bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Four Different Culture Media for Isolation and Growth of Type II and Type I/III
Mycobacterium avium
subsp.
paratuberculosis
Strains Isolated from Cattle and Goats
Authors: de Juan Lucía , Álvarez Julio , Romero Beatriz , Bezos Javier , Castellanos Elena , Aranaz Alicia , Mateos Ana , Domínguez Lucas
Published: September 2006

Culture is considered the definitive technique for Johne's disease diagnosis, and it is essential for later applications of certain molecular typing techniques. In this study, we have tested four solid media (Herrold's egg yolk medium [HEYM] with sodium pyruvate and mycobactin [HEYMm-SP], HEYM with mycobactin and without sodium pyruvate [HEYMm], Middlebrook 7H11 with mycobactin [Mm], and Löwenstein-Jensen with mycobactin [LJm]) for isolation of
Mycobacterium avium
subsp.
paratuberculosis
strains in 319 tissue samples from cattle herds and goat flocks. We have shown that each of the two main groups of
M. avium
subsp.
paratuberculosis
(type II and type I/III) has different requirements for growth in the culture media studied. The recommended solid media for isolation of type I/III strains are LJm and Mm, since the combination of both media allowed the recovery of all these strains. The most widespread culture medium, HEYM, is not suitable for the isolation of this group of
M. avium
subsp.
paratuberculosis
strains. Regarding the type II strains, HEYMm-SP was the medium where more strains were isolated, but the other three media are also needed in order to recover all type II strains. The incubation period is also related to the strain type. In conclusion, because the type of strain cannot be known in advance of culture, coupled with the fact that cattle and goats can be infected with both groups of strains, we recommend the use of the four solid media and the prolongation of the incubation period to more than 6 months to detect paratuberculous herds/flocks and to determine the true prevalence of the infection.
Complete Genome Sequence of the Marine, Chemolithoautotrophic, Ammonia-Oxidizing Bacterium
Nitrosococcus oceani
ATCC 19707
Authors: Klotz Martin G. , Arp Daniel J. , Chain Patrick S. G. , El-Sheikh Amal F. , Hauser Loren J. , Hommes Norman G. , Larimer Frank W. , Malfatti Stephanie A. , Norton Jeanette M. , Poret-Peterson Amisha T. , Vergez Lisa M. , Ward Bess B.
Published: September 2006

The gammaproteobacterium
Nitrosococcus oceani
(ATCC 19707) is a gram-negative obligate chemolithoautotroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing and annotation of the genome revealed a single circular chromosome (3,481,691 bp; G+C content of 50.4%) and a plasmid (40,420 bp) that contain 3,052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. Contrary to betaproteobacterial nitrifier genomes, the
N. oceani
genome contained two complete
rrn
operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxylamine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor, were identified. The
N. oceani
genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle, and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The
N. oceani
genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophosphate appear to be integrated in this bacterium's energy metabolism, stress tolerance, and ability to assimilate carbon via gluconeogenesis. One set of genes for type I ribulose-1,5-bisphosphate carboxylase/oxygenase was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The
N. oceani
genome contains two copies each of the genes or operons necessary to assemble functional complexes I and IV as well as ATP synthase (one H
+
-dependent F
0
F
1
type, one Na
+
-dependent V type).
Use of DNA Quantification To Measure Growth and Autolysis of
Lactococcus
and
Propionibacterium
spp. in Mixed Populations
Authors: Treimo Janneke , Vegarud Gerd , Langsrud Thor , Rudi Knut
Published: September 2006

Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of
Lactococcus lactis
subsp.
lactis
and three different species of
Propionibacterium
. Results showed that
L. lactis
subsp.
lactis
INF L2 autolyzed first, followed by
Propionibacterium acidipropionici
ATCC 4965,
Propionibacterium freudenreichii
ISU P59, and then
Propionibacterium jensenii
INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of
L. lactis
subsp.
lactis
INF L2 and
P. freudenreichii
ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.
Distinct Bacterial Communities Exist beneath a High Arctic Polythermal Glacier
Authors: Bhatia Maya , Sharp Martin , Foght Julia
Published: September 2006

Bacterial communities reside in basal ice, sediment, and meltwater in the supra-, sub-, and proglacial environments of John Evans Glacier, Nunavut, Canada. We examined whether the subglacial bacterial community shares common members with the pro- and supraglacial communities, and by inference, whether it could be derived from communities in either of these environments (e.g., by ice overriding proglacial sediments or by in-wash of surface meltwaters). Terminal restriction fragment length polymorphism analysis of bacterial 16S rRNA genes amplified from these environments revealed that the subglacial water, basal ice, and sediment communities were distinct from those detected in supraglacial meltwater and proglacial sediments, with 60 of 142 unique terminal restriction fragments (T-RFs) detected exclusively in subglacial samples and only 8 T-RFs detected in all three environments. Supraglacial waters shared some T-RFs with subglacial water and ice, likely reflecting the seasonal flow of surface meltwater into the subglacial drainage system, whereas supraglacial and proglacial communities shared the fewest T-RFs. Thus, the subglacial community at John Evans Glacier appears to be predominantly autochthonous rather than allochthonous, and it may be adapted to subglacial conditions. Chemical analysis of water and melted ice also revealed differences between the supraglacial and proglacial environments, particularly regarding electrical conductivity and nitrate, sulfate, and dissolved organic carbon concentrations. Whereas the potential exists for common bacterial types to be broadly distributed throughout the glacial system, we have observed distinct bacterial communities in physically and chemically different glacial environments.
Effect of Natural Sunlight on Bacterial Activity and Differential Sensitivity of Natural Bacterioplankton Groups in Northwestern Mediterranean Coastal Waters
Authors: Alonso-Sáez Laura , Gasol Josep M. , Lefort Thomas , Hofer Julia , Sommaruga Ruben
Published: September 2006

