bluuebunny/crossref_metadata_2025 huggingface dataset:
Testing the potential of proposed DNA barcodes for species identification of Zingiberaceae
Authors: SHI Lin‐Chun , ZHANG Jin , HAN Jian‐Ping , SONG Jing‐Yuan , YAO Hui , ZHU Ying‐Jie , LI Jia‐Chun , WANG Zhen‐Zhong , XIAO Wei , LIN Yu‐Lin , XIE Cai‐Xiang , QIAN Zhong‐Zhi , CHEN Shi‐Lin
Published: March 2011

Abstract  In 2009, the Consortium for the Barcode of Life (CBOL) recommended the combination of rbcL and matK as the plant barcode based on assessments of recoverability, sequencing quality, and levels of species discrimination. Subsequently, based on a study of more than 6600 samples belonging to 193 families from seven phyla, the internal transcribed spacer (ITS) 2 locus was proposed as a universal barcode sequence for all major plant taxa used in traditional herbal medicine. Neither of these two studies was based on a detailed analysis of a particular family. Here, Zingiberaceae plants, including many closely related species, were used to compare the genetic divergence and species identification efficiency of ITS2, rbcL, matK, psbK–psbI, trnH–psbA, and rpoB. The results indicate that ITS2 has the highest interspecific divergence and significant differences between inter‐ and intraspecific divergence, whereas matK and rbcL have much lower divergence values. Among 260 species belonging to 30 genera in Zingiberaceae, the discrimination ability of the ITS2 locus was 99.5% at the genus level and 73.1% at the species level. Thus, we propose that ITS2 is the preferred DNA barcode sequence for identifying Zingiberaceae plants.
New universalmatKprimers for DNA barcoding angiosperms
Authors: YU Jing , XUE Jian‐Hua , ZHOU Shi‐Liang
Published: March 2011

Abstract The chloroplast maturase K gene (matK) is one of the most variable coding genes of angiosperms and has been suggested to be a “barcode” for land plants. However,matKexhibits low amplification and sequencing rates due to low universality of currently available primers and mononucleotide repeats. To resolve these technical problems, we evaluated the entirematKregion to find a region of 600–800 bp that is highly variable, represents the best of allmatKregions with priming sites conservative enough to design universal primers, and avoids the mononucleotide repeats. After careful evaluation, a region in the middle was chosen and a pair of primers namedmatK472F andmatK1248R was designed to amplify and sequence thematKfragment of approximately 776 bp. This region encompasses the most variable sites, represents the entirematKregion best, and also exhibits high amplification rates and quality of sequences. The universality of this primer pair was tested using 58 species from 47 families of angiosperm plants. The primers showed a strong amplification (93.1%) and sequencing (92.6%) successes in the species tested. We propose that the new primers will solve, in part, the problems encountered when usingmatKand promote the adoption ofmatKas a DNA barcode for angiosperms.
Long‐term outcome and hepatocellular carcinoma development in chronic hepatitis B or cirrhosis patients after nucleoside analog treatment with entecavir or lamivudine
Authors: Kobashi Haruhiko , Miyake Yasuhiro , Ikeda Fusao , Yasunaka Tetsuya , Nishino Ken , Moriya Akio , Kubota Jyunichi , Nakamura Shinichiro , Takaki Akinobu , Nouso Kazuhiro , Yamada Gotaro , Yamamoto Kazuhide
Published: March 2011

Aim:  We conducted this prospective study to elucidate the long‐term outcome and incidence of hepatocellular carcinoma (HCC) development after nucleos(t)ide analog (NA) treatment in patients with chronic hepatitis B (CHB) or cirrhosis.

Methods:  CHB or cirrhosis patients without past NA treatment or HCC were started on entecavir (ETV) or lamivudine (LVD), and prospectively followed up with monthly blood tests, and with abdominal imaging every 6 months in CHB and every 3 months in cirrhosis patients.

