bluuebunny/crossref_metadata_2025 huggingface dataset:
Radiographic findings of Wistar rats fed with T‐2 toxin and Kashin‐Beck disease‐affected diet
Authors: YAN Denglu , KANG Pengde , LI Yansheng , YANG Jing , SHEN Bing , ZHOU Zongke , DENG Jianyun , PEI Fuxing
Published: August 2010

Objective:  To characterize the features of radiographic abnormalities of the tibial bone in Wistar rats which have been fed T‐2 toxin and Kashin‐Beck disease (KBD) epidemic district food.

Methods:  Ninety Wistar rats were divided into five groups. Group A was fed with a normal diet as control; group B was fed with a normal diet and T‐2 toxin; group C was fed with a low‐nutrition diet and T‐2 toxin; group D was fed with a low‐nutrition diet; and group E was fed with a KBD‐affected diet. At 4, 8 and 12 weeks, six rats from each group were radiographed. After radiographic examination, samples of left knees were harvested and stained with hematoxylin and eosin.

Results:  At 8 and 12 weeks, there were radiological changes in the epiphyseal plate. Abnormal radiological signs of blurring, thinning and irregularity were seen in groups C and E rats, and the length of tibial bones showed significant difference in the KBD‐fed rats and low‐nutrition diet combined T‐2 toxin rats, compared to the control group rats (P < 0.05). The epiphyseal plates showed more obvious necrosis of chondrocytes in groups C and E.

Conclusions:  A rat model of KBD can be established by a KBD‐affected diet; proximal epiphyseal plate and metaphyseal bone of the tibia abnormalities on radiographs and histopathology were present in KBD model rats. A low nutrition diet may be involved the aetiology of KBD, and determination of this should be studied in the future.
Using false discovery rates for multiple comparisons in ecology and evolution
Authors: Pike Nathan
Published: August 2010

1. Ecologists and evolutionary biologists often need to simultaneously evaluate the significance of multiple related hypotheses. Multiple comparisons need to be corrected to avoid inappropriately increasing the number of null hypotheses that are wrongly rejected. The traditional method of correction involves Bonferroni‐type multiple comparison procedures which are highly conservative, tending to increase the number of wrong rejections of true hypotheses as the number of hypotheses being simultaneously tested increases.

2. Newer procedures which are based on False Discovery Rates and which do not suffer the same loss of power as traditional methods are described. Algorithms and spreadsheet‐based software routines for three procedures which are especially useful in ecology and evolution are provided.

3. The strengths and potential pitfalls of FDR‐based analysis and of presenting results as FDR‐adjusted P‐values are discussed with reference to traditional methods such as the sequential Bonferroni correction.

4. FDR‐based multiple comparison procedures should be more widely adopted because they are often more appropriate than traditional methods for identifying truly significant results.
A unified approach to model selection using the likelihood ratio test
Authors: Lewis Fraser , Butler Adam , Gilbert Lucy
Published: August 2010

1. Ecological count data typically exhibit complexities such as overdispersion and zero‐inflation, and are often weakly associated with a relatively large number of correlated covariates. The use of an appropriate statistical model for inference is therefore essential. A common selection criteria for choosing between nested models is the likelihood ratio test (LRT). Widely used alternatives to the LRT are based on information‐theoretic metrics such as the Akaike Information Criterion.

2. It is widely believed that the LRT can only be used to compare the performance of nested models – i.e. in situations where one model is a special case of another. There are many situations in which it is important to compare non‐nested models, so, if true, this would be a substantial drawback of using LRTs for model comparison. In reality, however, it is actually possible to use the LRT for comparing both nested and non‐nested models. This fact is well‐established in the statistical literature, but not widely used in ecological studies.

3. The main obstacle to the use of the LRT with non‐nested models has, until relatively recently, been the fact that it is difficult to explicitly write down a formula for the distribution of the LRT statistic under the null hypothesis that one of the models is true. With modern computing power it is possible to overcome this difficulty by using a simulation‐based approach.

4. To demonstrate the practical application of the LRT to both nested and non‐nested model comparisons, a case study involving data on questing tick (Ixodes ricinus) abundance is presented. These data contain complexities typical in ecological analyses, such as zero‐inflation and overdispersion, for which comparison between models of differing structure – e.g. non‐nested models – is of particular importance.

5. Choosing between competing statistical models is an essential part of any applied ecological analysis. The LRT is a standard statistical test for comparing nested models. By use of simulation the LRT can also be used in an analogous fashion to compare non‐nested models, thereby providing a unified approach for model comparison within the null hypothesis testing paradigm. A simple practical guide is provided in how to apply this approach to the key models required in the analyses of count data.
A cross‐sectional study of geriatric horses in the United Kingdom. Part 2: Health care and disease
Authors: IRELAND J. L. , CLEGG P. D. , McGOWAN C. M. , McKANE S. A. , PINCHBECK G. L.
Published: August 2010

Reasons for performing study: Geriatric horses (aged ≥15 years) represent a substantial proportion of the equine population, yet very few studies have investigated the prevalence of diseases within the UK equine geriatric population.

Objectives: To describe the provision of routine preventive health care measures, prevalence of clinical signs of disease and the prevalence of owner reported diseases. Additionally, the effect of increasing age on the provision of preventive health care and the presence or absence of clinical signs and disease was assessed.

Methods: A cross‐sectional study was conducted, surveying a randomly selected sample of veterinary registered owners with horses aged ≥15 years, using a self‐administered postal questionnaire.