We studied the effects of natural sunlight on heterotrophic marine bacterioplankton in short-term experiments. We used a single-cell level approach involving flow cytometry combined with physiological probes and microautoradiography to determine sunlight effects on the activity and integrity of the cells. After 4 h of sunlight exposure, most bacterial cells maintained membrane integrity and viability as assessed by the simultaneous staining with propidium iodide and SYBR green I. In contrast, a significant inhibition of heterotrophic bacterial activity was detected, measured by 5-cyano-2,3 ditolyl tetrazolium chloride reduction and leucine incorporation. We applied microautoradiography combined with catalyzed reporter deposition-fluorescence in situ hybridization to test the sensitivity of the different bacterial groups naturally occurring in the Northwestern Mediterranean to sunlight. Members of the
Gammaproteobacteria
and
Bacteroidetes
groups appeared to be highly resistant to solar radiation, with small changes in activity after exposure. On the contrary,
Alphaproteobacteria
bacteria were more sensitive to radiation as measured by the cell-specific incorporation of labeled amino acids, leucine, and ATP. Within
Alphaproteobacteria
, bacteria belonging to the
Roseobacter
group showed higher resistance than members of the SAR11 cluster. The activity of
Roseobacter
was stimulated by exposure to photosynthetic available radiation compared to the dark treatment. Our results suggest that UV radiation can significantly affect the in situ single-cell activity of bacterioplankton and that naturally dominating phylogenetic bacterial groups have different sensitivity to natural levels of incident solar radiation.
Attachment of and Biofilm Formation by
Enterobacter sakazakii
on Stainless Steel and Enteral Feeding Tubes
Authors: Kim Hoikyung , Ryu Jee-Hoon , Beuchat Larry R.
Published: September 2006

Enterobacter sakazakii
has been reported to form biofilms, but environmental conditions affecting attachment to and biofilm formation on abiotic surfaces have not been described. We did a study to determine the effects of temperature and nutrient availability on attachment and biofilm formation by
E. sakazakii
on stainless steel and enteral feeding tubes. Five strains grown to stationary phase in tryptic soy broth (TSB), infant formula broth (IFB), or lettuce juice broth (LJB) at 12 and 25°C were examined for the extent to which they attach to these materials. Higher populations attached at 25°C than at 12°C. Stainless steel coupons and enteral feeding tubes were immersed for 24 h at 4°C in phosphate-buffered saline suspensions (7 log CFU/ml) to facilitate the attachment of 5.33 to 5.51 and 5.03 to 5.12 log CFU/cm
2
, respectively, before they were immersed in TSB, IFB, or LJB, followed by incubation at 12 or 25°C for up to 10 days. Biofilms were not produced at 12°C. The number of cells of test strains increased by 1.42 to 1.67 log CFU/cm
2
and 1.16 to 1.31 log CFU/cm
2
in biofilms formed on stainless steel and feeding tubes, respectively, immersed in IFB at 25°C; biofilms were not formed on TSB and LJB at 25°C, indicating that nutrient availability plays a major role in processes leading to biofilm formation on the surfaces of these inert materials. These observations emphasize the importance of temperature control in reconstituted infant formula preparation and storage areas in preventing attachment and biofilm formation by
E. sakazakii
.
Cloning of a Heavy-Metal-Binding Protein Derived from Activated-Sludge Microorganisms
Authors: Sano Daisuke , Myojo Ken , Omura Tatsuo
Published: September 2006

A gene of the heavy-metal-binding protein (HMBP) was newly isolated from a genetic DNA library of activated-sludge microorganisms. HMBP was produced by transformed
Escherichia coli
, and the copper-binding ability of HMBP was confirmed. HMBP derived from activated sludge could be available as heavy metal adsorbents in water and wastewater treatments.
Microbially Mediated Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5- Triazine by Extracellular Electron Shuttling Compounds
Authors: Kwon Man Jae , Finneran Kevin T.
Published: September 2006