Results:  A total of 256 subjects with CHB (n = 194) or cirrhosis (n = 62) received ETV (n = 129) or LVD (n = 127) for 4.25 years (range: 0.41–10.0). After NA treatment, serum HBV DNA, alanine aminotransferase and α‐fetoprotein (AFP) dropped significantly, along with significant increases in serum albumin and prothrombin time. Drug‐resistance developed in 60 cases in the LVD group and in only one case in the ETV group. HCC developed in 35 patients, and the incidence at years 1, 3, 5, 7 and 10 was significantly higher in patients with cirrhosis (8.1%, 17.5%, 43.2%, 46.7% and 53.4%, respectively) than chronic hepatitis (1.6%, 3.5%, 3.5%, 7.1% and 29.6%, respectively), with no difference between ETV and LVD. After NA treatment, the sensitivity/specificity for HCC of AFP and des‐γ‐carboxy prothrombin (DCP) was 45.7%/97.3% and 33.3%/96.2%, respectively, with the specificity of AFP being higher than at baseline (64.4%), at the cut‐off of 10 ng/mL.

Conclusion:  NA exerted a long‐term efficacy and improved hepatic reservation in CHB and cirrhosis. After NA treatment, AFP dropped to lower than 10 ng/mL with marked elevation of specificity, leading to an earlier detection of HCC.
Osteopontin plays an anti‐nucleation role in cholesterol gallstone formation
Authors: Yang Lin , Chen Jin‐Hong , Cai Duan , Wang Li‐Ying , Zha Xi‐Liang
Published: March 2011

Aim:  To investigate the role of osteopontin in cholesterol gallstone formation.

Methods:  Nucleation time was determined in model and human gallbladder bile in vitro. Effect of osteopontin on vesicles of bile was investigated via transmission electron microscopy. The mRNA and protein expression of osteopontin were detected in human calculus and normal gallbladder tissues, and then lipid compositions of human bile were determined via commercial kits.

Results:  Osteopontin could prolong the nucleation time in a dose‐dependent manner, and inhibit the pro‐nucleation effect induced by calcium ion. Cholesterol crystal growth was inhibited by osteopontin in a dose‐dependent manner in model and human gallbladder bile, but not affected by calcium. Furthermore, the formation, aggregation and fusion of vesicles were suppressed by osteopontin in model and human bile as demonstrated by transmission electron microscopy. The mRNA and protein expression of osteopontin in calculus gallbladder tissues were lower than those in normal tissues. The concentrations of cholesterol, phospholipid and bile acids, and cholesterol saturated index were higher and the contents of osteopontin and calcium, nevertheless, were found to be lower in lithogenic bile than those in normal controls.

Conclusion:  These findings indicated that osteopontin could inhibit the cholesterol gallstone formation as an anti‐nucleation factor, which may be involved in the pathogenesis of cholesterol gallstone formation.
Holter Monitoring in Clinically Healthy Cavalier King Charles Spaniels, Wire‐Haired Dachshunds, and Cairn Terriers
Authors: Rasmussen C.E. , Vesterholm S. , Ludvigsen T.P. , Häggström J. , Pedersen H.D. , Moesgaard S.G. , Olsen L.H.
Published: March 2011

Background:Few reported studies describe normal values from 24‐hour ECG (Holter) recordings of small breed dogs.

Objectives:To investigate influence of breed, age, sex, body weight, degree of recording artifact, and mitral valve prolapse (MVP) on Holter recordings of 3 breeds of small dogs that have differing predispositions for myxomatous mitral valve disease. The study also assessed if heart rate (HR) at clinical examination (HRex) was associated with HR during Holter monitoring and evaluated the reproducibility of Holter variables.

Animals:Fifty clinically healthy, privately owned dogs of the breeds Cavalier King Charles Spaniel (CKCS), Wire‐haired Dachshund (wD), or Cairn Terrier (CT).

Methods:Prospective, longitudinal observational study. Dogs were recruited for clinical examination, echocardiography, and Holter monitoring. In 8 CKCS, Holter recordings were performed twice with a 7‐day interval. Arrhythmia and heart rate variability (HRV) analysis (time and frequency domain analysis) were performed on Holter recordings.

Results:Fifteen out of 27 Holter derived variables were significantly associated with breed (P< .03), but not with age (P> .7), sex (P> .2), body weight (P> .7), degree of recording artifact (P> .4), or MVP (P> .6). During Holter recording, minimum (P= .0001) and mean HR (P= .0001) were higher in CKCS compared with wD. CKCS had significantly lower values than wD, CT, or both in 10 out of 13 HRV variables (P< .03). Minimum and mean HR during Holter recording were correlated with HRex (r= 0.55,P= .0003). HR and time domain variables had a coefficient of variation <10%.