Results: As geriatric horses increased in age, there was a reduction in the provision of preventive health care measures, such as vaccination, farrier care and routine veterinary checks. Only 68.7% of horses had received a routine veterinary visit within the previous 12 months. Owners frequently observed clinical signs in their animals, with 77% reporting at least one clinical sign of disease. Increasing age was associated with increased reporting of many clinical signs of disease. Over half (58%) of horses had at least one episode of disease within the previous 12 months, yet only 31% of owners reported that their animal currently suffered from a known disease or disorder.

Conclusions and potential relevance: Although owners frequently observed clinical signs in their aged horse, there may be incorrect or under recognition of many diseases and health problems. Reduced frequency of routine preventive health care measures, along with suboptimal owner recognition of health and welfare problems may lead to compromised welfare in the geriatric population.
Laparoscopic techniques for investigating the equine oviduct
Authors: KÖLLMANN M. , RÖTTING A. , HEBERLING A. , SIEME H.
Published: August 2010

Reasons for performing study:The diagnostic and therapeutic options for oviduct disorders in the mare are limited. The current best techniques require exploratory surgery under general anaesthesia or flank laparotomy.

Hypothesis:The orthograde flushing of the oviduct for diagnostic or therapeutic options is possible using laparoscopic techniques in the standing sedated mare.

Methods:Development of a laparoscopic technique for catheterisation of the infundibulum and flushing of the oviduct (sterile methylene blue solution) in the standing sedated mare was examined in 2 experiments. The first involved a transvaginal laparoscopic approach, the second a laparoscopic flank approach. Passage of fluid into the uterus was assessed by post operative hysteroscopy.

Results:InExperiment I, visualisation of the infundibulum was possible (left side 7/8 cases, right side in 6/8 cases). The beginning of the oviductal ampulla could be seen in 3 of 8 cases on the left side. An adequate opening of the infundibulum and visualisation or catheterisation of the abdominal ostium were not possible. InExperiment II, catheterisation of the ampulla was successful in 7 of 11 cases, and in 5 of these 7 cases the injected fluid could be identified in the uterus by post operative hysteroscopy.

Conclusion:A transvaginal laparoscopic approach to the oviduct is not appropriate for oviductal flushing in the mare. However, a laparoscopic flank‐approach permits investigation and flushing of the oviduct.

Potential relevance:Laparoscopic flushing could become a practical method for diagnosis and therapy of oviduct disorders and a minimally invasive technique for collection of young embryos or the transfer of gametes (GIFT).
A cross‐sectional study of geriatric horses in the United Kingdom. Part 1: Demographics and management practices
Authors: IRELAND J. L. , CLEGG P. D. , McGOWAN C. M. , McKANE S. A. , PINCHBECK G. L.
Published: August 2010

Reasons for performing study:With growing numbers of aged horses, geriatric medicine is becoming increasingly important in equine veterinary practice; however, there is a paucity of information on the UK equine geriatric population.

Objectives:To describe the demographic characteristics of the equine geriatric population and to assess management practices undertaken by owners of geriatric horses (aged ≥15 years).

Methods:A cross‐sectional study was conducted, surveying a randomly selected sample of veterinary registered owners with horses aged ≥15 years, using a self‐administered postal questionnaire.

Results:Horses aged ≥15 years represented 29% of the equine population in the study area. The response rate to the questionnaire survey was 80.2%. Management practices were similar to those previously described for the general equine population. However, the level of exercise and feeding practices changed significantly with increasing age. A summary of the demographic characteristics, feeding and management of horses in a sample of the geriatric equine population is presented.

Conclusions and potential relevance:The management of geriatric horses has a direct effect on their health and welfare and this study has provided valuable information on the demographics and management of geriatric horses and ponies in the UK. The information can be used to form targeted owner education programmes for geriatric health and should aid in the investigation and prevention of disease.
Enhancement of Targeted Homologous Recombination inMycoplasma mycoidessubsp.capriby Inclusion of HeterologousrecA
Authors: Allam Ayman B. , Reyes Leticia , Assad-Garcia Nacyra , Glass John I. , Brown Mary B.
Published: August 2010

A suicide plasmid, pExp1-ctpA::tetM-recAec, employingrecAfromEscherichia coliandtetMas a selection marker, was used to generatectpAknockout mutants inMycoplasma mycoidessubsp.caprithrough targeted gene disruption. Inclusion ofE. coli recAgreatly enhanced both the consistency and the recovery of mutants generated by homologous recombination.
Engineered Lactic Acid Bacterium
Lactococcus lactis
Capable of Binding Antibodies and Tumor Necrosis Factor Alpha
Authors: Ravnikar Matjaž , Štrukelj Borut , Obermajer Nataša , Lunder Mojca , Berlec Aleš
Published: August 2010

We have optimized the display of the B domain of staphylococcal protein A on the surface of
Lactococcus lactis.
The maximum binding capacity was estimated at 0.146 μg of antibody per 10
8
cells and was sustained at 86% after treatment with simulated gastric juice. A tumor necrosis factor alpha (TNF-α)-binding affibody was also displayed and bound TNF-α, which could be useful in the treatment of inflammatory bowel disease.
Quantitative Fluorescence
In Situ
Hybridization of Microbial Communities in the Rumens of Cattle Fed Different Diets
Authors: Kong Yunhong , He Maolong , McAlister Tim , Seviour Robert , Forster Robert
Published: August 2010