The potential for humic substances to stimulate the reduction of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was investigated. This study describes a novel approach for the remediation of RDX-contaminated environments using microbially mediated electron shuttling. Incubations without cells demonstrated that reduced AQDS transfers electrons directly to RDX, which was reduced without significant accumulation of the nitroso intermediates. Three times as much reduced AQDS (molar basis) was needed to completely reduce RDX. The rate and extent of RDX reduction differed greatly among electron shuttle/acceptor amendments for resting cell suspensions of
Geobacter metallireducens
and
G. sulfurreducens
with acetate as the sole electron donor. AQDS and purified humic substances stimulated the fastest rate of RDX reduction. The nitroso metabolites did not significantly accumulate in the presence of AQDS or humic substances. RDX reduction in the presence of poorly crystalline Fe(III) was relatively slow and metabolites transiently accumulated. However, adding humic substances or AQDS to Fe(III)-containing incubations increased the reduction rates. Cells of
G. metallireducens
alone reduced RDX; however, the rate of RDX reduction was slow relative to AQDS-amended incubations. These data suggest that extracellular electron shuttle-mediated RDX transformation is not organism specific but rather is catalyzed by multiple Fe(III)- and humic-reducing species. Electron shuttle-mediated RDX reduction may eventually become a rapid and effective cleanup strategy in both Fe(III)-rich and Fe(III)-poor environments.
Diversity of Telomere Palindromic Sequences and Replication Genes among
Streptomyces
Linear Plasmids
Authors: Zhang Ran , Yang Yong , Fang Ping , Jiang Chenglin , Xu Lihua , Zhu Yingmin , Shen Meijuan , Xia Haiyang , Zhao Jianfu , Chen Tongbin , Qin Zhongjun
Published: September 2006

Streptomyces
sp. linear plasmids and linear chromosomes usually contain
conserved terminal palindromic sequences bound by the conserved
telomeric proteins Tap and Tp, encoded by the
tap
and
tpg
genes, respectively, as well as plasmid loci required for
DNA replication in circular mode when the telomeres are deleted. These
consist of iterons and an adjacent
rep
gene. By using PCR, we
found that 8 of 17 newly detected linear plasmids in
Streptomyces
strains lack typical telomeric
tap
and
tpg
sequences. Instead, two novel telomeres in plasmids pRL1
and pRL2 from the eight strains and one conserved telomere in pFRL1
from the other strains were identified, while multiple short
palindromes were also found in the plasmids. The complete nucleotide
sequence of pRL2 revealed a gene encoding a protein containing two
domains, resembling Tap of
Streptomyces
and a helicase of
Thiobacillus
, and an adjacent gene encoding a protein similar
to Tpg of
Streptomyces
and a portion of the telomere terminal
protein pTP of adenoviruses. No typical iterons-
rep
loci were
found in the three plasmids. These results indicate an unexpected
diversity of telomere palindromic sequences and replication genes among
Streptomyces
linear
plasmids.
Halotolerant Cyanobacterium
Aphanothece halophytica
Contains a Betaine Transporter Active at Alkaline pH and High Salinity
Authors: Laloknam Surasak , Tanaka Kimihiro , Buaboocha Teerapong , Waditee Rungaroon , Incharoensakdi Aran , Hibino Takashi , Tanaka Yoshito , Takabe Teruhiro
Published: September 2006

Aphanothece halophytica
is a halotolerant alkaliphilic cyanobacterium which can grow in media of up to 3.0 M NaCl and pH 11. This cyanobacterium can synthesize betaine from glycine by three-step methylation using
S
-adenosylmethionine as a methyl donor. To unveil the mechanism of betaine uptake and efflux in this alkaliphile, we isolated and characterized a betaine transporter. A gene encoding a protein (BetT

A. halophytica

) that belongs to the betaine-choline-carnitine transporter (BCCT) family was isolated. Although the predicted isoelectric pH of a typical BCCT family transporter, OpuD of
Bacillus subtilis
, is basic, 9.54, that of BetT

A. halophytica

is acidic, 4.58. BetT

A. halophytica

specifically catalyzed the transport of betaine. Choline, γ-aminobutyric acid, betaine aldehyde, sarcosine, dimethylglycine, and amino acids such as proline did not compete for the uptake of betaine by BetT

A. halophytica

. Sodium markedly enhanced betaine uptake rates, whereas potassium and other cations showed no effect, suggesting that BetT

A. halophytica

is a Na
+
-betaine symporter. Betaine uptake activities of BetT

A. halophytica

were high at alkaline pH values, with the optimum pH around 9.0. Freshwater
Synechococcus
cells overexpressing BetT

A. halophytica

showed NaCl-activated betaine uptake activities with enhanced salt tolerance, allowing growth in seawater supplemented with betaine. Kinetic properties of betaine uptake in
Synechococcus
cells overexpressing BetT

A. halophytica

were similar to those in
A. halophytica
cells. These findings indicate that
A. halophytica
contains a Na
+
-betaine symporter that contributes to the salt stress tolerance at alkaline pH. BetT

A. halophytica

is the first identified transporter for compatible solutes in cyanobacteria.
Persistence of Nontuberculous Mycobacteria in a Drinking Water System after Addition of Filtration Treatment
Authors: Hilborn Elizabeth D. , Covert Terry C. , Yakrus Mitchell A. , Harris Stephanie I. , Donnelly Sandra F. , Rice Eugene W. , Toney Sean , Bailey Stephanie A. , Stelma Gerard N.
Published: September 2006