Conclusions and Clinical Importance:There is an influence of breed on Holter‐derived variables in 3 breeds of small dogs. Arrhythmia and HRV analysis can be performed on 24‐hour ambulatory ECG (Holter) recordings. Arrhythmia analysis includes HR measurements and identification of arrhythmias.
Novel Model for Multispecies Biofilms That Uses Rigid Gas-Permeable Lenses
Authors: Peyyala Rebecca , Kirakodu Sreenatha S. , Ebersole Jeffrey L. , Novak Karen F.
Published: March 2011

Oral biofilms comprise complex multispecies consortia aided by specific inter- and intraspecies interactions occurring among commensals and pathogenic bacterial species. Oral biofilms are primary initiating factors of periodontal disease, although complex multifactorial biological influences, including host cell responses, contribute to the individual outcome of the disease. To provide a system to study initial stages of interaction between oral biofilms and the host cells that contribute to the disease process, we developed a novel
in vitro
model system to grow biofilms on rigid gas-permeable contact lenses (RGPLs), which enable oxygen to permeate through the lens material. Bacterial species belonging to early- and late-colonizing groups were successfully established as single- or three-species biofilms, with each group comprising
Streptococcus gordonii
,
Streptococcus oralis
, and
Streptococcus sanguinis
;
S. gordonii
,
Actinomyces naeslundii
, and
Fusobacterium nucleatum
; or
S. gordonii
,
F. nucleatum
, and
Porphyromonas gingivalis
. Quantification of biofilm numbers by quantitative PCR (qPCR) revealed substantial differences in the magnitude of bacterial numbers in single-species and multispecies biofilms. We evaluated cell-permeable conventional nucleic acid stains acridine orange, hexidium iodide, and Hoechst 33258 and novel SYTO red, blue, and green fluorochromes for their effect on bacterial viability and fluorescence yield to allow visualization of the aggregates of individual bacterial species by confocal laser scanning microscopy (CLSM). Substantial differences in the quantity and distribution of the species in the multispecies biofilms were identified. The specific features of these biofilms may help us better understand the role of various bacteria in local challenge of oral tissues.
Protection of Arabidopsis thaliana against Leaf-Pathogenic Pseudomonas syringae by Sphingomonas Strains in a Controlled Model System
Authors: Innerebner Gerd , Knief Claudia , Vorholt Julia A.
Published: March 2011

Diverse bacterial taxa live in association with plants without causing deleterious effects. Previous analyses of phyllosphere communities revealed the predominance of few bacterial genera on healthy dicotyl plants, provoking the question of whether these commensals play a particular role in plant protection. Here, we tested two of them,
Methylobacterium
and
Sphingomonas
, with respect to their ability to diminish disease symptom formation and the proliferation of the foliar plant pathogen
Pseudomonas syringae
pv. tomato DC3000 on
Arabidopsis thaliana
. Plants were grown under gnotobiotic conditions in the absence or presence of the potential antagonists and then challenged with the pathogen. No effect of
Methylobacterium
strains on disease development was observed. However, members of the genus
Sphingomonas
showed a striking plant-protective effect by suppressing disease symptoms and diminishing pathogen growth. A survey of different
Sphingomonas
strains revealed that most plant isolates protected
A. thaliana
plants from developing severe disease symptoms. This was not true for
Sphingomonas
strains isolated from air, dust, or water, even when they reached cell densities in the phyllosphere comparable to those of the plant isolates. This suggests that plant protection is common among plant-colonizing
Sphingomonas
spp. but is not a general trait conserved within the genus
Sphingomonas
. The carbon source profiling of representative isolates revealed differences between protecting and nonprotecting strains, suggesting that substrate competition plays a role in plant protection by
Sphingomonas
. However, other mechanisms cannot be excluded at this time. In conclusion, the ability to protect plants as shown here in a model system may be an unexplored, common trait of indigenous
Sphingomonas
spp. and may be of relevance under natural conditions.
Production of Hydrogen from α-1,4- and β-1,4-Linked Saccharides by Marine Hyperthermophilic Archaea
Authors: Oslowski Daniel M. , Jung Jong-Hyun , Seo Dong-Ho , Park Cheon-Seok , Holden James F.
Published: March 2011