At present there is little quantitative information on the identity and composition of bacterial populations in the rumen microbial community. Quantitative fluorescence
in situ
hybridization using newly designed oligonucleotide probes was applied to identify the microbial populations in liquid and solid fractions of rumen digesta from cows fed barley silage or grass hay diets with or without flaxseed.
Bacteroidetes
,
Firmicutes
, and
Proteobacteria
were abundant in both fractions, constituting 31.8 to 87.3% of the total cell numbers. They belong mainly to the order
Bacteroidales
(0.1 to 19.2%), hybridizing with probe BAC1080; the families
Lachnospiraceae
(9.3 to 25.5%) and
Ruminococcaceae
(5.5 to 23.8%), hybridizing with LAC435 and RUM831, respectively; and the classes
Deltaproteobacteria
(5.8 to 28.3%) and
Gammaproteobacteria
(1.2 to 8.2%). All were more abundant in the rumen communities of cows fed diets containing silage (75.2 to 87.3%) than in those of cows fed diets containing hay (31.8 to 49.5%). The addition of flaxseed reduced their abundance in the rumens of cows fed silage-based diets (to 45.2 to 58.7%) but did not change markedly their abundance in the rumens of cows fed hay-based diets (31.8 to 49.5%). Fibrolytic species, including
Fibrobacter succinogenes
and
Ruminococcus
spp., and archaeal methanogens accounted for only a small proportion (0.4 to 2.1% and 0.2 to 0.6%, respectively) of total cell numbers. Depending on diet, between 37.0 and 91.6% of microbial cells specifically hybridized with the probes used in this study, allowing them to be identified
in situ
. The identities of other microbial populations (8.4 to 63.0%) remain unknown.
Isolation and Characterization of 4-
tert
-Butylphenol-Utilizing
Sphingobium fuliginis
Strains from
Phragmites australis
Rhizosphere Sediment
Authors: Toyama Tadashi , Momotani Naonori , Ogata Yuka , Miyamori Yuji , Inoue Daisuke , Sei Kazunari , Mori Kazuhiro , Kikuchi Shintaro , Ike Michihiko
Published: August 2010

We isolated three
Sphingobium fuliginis
strains from
Phragmites australis
rhizosphere sediment that were capable of utilizing 4-
tert
-butylphenol as a sole carbon and energy source. These strains are the first 4-
tert
-butylphenol-utilizing bacteria. The strain designated TIK-1 completely degraded 1.0 mM 4-
tert
-butylphenol in basal salts medium within 12 h, with concomitant cell growth. We identified 4-
tert
-butylcatechol and 3,3-dimethyl-2-butanone as internal metabolites by gas chromatography-mass spectrometry. When 3-fluorocatechol was used as an inactivator of
meta
-cleavage enzymes, strain TIK-1 could not degrade 4-
tert
-butylcatechol and 3,3-dimethyl-2-butanone was not detected. We concluded that metabolism of 4-
tert
-butylphenol by strain TIK-1 is initiated by hydroxylation to 4-
tert
-butylcatechol, followed by a
meta
-cleavage pathway. Growth experiments with 20 other alkylphenols showed that 4-isopropylphenol, 4-
sec
-butylphenol, and 4-
tert
-pentylphenol, which have alkyl side chains of three to five carbon atoms with α-quaternary or α-tertiary carbons, supported cell growth but that 4-
n
-alkylphenols, 4-
tert
-octylphenol, technical nonylphenol, 2-alkylphenols, and 3-alkylphenols did not. The rate of growth on 4-
tert
-butylphenol was much higher than that of growth on the other alkylphenols. Degradation experiments with various alkylphenols showed that strain TIK-1 cells grown on 4-
tert
-butylphenol could degrade 4-alkylphenols with variously sized and branched side chains (ethyl,
n
-propyl, isopropyl,
n
-butyl,
sec
-butyl,
tert
-butyl,
n
-pentyl,
tert
-pentyl,
n
-hexyl,
n
-heptyl,
n
-octyl,
tert
-octyl,
n
-nonyl, and branched nonyl) via a
meta
-cleavage pathway but not 2- or 3-alkylphenols. Along with the degradation of these alkylphenols, we detected methyl alkyl ketones that retained the structure of the original alkyl side chains. Strain TIK-1 may be useful in the bioremediation of environments polluted by 4-
tert
-butylphenol and various other 4-alkylphenols.
Sensitization of Microcin J25-Resistant Strains by a Membrane-Permeabilizing Peptide
Authors: Pomares María Fernanda , Delgado Mónica A. , Corbalán Natalia S. , Farías Ricardo N. , Vincent Paula A.
Published: August 2010

Microcin J25 (MccJ25) is a plasmid-encoded, 21-amino-acid, antibacterial peptide produced byEscherichia coli. MccJ25 inhibits RNA polymerase and the membrane respiratory chain. MccJ25 uptake intoE. coli-sensitive strains is mediated by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. This peptide is active on someE. coli,Salmonella, andShigellaspecies strains, while other Gram-negative bacteria, such as clinical isolates ofEnterobacter cloacae,Citrobacter freundii,Klebsiella pneumoniae,Pseudomonas aeruginosa,Acinetobacter baumannii,Moraxella catarrhalis, andSalmonella entericaserovar Typhimurium, are completely resistant. In the present work, we demonstrated that the membrane-permeabilizing peptide (KFF)3K made some resistant strains sensitive to MccJ25, among themS.Typhimurium, where the antibiotic inhibitsin vitrocell growth and bacterial replication within macrophages. The results demonstrate that the membrane permeabilization induced by (KFF)3K allows MccJ25 penetration in an FhuA and SbmA-independent manner and suggest that the combination of both peptides could be considered as a therapeutic agent against pathogenicSalmonellastrains.
Use of Purified
Clostridium difficile
Spores To Facilitate Evaluation of Health Care Disinfection Regimens
Authors: Lawley Trevor D. , Clare Simon , Deakin Laura J. , Goulding David , Yen Jennifer L. , Raisen Claire , Brandt Cordelia , Lovell Jon , Cooke Fiona , Clark Taane G. , Dougan Gordon
Published: August 2010