There is evidence that drinking water may be a source of infections with pathogenic nontuberculous mycobacteria (NTM) in humans. One method by which NTM are believed to enter drinking water distribution systems is by their intracellular colonization of protozoa. Our goal was to determine whether we could detect a reduction in the prevalence of NTM recovered from an unfiltered surface drinking water system after the addition of ozonation and filtration treatment and to characterize NTM isolates by using molecular methods. We sampled water from two initially unfiltered surface drinking water treatment plants over a 29-month period. One plant received the addition of filtration and ozonation after 6 months of sampling. Sample sites included those at treatment plant effluents, distributed water, and cold water taps (point-of-use [POU] sites) in public or commercial buildings located within each distribution system. NTM were recovered from 27% of the sites. POU sites yielded the majority of NTM, with >50% recovery despite the addition of ozonation and filtration. Closely related electrophoretic groups ofMycobacterium aviumwere found to persist at POU sites for up to 26 months. Water collected from POU cold water outlets was persistently colonized with NTM despite the addition of ozonation and filtration to a drinking water system. This suggests that cold water POU outlets need to be considered as a potential source of chronic human exposure to NTM.
Biotyping of Enterotoxigenic
Staphylococcus aureus
by Enterotoxin Gene Cluster (
egc
) Polymorphism and
spa
Typing Analyses
Authors: Blaiotta Giuseppe , Fusco Vincenzina , von Eiff Christof , Villani Francesco , Becker Karsten
Published: September 2006

Thirty-five
Staphylococcus aureus
strains, including 10 reference strains and 25 strains recovered from clinical specimens and food samples, were analyzed by PCR REA (restriction endonucleases analysis) of the
egc
operon and
spa
typing. Nineteen
spa
types and seven different
egc
operons, including four putative new
egc
variants, were revealed. In 13 strains, allelic variants of
sei
and/or
seg
were found. By an analysis of their nucleotide sequence identities, a new homogeneous cluster of a
sei
variant, called the
sei
variant, was detected in six strains. In addition, the prototype
sei
was shown to be more polymorphic than assumed so far. Seven strains possessed the recently described
seg
variant, also exhibiting several nucleotide exchanges.
spa
typing was more effective than REA
egc
grouping as a typing technique. Since, in some cases, the REA typing method was able to discriminate strains showing the same
spa
type, it must be considered for PCR approaches involved in diagnostic procedures and may be useful for epidemiological studies. Hence, the polyphasic approach used in this study can be reliably and advantageously applied for typing
egc
-positive
S. aureus
strains.
Global Molecular and Morphological Effects of 24-Hour Chromium(VI) Exposure on
Shewanella oneidensis
MR-1
Authors: Chourey Karuna , Thompson Melissa R. , Morrell-Falvey Jennifer , VerBerkmoes Nathan C. , Brown Steven D. , Shah Manesh , Zhou Jizhong , Doktycz Mitchel , Hettich Robert L. , Thompson Dorothea K.
Published: September 2006

The biological impact of 24-h (“chronic”) chromium(VI)
[Cr(VI) or chromate] exposure on
Shewanella oneidensis
MR-1
was assessed by analyzing cellular morphology as well as genome-wide
differential gene and protein expression profiles. Cells challenged
aerobically with an initial chromate concentration of 0.3 mM in complex
growth medium were compared to untreated control cells grown in the
absence of chromate. At the 24-h time point at which cells were
harvested for transcriptome and proteome analyses, no residual Cr(VI)
was detected in the culture supernatant, thus suggesting the complete
uptake and/or reduction of this metal by cells. In contrast to the
untreated control cells, Cr(VI)-exposed cells formed apparently
aseptate, nonmotile filaments that tended to aggregate. Transcriptome
profiling and mass spectrometry-based proteomic characterization
revealed that the principal molecular response to 24-h Cr(VI) exposure
was the induction of prophage-related genes and their encoded products
as well as a number of functionally undefined hypothetical genes that
were located within the integrated phage regions of the MR-1 genome. In
addition, genes with annotated functions in DNA metabolism, cell
division, biosynthesis and degradation of the murein (peptidoglycan)
sacculus, membrane response, and general environmental stress
protection were upregulated, while genes encoding chemotaxis, motility,
and transport/binding proteins were largely repressed under conditions
of 24-h chromate
treatment.
Microbial Communities in the Surface Mucopolysaccharide Layer and the Black Band Microbial Mat of Black Band-Diseased
Siderastrea siderea
Authors: Sekar Raju , Mills DeEtta K. , Remily Elizabeth R. , Voss Joshua D. , Richardson Laurie L.
Published: September 2006