Nineteen hyperthermophilic heterotrophs from deep-sea hydrothermal vents, plus the control organism
Pyrococcus furiosus
, were examined for their ability to grow and produce H
2
on maltose, cellobiose, and peptides and for the presence of the genes encoding proteins that hydrolyze starch and cellulose. All of the strains grew on these disaccharides and peptides and converted maltose and peptides to H
2
even when elemental sulfur was present as a terminal electron acceptor. Half of the strains had at least one gene for an extracellular starch hydrolase, but only
P. furiosus
had a gene for an extracellular β-1,4-endoglucanase.
P. furiosus
was serially adapted for growth on CF11 cellulose and H
2
production, which is the first reported instance of hyperthermophilic growth on cellulose, with a doubling time of 64 min. Cell-specific H
2
production rates were 29 fmol, 37 fmol, and 54 fmol of H
2
produced cell
−1
doubling
−1
on α-1,4-linked sugars, β-1,4-linked sugars, and peptides, respectively. The highest total community H
2
production rate came from growth on starch (2.6 mM H
2
produced h
−1
). Hyperthermophilic heterotrophs may serve as an important alternate source of H
2
for hydrogenotrophic microorganisms in low-H
2
hydrothermal environments, and some are candidates for H
2
bioenergy production in bioreactors.
Link between Genotype and Antimicrobial Resistance in Bovine Mastitis-Related Staphylococcus aureus Strains, Determined by Comparing Swiss and French Isolates from the Rhône Valley
Authors: Sakwinska Olga , Morisset Delphine , Madec Jean-Yves , Waldvogel Andreas , Moreillon Philippe , Haenni Marisa
Published: March 2011

Staphylococcus aureus
is a major bovine mastitis pathogen. Although the reported antimicrobial resistance was generally low, the emergence of new genetic clusters in bovine mastitis requires examination of the link between antimicrobial resistance and genotypes. Here, amplified fragment length polymorphism (AFLP) profiles and standard antimicrobial resistance profiles were determined in order to characterize a total of 343
S. aureus
cow mastitis isolates from two geographically close regions of Switzerland and France. AFLP profiles revealed similar population compositions in the two regions, with 4 major clusters (C8, C20, C97, and C151), but the proportions of isolates in each cluster significantly diverged between the two countries (
P
= 9.2 × 10
−9
). Antimicrobial resistance was overall low (<5% resistance to all therapeutically relevant molecules), with the exception of penicillin resistance, which was detected in 26% of the isolates. Penicillin resistance proportions differed between clusters, with only 1 to 2% of resistance associated with C20 and C151 and up to 70% associated with bovine C97. The prevalence of C20 and C8 was unexpectedly high and requires further investigation into the mechanism of adaptation to the bovine host. The strong association of penicillin resistance with few clusters highlights the fact that the knowledge of local epidemiology is essential for rational choices of antimicrobial treatment in the absence of susceptibility testing. Taken together, these observations argue in favor of more routine scrutiny of antimicrobial resistance and antibiotic-resistant clones in cattle and the farm environment.
Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris
Authors: To Thi Mai Huong , Grandvalet Cosette , Tourdot-Maréchal Raphaëlle
Published: March 2011

Cyclopropane fatty acids (CFAs) are synthetized
in situ
by the transfer of a methylene group from
S
-adenosyl-
l
-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium
Lactococcus lactis
MG1363. A mutant of the
cfa
gene was constructed by allelic exchange. The
cfa
gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the
cfa
gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for
L. lactis
subsp.
cremoris
survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells.
Insights into Physiological and Genetic Mupirocin Susceptibility in Bifidobacteria
Authors: Serafini Fausta , Bottacini Francesca , Viappiani Alice , Baruffini Enrico , Turroni Francesca , Foroni Elena , Lodi Tiziana , van Sinderen Douwe , Ventura Marco
Published: March 2011

