S. , Wu Liyou , Zhou Jizhong , Tebo Bradley M.
Published: November 2005

Whole-genome DNA microarrays were used to examine the gene expression profile of
Shewanella oneidensis
MR-1 during U(VI) and Cr(VI) reduction. The same control, cells pregrown with nitrate and incubated with no electron acceptor, was used for the two time points considered and for both metals. U(VI)-reducing conditions resulted in the upregulation (≥3-fold) of 121 genes, while 83 genes were upregulated under Cr(VI)-reducing conditions. A large fraction of the genes upregulated [34% for U(VI) and 29% for Cr(VI)] encode hypothetical proteins of unknown function. Genes encoding proteins known to reduce alternative electron acceptors [fumarate, dimethyl sulfoxide, Mn(IV), or soluble Fe(III)] were upregulated under both U(VI)- and Cr(VI)-reducing conditions. The involvement of these upregulated genes in the reduction of U(VI) and Cr(VI) was tested using mutants lacking one or several of the gene products. Mutant testing confirmed the involvement of several genes in the reduction of both metals:
mtrA
,
mtrB
,
mtrC
, and
menC
, all of which are involved in Fe(III) citrate reduction by MR-1. Genes encoding efflux pumps were upregulated under Cr(VI)- but not under U(VI)-reducing conditions. Genes encoding proteins associated with general (e.g.,
groL
and
dnaJ
) and membrane (e.g.,
pspBC
) stress were also upregulated, particularly under U(VI)-reducing conditions, pointing to membrane damage by the solid-phase reduced U(IV) and Cr(III) and/or the direct effect of the oxidized forms of the metals. This study sheds light on the multifaceted response of MR-1 to U(VI) and Cr(VI) under anaerobic conditions and suggests that the same electron transport pathway can be used for more than one electron acceptor.
Colonization of Mucin by Human Intestinal Bacteria and Establishment of Biofilm Communities in a Two-Stage Continuous Culture System
Authors: Macfarlane Sandra , Woodmansey Emma J. , Macfarlane George T.
Published: November 2005

The human large intestine is covered with a protective mucus coating, which is heavily colonized by complex bacterial populations that are distinct from those in the gut lumen. Little is known of the composition and metabolic activities of these biofilms, although they are likely to play an important role in mucus breakdown. The aims of this study were to determine how intestinal bacteria colonize mucus and to study physiologic and enzymatic factors involved in the destruction of this glycoprotein. Colonization of mucin gels by fecal bacteria was studied in vitro, using a two-stage continuous culture system, simulating conditions of nutrient availability and limitation characteristic of the proximal (vessel 1) and distal (vessel 2) colon. The establishment of bacterial communities in mucin gels was investigated by selective culture methods, scanning electron microscopy, and confocal laser scanning microscopy, in association with fluorescently labeled 16S rRNA oligonucleotide probes. Gel samples were also taken for analysis of mucin-degrading enzymes and measurements of residual mucin sugars. Mucin gels were rapidly colonized by heterogeneous bacterial populations, especially members of the
Bacteroides fragilis
group, enterobacteria, and clostridia. Intestinal bacterial populations growing on mucin surfaces were shown to be phylogenetically and metabolically distinct from their planktonic counterparts.
Distribution, Localization, and Phylogeny of Abundant Populations of
Crenarchaeota
in Anaerobic Granular Sludge
Authors: Collins Gavin , O'Connor Leanne , Mahony Thérèse , Gieseke Armin , de Beer Dirk , O'Flaherty Vincent
Published: November 2005

Eight anaerobic granular sludges were surveyed for
Crenarchaeota
using rRNA gene cloning. Microbial arrangement and substrate uptake patterns were elucidated by fluorescent in situ hybridization and beta imaging. Group 1.3
Crenarchaeota
represented up to 50% of
Archaea
and 25% of the total microbiota in five sludges.
Crenarchaeota
were localized in close association with methanogenic
Archaea.
Application of Measurements of Transepithelial Electrical Resistance of Intestinal Epithelial Cell Monolayers To Evaluate Probiotic Activity
Authors: Klingberg Trine Danø , Pedersen Maja Herold , Cencic Avrelija , Budde Birgitte Bjørn
Published: November 2005

Among five potentially probiotic lactobacilli investigated,
Lactobacillus plantarum
MF1298 and
Lactobacillus salivarius
DC5 showed the highest increase in the transepithelial electrical resistance (TER) of polarized monolayers of Caco-2 cells, and this increase was shown to be dose dependent. Furthermore, preincubation with MF1298 attenuated a decrease in TER induced by
Listeria monocytogenes
.
Strain- and Genotype-Specific Differences in Virulence of
Paenibacillus larvae
subsp.
larvae
, a Bacterial Pathogen Causing American Foulbrood Disease in Honeybees
Authors: Genersch Elke , Ashiralieva Ainura , Fries Ingemar
Published: November 2005