Clostridium difficile
is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of
C. difficile
that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious
C. difficile
spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified
C. difficile
spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure spores and blocking spore-mediated transmission. Complete inactivation of 10
6
pure
C. difficile
spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with
C. difficile
spores (17 to 29 spores/cm
2
) and block host transmission. Thus, pure
C. difficile
spores facilitate practical methods for evaluating the efficacy of
C. difficile
spore disinfection regimens and bringing scientific acumen to
C. difficile
infection control.
Characterization of
Lactococcus lactis
Phage 949 and Comparison with Other Lactococcal Phages
Authors: Samson Julie E. , Moineau Sylvain
Published: August 2010

The virulent
Lactococcus lactis
phage 949 was isolated in 1975 from cheese whey in New Zealand. This phage is a member of the
Siphoviridae
family and of a rare lactococcal phage group that bears its name (949 group). It has an icosahedral capsid (79-nm diameter) and a very long noncontractile tail (length, 500 nm; width, 12 nm). It infected 7 of 59 tested
L. lactis
strains, a somewhat expanded host range for a rare lactococcal phage. The abortive phage infection defense mechanisms AbiQ and AbiT strongly inhibited the multiplication of phage 949, but AbiK and AbiV did not. Its double-stranded DNA (dsDNA) genome of 114,768 bp is, to date, the largest among lactococcal phages. Its GC content was calculated at 32.7%, which is the lowest reported for a lactococcal phage. Its 154 open reading frames (ORFs) share limited identity with database sequences. In addition, terminal redundancy was observed as well as the presence of six tRNAs, one group I intron, and putative recombinases. SDS-PAGE coupled with mass spectrometry identified 13 structural proteins. The genomes of the members of the 10 currently known
L. lactis
phage groups were used to construct a proteomic tree. Each
L. lactis
phage group separated into distinct genetic clusters, validating the current classification scheme. Of note, members of the polythetic P335 groups were clearly separated into subgroups.
Acquisition of Iron by Alkaliphilic
Bacillus
Species
Authors: McMillan Duncan G. G. , Velasquez Imelda , Nunn Brook L. , Goodlett David R. , Hunter Keith A. , Lamont Iain , Sander Sylvia G. , Cook Gregory M.
Published: August 2010

The biochemical and molecular mechanisms used by alkaliphilic bacteria to acquire iron are unknown. We demonstrate that alkaliphilic (pH > 9)
Bacillus
species are sensitive to artificial iron (Fe
3+
) chelators and produce iron-chelating molecules. These alkaliphilic siderophores contain catechol and hydroxamate moieties, and their synthesis is stimulated by manganese(II) salts and suppressed by FeCl
3
addition. Purification and mass spectrometric characterization of the siderophore produced by
Caldalkalibacillus thermarum
failed to identify any matches to previously observed fragmentation spectra of known siderophores, suggesting a novel structure.
Effect of Estuarine Wetland Degradation on Transport of
Toxoplasma gondii
Surrogates from Land to Sea
Authors: Shapiro Karen , Conrad Patricia A. , Mazet Jonna A. K. , Wallender Wesley W. , Miller Woutrina A. , Largier John L.
Published: August 2010

The flux of terrestrially derived pathogens to coastal waters presents a significant health risk to marine wildlife, as well as to humans who utilize the nearshore for recreation and seafood harvest. Anthropogenic changes in natural habitats may result in increased transmission of zoonotic pathogens to coastal waters. The objective of our work was to evaluate how human-caused alterations of coastal landscapes in California affect the transport of
Toxoplasma gondii
to estuarine waters.
Toxoplasma gondii
is a protozoan parasite that is excreted in the feces of infected felids and is thought to reach coastal waters in contaminated runoff. This zoonotic pathogen causes waterborne toxoplasmosis in humans and is a significant cause of death in threatened California sea otters. Surrogate particles that mimic the behavior of
T. gondii
oocysts in water were released in transport studies to evaluate if the loss of estuarine wetlands is contributing to an increased flux of oocysts into coastal waters. Compared to vegetated sites, more surrogates were recovered from unvegetated mudflat habitats, which represent degraded wetlands. Specifically, in Elkhorn Slough, where a large proportion of otters are infected with
T. gondii
, erosion of 36% of vegetated wetlands to mudflats may increase the flux of oocysts by more than 2 orders of magnitude. Total degradation of wetlands may result in increased
Toxoplasma
transport of 6 orders of magnitude or more. Destruction of wetland habitats along central coastal California may thus facilitate pathogen pollution in coastal waters with detrimental health impacts to wildlife and humans.
Proteolytic Enzyme Production by Strains of the Insect Pathogen
Xenorhabdus
and Characterization of an Early-Log-Phase-Secreted Protease as a Potential Virulence Factor
Authors: Massaoud Mustafa K. , Marokházi Judit , Fodor András , Venekei István
Published: August 2010