Mupirocin is an antibiotic commonly used in selective media for the isolation of bifidobacteria. However, little is known about the genetic traits responsible for bifidobacterial resistance to mupirocin. Our investigation demonstrates that all of the bifidobacteria tested exhibit a phenotype of generally high resistance to this antibiotic. The genotypic reason for bifidobacterial mupirocin resistance was further characterized by sequencing of the isoleucyl-tRNA synthetase gene (
ileS
) coupled with three-dimensional modeling of the encoded protein and cloning of the
ileS
gene of
Bifidobacterium bifidum
PRL2010 in a mupirocin-sensitive
Escherichia coli
strain. These analyses revealed key amino acid residues of the IleS protein that apparently are crucial for conferring a mupirocin resistance phenotype to bifidobacteria.
In Silico
Evidence for the Horizontal Transfer of
gsiB
, a σ
Β
-Regulated Gene in Gram-Positive Bacteria, to Lactic Acid Bacteria
Authors: Asteri Ioanna-Areti , Boutou Effrossyni , Anastasiou Rania , Pot Bruno , Vorgias Constantinos E. , Tsakalidou Effie , Papadimitriou Konstantinos
Published: March 2011

gsiB
, coding for glucose starvation-inducible protein B, is a characteristic member of the σ
Β
stress regulon of
Bacillus subtilis
and several other Gram-positive bacteria. Here we provide
in silico
evidence for the horizontal transfer of
gsiB
in lactic acid bacteria that are devoid of the σ
Β
factor.
Genotypic-Phenotypic Discrepancies between Antibiotic Resistance Characteristics of Escherichia coli Isolates from Calves in Management Settings with High and Low Antibiotic Use
Authors: Davis Margaret A. , Besser Thomas E. , Orfe Lisa H. , Baker Katherine N. K. , Lanier Amelia S. , Broschat Shira L. , New Daniel , Call Douglas R.
Published: March 2011

We hypothesized that bacterial populations growing in the absence of antibiotics will accumulate more resistance gene mutations than bacterial populations growing in the presence of antibiotics. If this is so, the prevalence of dysfunctional resistance genes (resistance pseudogenes) could provide a measure of the level of antibiotic exposure present in a given environment. As a proof-of-concept test, we assayed field strains of
Escherichia coli
for their resistance genotypes using a resistance gene microarray and further characterized isolates that had resistance phenotype-genotype discrepancies. We found a small but significant association between the prevalence of isolates with resistance pseudogenes and the lower antibiotic use environment of a beef cow-calf operation versus a higher antibiotic use dairy calf ranch (Fisher's exact test,
P
= 0.044). Other significant findings include a very strong association between the dairy calf ranch isolates and phenotypes unexplained by well-known resistance genes (Fisher's exact test,
P
< 0.0001). Two novel resistance genes were discovered in
E. coli
isolates from the dairy calf ranch, one associated with resistance to aminoglycosides and one associated with resistance to trimethoprim. In addition, isolates resistant to expanded-spectrum cephalosporins but negative for
bla
CMY-2
had mutations in the promoter regions of the chromosomal
E. coli
ampC
gene consistent with reported overexpression of native AmpC beta-lactamase. Similar mutations in hospital
E. coli
isolates have been reported worldwide. Prevalence or rates of
E. coli
ampC
promoter mutations may be used as a marker for high expanded-spectrum cephalosporin use environments.
Mechanisms of Resistance to Bacteriocins Targeting the Mannose Phosphotransferase System
Authors: Kjos Morten , Nes Ingolf F. , Diep Dzung B.
Published: March 2011

The membrane proteins IIC and IID of the mannose phosphotransferase system (Man-PTS) together form a membrane-located complex that serves as a receptor for several different bacteriocins, including the pediocin-like class IIa bacteriocins and the class IIc bacteriocin lactococcin A. Bacterial strains sensitive to class IIa bacteriocins readily give rise to resistant mutants upon bacteriocin exposure. In the present study, we have therefore investigated lactococcin A-resistant mutants of
Lactococcus lactis
as well as natural food isolates of
Listeria monocytogenes
with different susceptibilities to class IIa bacteriocins. We found two major mechanisms of resistance. The first involves downregulation of Man-PTS gene expression, which takes place both in spontaneous resistant mutants and in natural resistant isolates. The second involves normal expression of the Man-PTS system, but the underlying mechanism of resistance for these cells is unknown. In some cases, the resistant phenotype was linked to a shift in the metabolism; i.e., reduced growth on glucose due to reduction in Man-PTS expression was accompanied by enhanced growth on another sugar, such as galactose. The implications of these findings in terms of metabolic heterogeneity are discussed.
Bacterial Hand Contamination and Transfer after Use of Contaminated Bulk-Soap-Refillable Dispensers
Authors: Zapka Carrie A. , Campbell Esther J. , Maxwell Sheri L. , Gerba Charles P. , Dolan Michael J. , Arbogast James W. , Macinga David R.
Published: March 2011