Microbial community profiles and species composition associated with two black band-diseased colonies of the coral
Siderastrea siderea
were studied by 16S rRNA-targeted gene cloning, sequencing, and amplicon-length heterogeneity PCR (LH-PCR). Bacterial communities associated with the surface mucopolysaccharide layer (SML) of apparently healthy tissues of the infected colonies, together with samples of the black band disease (BBD) infections, were analyzed using the same techniques for comparison. Gene sequences, ranging from 424 to 1,537 bp, were retrieved from all positive clones (
n
= 43 to 48) in each of the four clone libraries generated and used for comparative sequence analysis. In addition to LH-PCR community profiling, all of the clone sequences were aligned with LH-PCR primer sequences, and the theoretical lengths of the amplicons were determined. Results revealed that the community profiles were significantly different between BBD and SML samples. The SML samples were dominated by γ-proteobacteria (53 to 64%), followed by β-proteobacteria (18 to 21%) and α-proteobacteria (5 to 11%). In contrast, both BBD clone libraries were dominated by α-proteobacteria (58 to 87%), followed by verrucomicrobia (2 to 10%) and 0 to 6% each of δ-proteobacteria, bacteroidetes, firmicutes, and cyanobacteria. Alphaproteobacterial sequence types related to the bacteria associated with toxin-producing dinoflagellates were observed in BBD clone libraries but were not found in the SML libraries. Similarly, sequences affiliated with the family
Desulfobacteraceae
and toxin-producing cyanobacteria, both believed to be involved in BBD pathogenesis, were found only in BBD libraries. These data provide evidence for an association of numerous toxin-producing heterotrophic microorganisms with BBD of corals.
Environmental Influences onVibrioPopulations in Northern Temperate and Boreal Coastal Waters (Baltic and Skagerrak Seas)
Authors: Eiler Alexander , Johansson Mona , Bertilsson Stefan
Published: September 2006

Even if manyVibriospp. are endemic to coastal waters, their distribution in northern temperate and boreal waters is poorly studied. To identify environmental factors regulatingVibriopopulations in a salinity gradient along the Swedish coastline, we combinedVibrio-specific quantitative competitive PCR with denaturant gradient gel electrophoresis-based genotyping. The totalVibrioabundance ranged from 4 × 103to 9.6 × 104cells liter−1, with the highest abundances in the more saline waters of the Skagerrak Sea. SeveralVibriopopulations were present throughout the salinity gradient, with abundances of single populations ranging from 5 × 102to 7 × 104cells liter−1. Clear differences were observed along the salinity gradient, where three populations dominated the more saline waters of the Skagerrak Sea and two populations containing mainly representatives ofV. anguillarumandV. aestuarianusgenotypes were abundant in the brackish waters of the Baltic Sea. Our results suggest that this apparent niche separation within the genusVibriomay also be influenced by alternate factors such as nutrient levels and high abundances of dinoflagellates. AV. cholerae/V. mimicuspopulation was detected in more than 50% of the samples, with abundances exceeding 103cells liter−1, even in the cold (annual average water temperature of around 5°C) and low-salinity (2 to 4‰) samples from the Bothnian Bay (latitude, 65°N). The unsuspected and widespread occurrence of this population in temperate and boreal coastal waters suggests that potentialVibriopathogens may also be endemic to cold and brackish waters and hence may represent a previously overlooked health hazard.
Mutational and Functional Analysis of the β-Carotene Ketolase Involved in the Production of Canthaxanthin and Astaxanthin
Authors: Ye Rick W. , Stead Kristen J. , Yao Henry , He Hongxian
Published: September 2006

Biosynthesis of the commercial carotenoids canthaxanthin and astaxanthin requires β-carotene ketolase. The functional importance of the conserved amino acid residues of this enzyme from
Paracoccus
sp. strain N81106 (formerly classified as
Agrobacterium aurantiacum
) was analyzed by alanine-scanning mutagenesis. Mutations in the three highly conserved histidine motifs involved in iron coordination abolished its ability to catalyze the formation of ketocarotenoids. This supports the hypothesis that the CrtW ketolase belongs to the family of iron-dependent integral membrane proteins. Most of the mutations generated at other highly conserved residues resulted in partial activity. All partially active mutants showed a higher amount of adonixanthin accumulation than did the wild type when expressed in
Escherichia coli
cells harboring the zeaxanthin biosynthetic gene cluster. Some of the partially active mutants also produced a significant amount of echinenone when expressed in cells producing β-carotene. In fact, expression of a mutant carrying D117A resulted in the accumulation of echinenone as the predominant carotenoid. These observations indicate that partial inactivation of the CrtW ketolase can often lead to the production of monoketolated intermediates. In order to improve the conversion rate of astaxanthin catalyzed by the CrtW ketolase, a color screening system was developed. Three randomly generated mutants, carrying L175M, M99V, and M99I, were identified to have improved activity. These mutants are potentially useful in pathway engineering for the production of astaxanthin.
Detection of
Cryptosporidium
Oocysts in Water: Effect of the Number of Samples and Analytic Replicates on Test Results
Authors: Xiao Lihua , Alderisio Kerri A. , Jiang Jianlin
Published: September 2006