Virulence variations of
Paenibacillus larvae
subsp.
larvae
, the causative agent of American foulbrood disease of honeybees, were investigated by analysis of 16 field isolates of this pathogen, belonging to three previously characterized genotypes, as well as the type strain (ATCC 9545) of
P. larvae
subsp.
larvae
, with exposure bioassays. We demonstrated that the strain-specific 50% lethal concentrations varied within an order of magnitude and that differences in amount of time for the pathogen to kill 100% of the infected hosts (LT
100
) correlated with genotype. One genotype killed rather quickly, with a mean LT
100
of 7.8 ± 1.7 days postinfection, while the other genotypes acted more slowly, with mean LT
100
s of 11.2 ± 0.8 and 11.6 ± 0.6 days postinfection.
Evidence for Horizontal Transfer of 1-Aminocyclopropane-1-Carboxylate Deaminase Genes
Authors: Hontzeas N. , Richardson A. O. , Belimov A. , Safronova V. , Abu-Omar M. M. , Glick B. R.
Published: November 2005

PCR was used to rapidly identify and isolate 1-aminocyclopropane-1-carboxylate (ACC) deaminase genes from bacteria. The Shimodaira-Hasegawa test was used to assess whether phylogenetically anomalous gene placements suggestive of horizontal gene transfer (HGT) were significantly favored over vertical transmission. The best maximum likelihood (ML) ACC deaminase tree was significantly more likely than four alternative ML trees, suggesting HGT.
Putative Exposed Aromatic and Hydroxyl Residues on the Surface of the N-Terminal Domains of Chi1 from
Aeromonas caviae
CB101 Are Essential for Chitin Binding and Hydrolysis
Authors: Li Qiang , Wang Fengping , Zhou Ying , Xiao Xiang
Published: November 2005

Chitinase Chi1 of
Aeromonas caviae
CB101 possesses chitin binding sites at both its N and C termini. Four putative exposed residues aligned in a line on the surface of the N-terminal domains of Chi1 were found to contribute to the enzyme-chitin binding and hydrolysis via site-directed mutagenesis. Also, it was found that Chi1 requires the cooperation of the N- and C-terminal domains to bind fully with crystalline and colloidal chitin.
Occurrence of
Listeria
spp. in Effluents of French Urban Wastewater Treatment Plants
Authors: Paillard Delphine , Dubois Véronique , Thiebaut Rodolphe , Nathier Fany , Hoogland Emilie , Caumette Pierre , Quentin Claudine
Published: November 2005

Listeria
spp. were found in most treated waters (84.4%) and raw sludge (89.2%) of six French urban wastewater treatment plants and one composting facility, examined monthly over a 1-year period. Most strains belonged to
Listeria monocytogenes
, serotypes 4b/4e being predominant. Sludge composting and liming reduced or prevented
Listeria
contamination.
Rhizobium leguminosarum
Biovar viciae Symbiotic Hydrogenase Activity and Processing Are Limited by the Level of Nickel in Agricultural Soils
Authors: Ureta Ana-Claudia , Imperial Juan , Ruiz-Argüeso Tomás , Palacios Jose M.
Published: November 2005

Analysis of levels of hydrogenase processing and activity in
Rhizobium leguminosarum
biovar viciae bacteroids from pea (
Pisum sativum
) plants showed that the oxidation of nitrogenase-evolved hydrogen is limited by the availability of nickel in agricultural soils. This limitation was overcome by using an inoculant strain engineered for higher hydrogenase expression.
When Coupled to Natural Transformation inAcinetobactersp. Strain ADP1, PCR Mutagenesis Is Made Less Random by Mismatch Repair
Authors: Buchan Alison , Ornston L. Nicholas
Published: November 2005

Random PCR mutagenesis is a powerful tool for structure-function analysis of targeted proteins, especially when coupled with DNA integration through natural transformation followed by selection for loss of function. The technique has been applied successfully to structure-function analysis of transcriptional regulators, enzymes, and transporters inAcinetobactersp. strain ADP1. However, the mismatch repair system prevents the full spectrum of nucleotide substitutions that may be selected at the level of protein function from being recovered. This barrier may be overcome by introducing PCR-mutagenized genes into strains in which the corresponding genes have been deleted.
Identification of Pilus-Like Structures and Genes in
Microcystis aeruginosa
PCC7806
Authors: Nakasugi Kenlee , Neilan Brett A.
Published: November 2005

Four putative type IV pilus genes from the toxic, naturally transformable
Microcystis aeruginosa
PCC7806 were identified. Three of these genes were clustered in an arrangement which is identical to that from other cyanobacterial genomes. Type IV pilus-like appendages were also observed by electron microscopy.
Parental consent for newborn screening in southern Taiwan
Authors: Huang M-C , Lee C-K , Lin S-J , Lu I-C
Published: November 2005

Objects:With the advent of genetic technologies, many genetic/metabolic disorders can be detected asymptomatically but might be untreatable, and the benefits and risks of screening for them are not fully known. The purpose of this study is to explore current practice with regard to the parental consent process in newborn screening (NBS).