As a comparison to a similar study on
Photorhabdus
strains, 15
Xenorhabdus
bacterial strains and secondary phenotypic variants of two strains were screened for proteolytic activity by five detection methods. Although the number and intensity of proteolytic activities were different, every strain was positive for proteolytic activity by several tests. Zymography following native PAGE detected two groups of activities with different substrate affinities and a higher and lower electrophoretic mobility that were distinguished as activity 1 and 2, respectively. Zymography following SDS-PAGE resolved three activities, which were provisionally named proteases A, B, and C. Only protease B, an ∼55-kDa enzyme, was produced by every strain. This enzyme exhibited higher affinity to the gelatin substrate than to the casein substrate. Of the chromogenic substrates used, three were hydrolyzed: furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY), Fua-LGPA (LGPA is Leu-Gly-Pro-Ala) (a substrate for collagen peptidases), and succinyl-Ala-Ala-Pro-Phe-thiobenzyl (Succ-AAPF-SBzl). All but the Fua-LGPA-ase activity seemed to be from secreted enzymes. According to their substrate preference profiles and inhibitor sensitivities, at least six such proteolytic enzymes could be distinguished in the culture medium of
Xenorhabdus
strains. The proteolytic enzyme that was secreted the earliest, protease B and the Succ-AAPF-SBzl-hydrolyzing enzyme, appeared from the early logarithmic phase of growth. Protease B could also be detected in the hemolymph of
Xenorhabdus
-infected
Galleria mellonella
larvae from 15 h postinfection. The purified protease B hydrolyzed
in vitro
seven proteins in the hemolymph of
Manduca sexta
that were also cleaved by PrtA peptidase from
Photorhabdus
. The N-terminal sequence of protease B showed similarity to a 55-kDa serralysin type metalloprotease in
Xenorhabdus nematophila
, which had been identified as an orthologue of
Photorhabdus
PrtA peptidase.
Vertical Distribution of Ammonia-Oxidizing Crenarchaeota and Methanogens in the Epipelagic Waters of Lake Kivu (Rwanda-Democratic Republic of the Congo)
Authors: Llirós Marc , Gich Frederic , Plasencia Anna , Auguet Jean-Christophe , Darchambeau François , Casamayor Emilio O. , Descy Jean-Pierre , Borrego Carles
Published: August 2010

Four stratified basins in Lake Kivu (Rwanda-Democratic Republic of the Congo) were sampled in March 2007 to investigate the abundance, distribution, and potential biogeochemical role of planktonic archaea. We used fluorescence
in situ
hybridization with catalyzed-reported deposition microscopic counts (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) fingerprinting, and quantitative PCR (qPCR) of signature genes for ammonia-oxidizing archaea (16S rRNA for marine
Crenarchaeota
group 1.1a [MCG1] and ammonia monooxygenase subunit A [
amoA
]). Abundance of archaea ranged from 1 to 4.5% of total DAPI (4′,6-diamidino-2-phenylindole) counts with maximal concentrations at the oxic-anoxic transition zone (∼50-m depth). Phylogenetic analysis of the archaeal planktonic community revealed a higher level of richness of crenarchaeal 16S rRNA gene sequences (21 of the 28 operational taxonomic units [OTUs] identified [75%]) over euryarchaeotal ones (7 OTUs). Sequences affiliated with the kingdom
Euryarchaeota
were mainly recovered from the anoxic water compartment and mostly grouped into methanogenic lineages (
Methanosarcinales
and
Methanocellales
). In turn, crenarchaeal phylotypes were recovered throughout the sampled epipelagic waters (0- to 100-m depth), with clear phylogenetic segregation along the transition from oxic to anoxic water masses. Thus, whereas in the anoxic hypolimnion crenarchaeotal OTUs were mainly assigned to the miscellaneous crenarchaeotic group, the OTUs from the oxic-anoxic transition and above belonged to
Crenarchaeota
groups 1.1a and 1.1b, two lineages containing most of the ammonia-oxidizing representatives known so far. The concomitant vertical distribution of both nitrite and nitrate maxima and the copy numbers of both MCG1 16S rRNA and
amoA
genes suggest the potential implication of
Crenarchaeota
in nitrification processes occurring in the epilimnetic waters of the lake.
Extracts of Edible and Medicinal Plants Damage Membranes of
Vibrio cholerae
Authors: Sánchez Eduardo , García Santos , Heredia Norma
Published: August 2010

The use of natural compounds from plants can provide an alternative approach against food-borne pathogens. The mechanisms of action of most plant extracts with antimicrobial activity have been poorly studied. In this work, changes in membrane integrity, membrane potential, internal pH (pH
in
), and ATP synthesis were measured in
V
ibrio
cholerae
cells after exposure to extracts of edible and medicinal plants. A preliminary screen of methanolic, ethanolic, and aqueous extracts of medicinal and edible plants was performed. Minimal bactericidal concentrations (MBCs) were measured for extracts showing high antimicrobial activity. Our results indicate that methanolic extracts of basil (
Ocimum basilicum
L.), nopal cactus (
Opuntia ficus-indica
var. Villanueva L.), sweet acacia (
Acacia farnesiana
L.), and white sagebrush (
Artemisia ludoviciana
Nutt.) are the most active against
V. cholera
, with MBCs ranging from 0.5 to 3.0 mg/ml. Using four fluorogenic techniques, we studied the membrane integrity of
V. cholerae
cells after exposure to these four extracts. Extracts from these plants were able to disrupt the cell membranes of
V. cholerae
cells, causing increased membrane permeability, a clear decrease in cytoplasmic pH, cell membrane hyperpolarization, and a decrease in cellular ATP concentration in all strains tested. These four plant extracts could be studied as future alternatives to control
V. cholerae
contamination in foods and the diseases associated with this microorganism.
A Novel Gene,ROA, Is Required for Normal Morphogenesis and Discharge of Ascospores in Gibberella zeae
Authors: Min Kyunghun , Lee Jungkwan , Kim Jin-Cheol , Kim Sang Gyu , Kim Young Ho , Vogel Steven , Trail Frances , Lee Yin-Won
Published: August 2010