Due to the small number of
Cryptosporidium
oocysts in water, the number of samples taken and the analyses performed can affect the results of detection. In this study, 42 water samples were collected from one watershed during 20 storm events over 1 year, including duplicate or quadruplicate samples from 16 storm events. Ten samples from four events had three to eight subsamples. They were processed by EPA method 1623, and
Cryptosporidium
oocysts present were detected by immunofluorescent microscopy or PCR. Altogether, 24 of 39 samples (47 of 67 samples and subsamples) analyzed by microscopy were positive for
Cryptosporidium
. In contrast, 36 of 42 samples (62 of 76 samples and subsamples) were positive by PCR, including 10 microscopy-negative samples (13 microscopy-negative samples and subsamples). Six of the 24 microscopy-positive samples were negative by PCR, and all samples had one or less oocyst in a 0.5-ml packed pellet volume calculated. Discordant results were obtained by microscopy and PCR from six and three of the storm events, respectively, with multiple samples. Discordant microscopy or PCR results were also obtained among subsamples. Most of the 14
Cryptosporidium
genotypes were found over a brief period.
Cryptosporidium
-positive samples had a mean of 1.9 genotypes per sample, with 39 of the 62 positive samples/subsamples having more than one genotype. Samples/subsamples with more than one genotype had an overall PCR-positive rate of 73%, compared to 34% for those with one genotype. The PCR amplification rate of samples was affected by the volume of DNA used in PCR.
Microbial Population Dynamics Associated with Crude-Oil Biodegradation in Diverse Soils
Authors: Hamamura Natsuko , Olson Sarah H. , Ward David M. , Inskeep William P.
Published: September 2006

Soil bacterial population dynamics were examined in several crude-oil-contaminated soils to identify those organisms associated with alkane degradation and to assess patterns in microbial response across disparate soils. Seven soil types obtained from six geographically distinct areas of the United States (Arizona, Oregon, Indiana, Virginia, Oklahoma, and Montana) were used in controlled contamination experiments containing 2% (wt/wt) crude oil spiked with [1-
14
C]hexadecane. Microbial populations present during hydrocarbon degradation were analyzed using both 16S rRNA gene sequence analysis and by traditional methods for cultivating hydrocarbon-oxidizing bacteria. After a 50-day incubation, all seven soils showed comparable hydrocarbon depletion, where >80% of added crude oil was depleted and approximately 40 to 70% of added [
14
C]hexadecane was converted to
14
CO
2
. However, the initial rates of hydrocarbon depletion differed up to 10-fold, and preferential utilization of shorter-chain-length
n
-alkanes relative to longer-chain-length
n-
alkanes was observed in some soils. Distinct microbial populations developed, concomitant with crude-oil depletion. Phylogenetically diverse bacterial populations were selected across different soils, many of which were identical to hydrocarbon-degrading isolates obtained from the same systems (e.g.,
Nocardioides albus
,
Collimonas
sp., and
Rhodococcus coprophilus
). In several cases, soil type was shown to be an important determinant, defining specific microorganisms responding to hydrocarbon contamination. However, similar
Rhodococcus erythropolis
-like populations were observed in four of the seven soils and were the most common hydrocarbon-degrading organisms identified via cultivation.
Contraselectable Streptomycin Susceptibility Determinant for Genetic Manipulation and Analysis of
Helicobacter pylori
Authors: Dailidiene Daiva , Dailide Giedrius , Kersulyte Dangeruta , Berg Douglas E.
Published: September 2006

Many
Helicobacter pylori
genetic studies would benefit from an ability to move DNA sequences easily between strains by transformation and homologous recombination, without needing to leave a conventional drug resistance determinant at the targeted locus. Presented here is a two-gene cassette that can be selected both (i) against, due to a
Campylobacter jejuni rpsL
gene (dominant streptomycin susceptibility in cells also carrying an
rpsL
-
str

r

allele), and (ii) for, due to an
erm
gene (erythromycin resistance). This
rpsL,erm
cassette's utility was assessed by using it to replace four gene loci (
mdaB
,
frxA
,
fur
, and
nikR
) in four streptomycin-resistant [Str
r
] strain backgrounds (derivatives of 26695, SS1, X47, and G27MA). The resultant 16 strains (phenotypically erythromycin resistant [Erm
r
] and Str
s
) were each transformed with wild-type genomic DNAs, and Str
r
derivatives were selected. The desired Erm
s
Str
r
isolates were obtained at frequencies that ranged from 17 to 96% among Str
r
transformants, with the Erm
s
yield apparently depending on the strain background and genome location of the targeted locus. The ease of isolating unmarked transformants described here should be valuable for many
H. pylori
molecular genetic and evolutionary analyses.
Enzymatic Activation of Lysine 2,3-Aminomutase from
Porphyromonas gingivalis
Authors: Brazeau Brian J. , Gort Steven J. , Jessen Holly J. , Andrew Amy J. , Liao Hans H.
Published: September 2006

The development of lysine 2,3-aminomutase as a robust biocatalyst hinges on the development of an in vivo activation system to trigger catalysis. This is the first report to show that, in the absence of chemical reductants, lysine 2,3-aminomutase activity is dependent upon the presence of flavodoxin, ferredoxin, or flavodoxin-NADP
+
reductase.
Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination
Authors: Gebauer Birgit , Jendrossek Dieter
Published: September 2006

Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a software-controlled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated fromRalstonia eutrophaH16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatization of 3HB oligomers into bromophenacyl derivates and separation by high-performance liquid chromatography. The method allows the separation and quantification of 3HB and 3HB oligomers up to the octamer. The two methods were applied to investigate the hydrolysis of different types of PHB by selected PHB depolymerases.
Complementation of a
Clostridium perfringens spo0A
Mutant with Wild-Type
spo0A
from Other
Clostridium
Species
Authors: Huang I-Hsiu , Sarker Mahfuzur R.
Published: September 2006