Design:Staff in 23 obstetric clinics/hospitals that conduct NBS in one city of southern Taiwan were interviewed. Using content analysis, 15 interview transcripts, eight completed questionnaires, and other relevant documents from the 23 clinics/hospitals were analysed to reveal the framework of the parental consent process in NBS in southern Taiwan.

Main measures:Three categories—informed consent, informed dissent, and no informed/consent—were developed to analyse the parental consent process in NBS.

Results:The parental consent procedures in NBS and the quality of the information provided before obtaining consent vary widely. Because the traditional NBS was incorporated into routine paediatric practices in most clinics/hospitals, the most frequently encountered consent model is “informed dissent” (60.9%) and “no informed/consent” (30.4%); while an “informed consent” model (45.5%) is the frequent model for screening rare metabolic/genetic disorders.

Conclusions:Specific guidelines to regulate the parental consent process for NBS are essential. Further studies should investigate parental responses to NBS, taking these as the basis on which to establish an informed consent model in Taiwan.
MAP kinase and calcium signaling mediate fluid flow-induced human mesenchymal stem cell proliferation
Authors: Riddle Ryan C. , Taylor Amanda F. , Genetos Damian C. , Donahue Henry J.
Published: November 2005

Mechanical signals are important regulators of skeletal homeostasis, and strain-induced oscillatory fluid flow is a potent mechanical stimulus. Although the mechanisms by which osteoblasts and osteocytes respond to fluid flow are being elucidated, little is known about the mechanisms by which bone marrow-derived mesenchymal stem cells respond to such stimuli. Here we show that the intracellular signaling cascades activated in human mesenchymal stem cells by fluid flow are similar to those activated in osteoblastic cells. Oscillatory fluid flow inducing shear stresses of 5, 10, and 20 dyn/cm2triggered rapid, flow rate-dependent increases in intracellular calcium that pharmacological studies suggest are inositol trisphosphate mediated. The application of fluid flow also induced the phosphorylation of extracellular signal-regulated kinase-1 and -2 as well as the activation of the calcium-sensitive protein phosphatase calcineurin in mesenchymal stem cells. Activation of these signaling pathways combined to induce a robust increase in cellular proliferation. These data suggest that mechanically induced fluid flow regulates not only osteoblastic behavior but also that of mesenchymal precursors, implying that the observed osteogenic response to mechanical loading may be mediated by alterations in the cellular behavior of multiple members of the osteoblast lineage, perhaps by a common signaling pathway.
Hindbrain administration of NMDA receptor antagonist AP-5 increases food intake in the rat
Authors: Hung Chun-Yi , Covasa M. , Ritter R. C. , Burns G. A.
Published: November 2005

Hindbrain administration of MK-801, a noncompetitive N-methyl-d-aspartate (NMDA) channel blocker, increases meal size, suggesting NMDA receptors in this location participate in control of food intake. However, dizocilpine (MK-801) reportedly antagonizes some non-NMDA ion channels. Therefore, to further assess hindbrain NMDA receptor participation in food intake control, we measured deprivation-induced intakes of 15% sucrose solution or rat chow after intraperitoneal injection of either saline vehicle or d(-)-2-amino-5-phosphonopentanoic acid (AP5), a competitive NMDA receptor antagonist, to the fourth ventricular, or nucleus of the solitary tract (NTS). Intraperitoneal injection of AP5 (0.05, 0.1, 1.0, 3.0, and 5.0 mg/kg) did not alter 30-min sucrose intake at any dose (10.7 ± 0.4 ml, saline control) (11.0 ± 0.8, 11.2 ± 1.0, 11.2 ± 1.0, 13.1 ± 2.2, and 11.0 ± 1.9 ml, AP5 doses, respectively). Fourth ventricular administration of both 0.2 μg (16.7 ± 0.6 ml) and 0.4 μg (14.9 ± 0.5 ml) but not 0.1 and 0.6 μg of AP5 significantly increased 60-min sucrose intake compared with saline (11.2 ± 0.4 ml). Twenty-four hour chow intake also was increased compared with saline (AP5: 31.5 ± 0.1 g vs. saline: 27.1 ± 0.6 g). Furthermore, rats did not increase intake of 0.2% saccharin after fourth ventricular AP5 administration (AP5: 9.8 ± 0.7ml, vs. saline: 10.5 ± 0.5ml). Finally, NTS AP5 (20 ng/30 nl) significantly increased 30- (AP5: 17.2 ± 0.7 ml vs. saline: 14.6 ± 1.7 ml), and 60-min (AP5: 19.4 ± 0.6 ml vs. saline: 15.5 ± 1.4 ml) sucrose intake, as well as 24-h chow intake (AP5: 31.6 ± 0.3 g vs. saline: 26.1 ± 1.2 g). These results support the hypothesis that hindbrain NMDA receptors participate in control of food intake and suggest that this participation also may contribute to control of body weight over a 24-h period.