Head blight, caused byGibberella zeae, is a significant disease among cereal crops, including wheat, barley, and rice, due to contamination of grain with mycotoxins.G. zeaeis spread by ascospores forcibly discharged from sexual fruiting bodies forming on crop residues. In this study, we characterized a novel gene,ROA, which is required for normal sexual development. Deletion ofROA(Δroa) resulted in an abnormal size and shape of asci and ascospores but did not affect vegetative growth. The Δroamutation triggered round ascospores and insufficient cell division after spore delimitation. The asci of the Δroastrain discharged fewer ascospores from the perithecia but achieved a greater dispersal distance than those of the wild-type strain. Turgor pressure within the asci was calculated through the analysis of osmolytes in the epiplasmic fluid. Deletion of theROAgene appeared to increase turgor pressure in the mutant asci. The higher turgor pressure of the Δroamutant asci and the mutant spore shape contributed to the longer distance dispersal. When the Δroamutant was outcrossed with a Δmat1-2mutant, a strain that contains a green fluorescence protein (GFP) marker in place of theMAT1-2gene, unusual phenotypic segregation occurred. The ratio of GFP to non-GFP segregation was 1:1; however, all eight spores had the same shape. Taken together, the results of this study suggest thatROAplays multiple roles in maintaining the proper morphology and discharge of ascospores inG. zeae.
Comparative Analysis of the Biochemical and Functional Properties of C-Terminal Domains of Autotransporters
Authors: Marín Elvira , Bodelón Gustavo , Fernández Luis Ángel
Published: August 2010

Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-terminal domain that is inserted in the outer membrane (OM) and is essential for the translocation of an N-terminal passenger domain to the extracellular milieu. Several mechanisms have been proposed for AT secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the β-barrel or by the oligomerization of C-terminal domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam complex. In this work we have investigated AT secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-terminal domains selected from ATs of gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria. Our results indicate that C-terminal domains having an N-terminal α-helix and a β-barrel constitute functional transport units for the translocation of peptides and immunoglobulin domains with disulfide bonds.In vivoandin vitroanalyses show that multimerization is not a conserved feature in AT C-terminal domains. Furthermore, we demonstrate that the deletion of the conserved α-helix severely impairs β-barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that the AT β-barrel without its α-helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for an understanding of AT secretion are discussed.
Mechanism of Positive Regulation by DsrA and RprA Small Noncoding RNAs: Pairing Increases Translation and ProtectsrpoSmRNA from Degradation
Authors: McCullen Colleen A. , Benhammou Jihane N. , Majdalani Nadim , Gottesman Susan
Published: August 2010

Small noncoding RNAs (sRNAs) regulate gene expression inEscherichia coliby base pairing with mRNAs and modulating translation and mRNA stability. The sRNAs DsrA and RprA stimulate the translation of the stress response transcription factor RpoS by base pairing with the 5′ untranslated region of therpoSmRNA. In the present study, we found that therpoSmRNA was unstable in the absence of DsrA and RprA and that expression of these sRNAs increased both the accumulation and the half-life of therpoSmRNA. Mutations indsrA,rprA, orrpoSthat disrupt the predicted pairing sequences and reduce translation of RpoS also destabilize therpoSmRNA. We found that therpoSmRNA accumulates in an RNase E mutant strain in the absence of sRNA expression and, therefore, is degraded by an RNase E-mediated mechanism. DsrA expression is required, however, for maximal translation even whenrpoSmRNA is abundant. This suggests that DsrA protectsrpoSmRNA from degradation by RNase E and that DsrA has a further activity in stimulating RpoS protein synthesis.rpoSmRNA is subject to degradation by an additional pathway, mediated by RNase III, which, in contrast to the RNase E-mediated pathway, occurs in the presence and absence of DsrA or RprA.rpoSmRNA and RpoS protein levels are increased in an RNase III mutant strain with or without the sRNAs, suggesting that the role of RNase III in this context is to reduce the translation of RpoS even when the sRNAs are acting to stimulate translation.
Biogenesis of
Salmonella enterica
Serovar Typhimurium Membrane Vesicles Provoked by Induction of PagC
Authors: Kitagawa Ryo , Takaya Akiko , Ohya Mai , Mizunoe Yoshimitsu , Takade Akemi , Yoshida Shin-ichi , Isogai Emiko , Yamamoto Tomoko
Published: August 2010