To evaluate whether
C. perfringens
can be used as a model organism for studying the sporulation process in other clostridia,
C. perfringens spo0A
mutant IH101 was complemented with wild-type
spo0A
from four different
Clostridium
species. Wild-type
spo0A
from
C. acetobutylicum
or
C. tetani
, but not from
C. botulinum
or
C. difficile
, restored sporulation and enterotoxin production in IH101. The ability of
spo0A
from
C. botulinum
or
C. difficile
to complement the lack of spore formation in IH101 might be due, at least in part, to the low levels of
spo0A
transcription and Spo0A production.
Integrated Analysis of Established and Novel Microbial and Chemical Methods for Microbial Source Tracking
Authors: Blanch Anicet R. , Belanche-Muñoz Lluís , Bonjoch Xavier , Ebdon James , Gantzer Christophe , Lucena Francisco , Ottoson Jakob , Kourtis Christos , Iversen Aina , Kühn Inger , Mocé Laura , Muniesa Maite , Schwartzbrod Janine , Skraber Sylvain , Papageorgiou Georgios T. , Taylor Huw , Wallis Jessica , Jofre Joan
Published: September 2006

Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting
Bacteroides fragilis
RYC2056 and
Bacteroides thetaiotaomicron
GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of
Bifidobacterium adolescentis
and
Bifidobacterium dentium
; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting
B. thetaiotaomicron
, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting
Bacteroides thetaiotaomicron
to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting
Bacteroides thetaiotaomicron
to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.
Visible-Light-Induced Bactericidal Activity of a Nitrogen-Doped Titanium Photocatalyst against Human Pathogens
Authors: Wong Ming-Show , Chu Wen-Chen , Sun Der-Shan , Huang Hsuan-Shun , Chen Jiann-Hwa , Tsai Pei-Jane , Lin Nien-Tsung , Yu Mei-Shiuan , Hsu Shang-Feng , Wang Shih-Lien , Chang Hsin-Hou
Published: September 2006

The antibacterial activity of photocatalytic titanium dioxide (TiO
2
) substrates is induced primarily by UV light irradiation. Recently, nitrogen- and carbon-doped TiO
2
substrates were shown to exhibit photocatalytic activities under visible-light illumination. Their antibacterial activity, however, remains to be quantified. In this study, we demonstrated that nitrogen-doped TiO
2
substrates have superior visible-light-induced bactericidal activity against
Escherichia coli
compared to pure TiO
2
and carbon-doped TiO
2
substrates. We also found that protein- and light-absorbing contaminants partially reduce the bactericidal activity of nitrogen-doped TiO
2
substrates due to their light-shielding effects. In the pathogen-killing experiment, a significantly higher proportion of all tested pathogens, including
Shigella flexneri
,
Listeria monocytogenes
,
Vibrio parahaemolyticus
,
Staphylococcus aureus
,
Streptococcus pyogenes
, and
Acinetobacter baumannii
, were killed by visible-light-illuminated nitrogen-doped TiO
2
substrates than by pure TiO
2
substrates. These findings suggest that nitrogen-doped TiO
2
has potential application in the development of alternative disinfectants for environmental and medical usages.
Identification and Differentiation of
Legionella pneumophila
and
Legionella
spp. with Real-Time PCR Targeting the 16S rRNA Gene and Species Identification by
mip
Sequencing
Authors: Stølhaug Anne , Bergh Kåre
Published: September 2006

Fluorescent resonance energy transfer probes targeting the 16S rRNA gene were constructed for a sensitive and specific real-time PCR for identification and differentiation of
Legionella pneumophila
from other
Legionella
spp. For identification of non-
L. pneumophila
spp. by direct amplicon sequencing, two conventional PCR assays targeting the
mip
gene were established.
Copper Resistance in
Enterococcus faecium
, Mediated by the
tcrB
Gene, Is Selected by Supplementation of Pig Feed with Copper Sulfate
Authors: Hasman Henrik , Kempf Isabelle , Chidaine Bérangère , Cariolet Roland , Ersbøll Annette Kjær , Houe Hans , Bruun Hansen Hans Christian , Aarestrup Frank Møller
Published: September 2006

The
tcr
gene cluster mediates in vitro copper resistance in
Enterococcus faecium
. Here we describe the selection of
tcr
-mediated copper resistance in
E. faecium
in an animal feeding experiment with young pigs fed 175 mg copper/kg feed (ppm), which is the concentration commonly used for piglets in European pig production.
tcr
-mediated copper resistance was not selected for in a control group fed low levels of copper (6 ppm). We also show coselection of macrolide- and glycopeptide-resistant
E. faecium
in the animal group fed the high level of copper. Finally, we identify the
tcr
genes in the enterococcal species
E. mundtii
,
E. casseliflavus
, and
E. gallinarum
for the first time.
Microbial Community Diversity Associated with Carbon and Nitrogen Cycling in Permeable Shelf Sediments
Authors: Hunter Evan M. , Mills Heath J. , Kostka Joel E.
Published: September 2006

Though a large fraction of primary production and organic matter cycling in the oceans occurs on continental shelves dominated by sandy deposits, the microbial communities associated with permeable shelf sediments remain poorly characterized. Therefore, in this study, we provide the first detailed characterization of microbial diversity in marine sands of the South Atlantic Bight through parallel analyses of small-subunit (SSU) rRNA gene (
Bacteria
),
nosZ
(denitrifying bacteria), and
amoA
(ammonia-oxidizing bacteria) sequences. Communities were analyzed by parallel DNA extractions and clone library construction from both sediment core material and manipulated sediment within column experiments designed for geochemical rate determinations. Rapid organic-matter degradation and coupled nitrification-denitrification were observed in column experiments at flow rates resembling in situ conditions over a range of oxygen concentrations. Numerous SSU rRNA phylotypes were affiliated with the phyla
Proteobacteria
(classes
Alpha
-,
Delta
-, and
Gammaproteobacteria
),
Planctomycetes
,
Cyanobacteria
,
Chloroflexi
, and
Bacteroidetes
. Detectable sequence diversity of
nosZ
and SSU rRNA genes increased in stratified redox-stabilized columns compared to in situ sediments, with the
Alphaproteobacteria
comprising the most frequently detected group. Alternatively, nitrifier communities showed a relatively low and stable diversity that did not covary with the other gene targets. Our results elucidate predominant phylotypes that are likely to catalyze carbon and nitrogen cycling in marine sands. Although overall diversity increased in response to redox stabilization and stratification in column experiments, the major phylotypes remained the same in all of our libraries, indicating that the columns sufficiently mimic in situ conditions.
Isolation and Characterization of a New T-Even Bacteriophage, CEV1, and Determination of Its Potential To Reduce
Escherichia coli
O157:H7 Levels in Sheep
Authors: Raya Raul R. , Varey Peter , Oot Rebecca A. , Dyen Michael R. , Callaway Todd R. , Edrington Tom S. , Kutter Elizabeth M. , Brabban Andrew D.
Published: September 2006

Bacteriophage CEV1 was isolated from sheep resistant to
Escherichia coli
O157:H7 colonization. In vitro, CEV1 efficiently infected
E.
coli
O157:H7 grown both aerobically and anaerobically. In vivo,
sheep receiving a single oral dose of CEV1 showed a 2-log-unit
reduction in intestinal
E. coli
O157:H7 levels within 2 days
compared to levels in the
controls.
Survival of Environmental Mycobacteria in
Acanthamoeba polyphaga
Authors: Adékambi Toïdi , Ben Salah Skandar , Khlif Mohamed , Raoult Didier , Drancourt Michel
Published: September 2006

Free-living amoebae in water are hosts to many bacterial species living in such an environment. Such an association enables bacteria to select virulence factors and survive in adverse conditions. Waterborne mycobacteria (WBM) are important sources of community- and hospital-acquired outbreaks of nontuberculosis mycobacterial infections. However, the interactions between WBM and free-living amoebae in water have been demonstrated for only few
Mycobacterium
spp. We investigated the ability of a number (
n
= 26) of
Mycobacterium
spp. to survive in the trophozoites and cysts of
Acanthamoeba polyphaga
. All the species tested entered the trophozoites of
A. polyphaga
and survived at this location over a period of 5 days. Moreover, all
Mycobacterium
spp. survived inside cysts for a period of 15 days. Intracellular
Mycobacterium
spp. within amoeba cysts survived when exposed to free chlorine (15 mg/liter) for 24 h. These data document the interactions between free-living amoebae and the majority of waterborne
Mycobacterium
spp. Further studies are required to examine the effects of various germicidal agents on the survival of WBM in an aquatic environment.
A pilot study on the effects of pre-event manipulation on jump height and running velocity
Authors: Shrier I , Macdonald D , Uchacz G
Published: September 2006

Purpose:To compare changes in jump height and running velocity with and without pre-event high-velocity, low-amplitude manipulation (HVLA).

Methods:A crossover study design with elite healthy athletes was used. After a 15 min warm-up, the subjects were tested for countermovement jump height (CMJ) and flying 40 m sprint time (SPRINT). A sport chiropractor then evaluated each subject. Subjects were randomised to either HVLA (applied to joints based on examination) or placebo (simulated performance-enhancement stickers). They then rested for 60 min, performed another 15 min warm-up, and were retested. The protocol was repeated 48 h later with the alternative intervention. The mean of two sprints and three jumps were analysed, as well as peak performances. The sample size was based on prior results from the effects of stretching.

Results:19 subjects involved in sprint sports were enrolled; two were too sore to participate on day 2, and one could only participate in the jump (all had HVLA on day 1). Of the 17 participants analysed, seven were female, age range was 19–35, and 17 were national or world-class athletes. The ranges for baseline measures were: SPRINT 4.1–5.5 s; CMJ 47.4–92.7 cm. Overall, the greater than expected variability in this pilot study led to the study being underpowered. Subjects tended to perform better after HVLA for both CMJ and SPRINT (both mean and peak results), but none of the results were statistically significant (p  =  0.30–0.61).

Conclusion:Although the larger than expected variability in the pilot study means that the observed clinically relevant differences were not statistically significant, the direction and magnitude of the changes associated with HVLA suggest that it may be beneficial. That said, the increased soreness after HVLA suggests that it may be detrimental. HVLA warrants further study.