Gram-negative bacteria ubiquitously release membrane vesicles (MVs) into the extracellular milieu. Although MVs are the product of growing bacteria, not of cell lysis or death, the regulatory mechanisms underlying MV formation remained unknown. We have found that MV biogenesis is provoked by the induction of PagC, a
Salmonella
-specific protein whose expression is activated by conditions that mimic acidified macrophage phagosomes. PagC is a major constituent of
Salmonella
MVs, and increased expression accelerates vesiculation. Expression of PagC is regulated at the posttranscriptional and/or posttranslational level in a sigmaS (RpoS)-dependent manner. Serial quantitative analysis has demonstrated that MV formation can accelerate when the quantity of the MV constituents, OmpX and PagC, rises. Overproduction of PagC dramatically impacts the difference in the relative amount of vesiculation, but the corresponding overproduction of OmpX was less pronounced. Quantitative examination of the ratios of PagC and OmpX in the periplasm, outer membrane, and MVs demonstrates that PagC is preferentially enriched in MVs released from
Salmonella
cells. This suggests that specific protein sorting mechanisms operate when MVs are formed. The possible role(s) of PagC-MV in host cells is discussed.
A Novel Six-Rhodopsin System in a Single Archaeon
Authors: Fu Hsu-Yuan , Lin Yu-Cheng , Chang Yung-Ning , Tseng Hsiaochu , Huang Ching-Che , Liu Kang-Cheng , Huang Ching-Shin , Su Che-Wei , Weng Rueyhung Roc , Lee Yin-Yu , Ng Wailap Victor , Yang Chii-Shen
Published: August 2010

Microbial rhodopsins, a diverse group of photoactive proteins found in
Archaea
,
Bacteria
, and
Eukarya
, function in photosensing and photoenergy harvesting and may have been present in the resource-limited early global environment. Four different physiological functions have been identified and characterized for nearly 5,000 retinal-binding photoreceptors, these being ion transporters that transport proton or chloride and sensory rhodopsins that mediate light-attractant and/or -repellent responses. The greatest number of rhodopsins previously observed in a single archaeon had been four. Here, we report a newly discovered six-rhodopsin system in a single archaeon,
Haloarcula marismortui
, which shows a more diverse absorbance spectral distribution than any previously known rhodopsin system, and, for the first time, two light-driven proton transporters that respond to the same wavelength. All six rhodopsins, the greatest number ever identified in a single archaeon, were first shown to be expressed in
H. marismortui
, and these were then overexpressed in
Escherichia coli
. The proteins were purified for absorption spectra and photocycle determination, followed by measurement of ion transportation and phototaxis. The results clearly indicate the existence of a proton transporter system with two isochromatic rhodopsins and a new type of sensory rhodopsin-like transducer in
H. marismortui
.
The Elemental Sulfur-Responsive Protein (SipA) from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Is Regulated by Sulfide in an Iron-Dependent Manner
Authors: Clarkson Sonya M. , Newcomer Elizabeth C. , Young Everett G. , Adams Michael W. W.
Published: August 2010

The gene (
sipA
) encoding the sulfur-induced protein A (PF2025) is highly upregulated during growth of
Pyrococcus furiosus
on elemental sulfur (S
0
). Expression of
sipA
is regulated by sulfide, the product of S
0
reduction, but in an iron-dependent manner. SipA is proposed to play a role in intracellular iron sulfide detoxification.
AglJ Adds the First Sugar of the N-Linked Pentasaccharide Decorating the
Haloferax volcanii
S-Layer Glycoprotein
Authors: Kaminski Lina , Abu-Qarn Mehtap , Guan Ziqiang , Naparstek Shai , Ventura Valeria V. , Raetz Christian R. H. , Hitchen Paul G. , Dell Anne , Eichler Jerry
Published: August 2010

Like the
Eukarya
and
Bacteria
, the
Archaea
also perform N glycosylation. Using the haloarchaeon
Haloferax volcanii
as a model system, a series of Agl proteins involved in the archaeal version of this posttranslational modification has been identified. In the present study, the participation of HVO_1517 in N glycosylation was considered, given its homology to a known component of the eukaryal N-glycosylation pathway and because of the genomic proximity of
HVO
_
1517
to
agl
genes encoding known elements of the
H. volcanii
N-glycosylation process. By combining the deletion of
HVO
_
1517
with mass spectrometric analysis of both dolichol phosphate monosaccharide-charged carriers and the S-layer glycoprotein, evidence was obtained showing the participation of HVO_1517, renamed AglJ, in adding the first hexose of the N-linked pentasaccharide decorating this reporter glycoprotein. The deletion of
aglJ
, however, did not fully prevent the attachment of a hexose residue to the S-layer glycoprotein. Moreover, in the absence of AglJ, the level of only one of the three monosaccharide-charged dolichol phosphate carriers detected in the cell was reduced. Nonetheless, in cells lacking AglJ, no further sugar subunits were added to the remaining monosaccharide-charged dolichol phosphate carriers or to the monosaccharide-modified S-layer glycoprotein, pointing to the importance of the sugar added through the actions of AglJ for proper N glycosylation. Finally, while
aglJ
can be deleted,
H. volcanii
surface layer integrity is compromised in the absence of the encoded protein.
A Commensal Gone Bad: Complete Genome Sequence of the Prototypical Enterotoxigenic
Escherichia coli
Strain H10407
Authors: Crossman Lisa C. , Chaudhuri Roy R. , Beatson Scott A. , Wells Timothy J. , Desvaux Mickael , Cunningham Adam F. , Petty Nicola K. , Mahon Vivienne , Brinkley Carl , Hobman Jon L. , Savarino Stephen J. , Turner Susan M. , Pallen Mark J. , Penn Charles W. , Parkhill Julian , Turner A. Keith , Johnson Timothy J. , Thomson Nicholas R. , Smith Stephen G. J. , Henderson Ian R.
Published: August 2010

In most cases,
Escherichia coli
exists as a harmless commensal organism, but it may on occasion cause intestinal and/or extraintestinal disease. Enterotoxigenic
E. coli
(ETEC) is the predominant cause of
E. coli
-mediated diarrhea in the developing world and is responsible for a significant portion of pediatric deaths. In this study, we determined the complete genomic sequence of
E. coli
H10407, a prototypical strain of enterotoxigenic
E. coli
, which reproducibly elicits diarrhea in human volunteer studies. We performed genomic and phylogenetic comparisons with other
E. coli
strains, revealing that the chromosome is closely related to that of the nonpathogenic commensal strain
E. coli
HS and to those of the laboratory strains
E. coli
K-12 and C. Furthermore, these analyses demonstrated that there were no chromosomally encoded factors unique to any sequenced ETEC strains. Comparison of the
E. coli
H10407 plasmids with those from several ETEC strains revealed that the plasmids had a mosaic structure but that several loci were conserved among ETEC strains. This study provides a genetic context for the vast amount of experimental and epidemiological data that have been published.
Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in
Micrococcus luteus
: Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C
50
Carotenoid Cyclases
Authors: Netzer Roman , Stafsnes Marit H. , Andreassen Trygve , Goksøyr Audun , Bruheim Per , Brautaset Trygve
Published: August 2010

We report the cloning and characterization of the biosynthetic gene cluster (
crtE
,
crtB
,
crtI
,
crtE2
,
crtYg
,
crtYh
, and
crtX
) of the γ-cyclic C
50
carotenoid sarcinaxanthin in
Micrococcus luteus
NCTC2665. Expression of the complete and partial gene cluster in
Escherichia coli
hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C
40
lycopene, C
45
nonaflavuxanthin, C
50
flavuxanthin, and C
50
sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the
crtX
gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C
50
carotenoid. Expression of the corresponding genes from the marine
M. luteus
isolate Otnes7 in a lycopene-producing
E. coli
host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ε-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C
50
carotenoid cyclase genes
crtYg
and
crtYh
from
M. luteus
replaced with the analogous ε-cyclic C
50
carotenoid cyclase genes
crtYe
and
crtYf
from the natural decaprenoxanthin producer
Corynebacterium glutamicum
. Surprisingly, expression of this hybrid gene cluster in an
E. coli
host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C
50
carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C
50
carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.
Radiation Desiccation Response Motif-Like Sequences Are Involved in Transcriptional Activation of the Deinococcal
ssb
Gene by Ionizing Radiation but Not by Desiccation
Authors: Ujaoney Aman Kumar , Potnis Akhilesh A. , Kane Pratiksha , Mukhopadhyaya Rita , Apte Shree Kumar
Published: August 2010

Single-stranded-DNA binding protein (SSB) levels during poststress recovery of
Deinococcus radiodurans
were significantly enhanced by
60
Co gamma rays or mitomycin C treatment but not by exposure to UV rays, hydrogen peroxide (H
2
O
2
), or desiccation. Addition of rifampin prior to postirradiation recovery blocked such induction.
In silico
analysis of the
ssb
promoter region revealed a 17-bp palindromic radiation/desiccation response motif (RDRM1) at bp −114 to −98 and a somewhat similar sequence (RDRM2) at bp −213 to −197, upstream of the
ssb
open reading frame. Involvement of these
cis
elements in radiation-responsive
ssb
gene expression was assessed by constructing transcriptional fusions of edited versions of the
ssb
promoter region with a nonspecific acid phosphatase encoding reporter gene,
phoN
. Recombinant
D. radiodurans
strains carrying such constructs clearly revealed (i) transcriptional induction of the
ssb
promoter upon irradiation and mitomycin C treatment but not upon UV or H
2
O
2
treatment and (ii) involvement of both RDRM-like sequences in such activation of SSB expression, in an additive manner.
Draft Genome Sequences of
Actinobacillus pleuropneumoniae
Serotypes 2 and 6
Authors: Zhan Bujie , Angen Øystein , Hedegaard Jakob , Bendixen Christian , Panitz Frank
Published: August 2010

Actinobacillus pleuropneumoniae
is a bacterial pathogen that causes highly contagious respiratory infection in pigs and has a serious impact on the production economy and animal welfare. As clear differences in virulence between serotypes have been observed, the genetic basis should be investigated at the genomic level. Here, we present the draft genome sequences of the
A. pleuropneumoniae
serotypes 2 (strain 4226) and 6 (strain Femo).
Complete Genome Sequence of
Staphylococcus aureus
Strain JKD6008, an ST239 Clone of Methicillin-Resistant
Staphylococcus aureus
with Intermediate-Level Vancomycin Resistance
Authors: Howden Benjamin P. , Seemann Torsten , Harrison Paul F. , McEvoy Chris R. , Stanton Jo-Ann L. , Rand Christy J. , Mason Chris W. , Jensen Slade O. , Firth Neville , Davies John K. , Johnson Paul D. R. , Stinear Timothy P.
Published: August 2010

We report here the complete 2.92-Mb genome sequence of a clinical isolate of methicillin-resistant
Staphylococcus aureus
subsp.
aureus
that demonstrates intermediate-level vancomycin resistance. The strain, named JKD6008, belongs to multilocus sequence type 239 and was isolated from the bloodstream of a patient in New Zealand in 2003.