Bulk-soap-refillable dispensers are prone to extrinsic bacterial contamination, and recent studies demonstrated that approximately one in four dispensers in public restrooms are contaminated. The purpose of this study was to quantify bacterial hand contamination and transfer after use of contaminated soap under controlled laboratory and in-use conditions in a community setting. Under laboratory conditions using liquid soap experimentally contaminated with 7.51 log
10
CFU/ml of
Serratia marcescens
, an average of 5.28 log
10
CFU remained on each hand after washing, and 2.23 log
10
CFU was transferred to an agar surface. In an elementary-school-based field study, Gram-negative bacteria on the hands of students and staff increased by 1.42 log
10
CFU per hand (26-fold) after washing with soap from contaminated bulk-soap-refillable dispensers. In contrast, washing with soap from dispensers with sealed refills significantly reduced bacteria on hands by 0.30 log
10
CFU per hand (2-fold). Additionally, the mean number of Gram-negative bacteria transferred to surfaces after washing with soap from dispensers with sealed-soap refills (0.06 log
10
CFU) was significantly lower than the mean number after washing with contaminated bulk-soap-refillable dispensers (0.74 log
10
CFU;
P
< 0.01). Finally, significantly higher levels of Gram-negative bacteria were recovered from students (2.82 log
10
CFU per hand) than were recovered from staff (2.22 log
10
CFU per hand) after washing with contaminated bulk soap (
P
< 0.01). These results demonstrate that washing with contaminated soap from bulk-soap-refillable dispensers can increase the number of opportunistic pathogens on the hands and may play a role in the transmission of bacteria in public settings.
Investigation of Genotypes of Borrelia burgdorferi in Ixodes scapularis Ticks Collected during Surveillance in Canada
Authors: Ogden N. H. , Margos G. , Aanensen D. M. , Drebot M. A. , Feil E. J. , Hanincová K. , Schwartz I. , Tyler S. , Lindsay L. R.
Published: March 2011

The genetic diversity ofBorrelia burgdorferisensu stricto, the agent of Lyme disease in North America, has consequences for the performance of serological diagnostic tests and disease severity. To investigateB. burgdorferidiversity in Canada, where Lyme disease is emerging, bacterial DNA in 309 infected adultIxodes scapularisticks collected in surveillance was characterized by multilocus sequence typing (MLST) and analysis of outer surface protein C gene (ospC) alleles. Six ticks carriedBorrelia miyamotoi, and one tick carried the novel speciesBorrelia kurtenbachii. 142 ticks carriedB. burgdorferisequence types (STs) previously described from the United States. Fifty-eight ticks carriedB. burgdorferiof 1 of 19 novel or undescribed STs, which were single-, double-, or triple-locus variants of STs first described in the United States. Clonal complexes with founder STs from the United States were identified. SeventeenospCalleles were identified in 309B. burgdorferi-infected ticks. Positive and negative associations in the occurrence of different alleles in the same tick supported a hypothesis of multiple-niche polymorphism forB. burgdorferiin North America. Geographic analysis of STs andospCalleles were consistent with south-to-north dispersion of infected ticks from U.S. sources on migratory birds. These observations suggest that the genetic diversity ofB. burgdorferiin eastern and central Canada corresponds to that in the United States, but there was evidence for founder events skewing the diversity in emerging tick populations. Further studies are needed to investigate the significance of these observations for the performance of diagnostic tests and clinical presentation of Lyme disease in Canada.
Functional Survey for Heterologous Sugar Transport Proteins, Using Saccharomyces cerevisiae as a Host
Authors: Young Eric , Poucher Ashley , Comer Austin , Bailey Alexandra , Alper Hal
Published: March 2011

Molecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism in
Saccharomyces cerevisiae
. As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinant
Saccharomyces cerevisiae
host unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes of
Scheffersomyces stipitis
and open reading frames from the yeasts
Yarrowia lipolytica
and
Debaryomyces hansenii.
Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised of
D. hansenii
XylHP
and
S. stipitis
XUT1
and
XUT3
demonstrated moderate transport efficiency and higher xylose preferences.
Substrate Specificity of the Bacillus licheniformis Lyxose Isomerase YdaE and Its Application in
In Vitro
Catalysis for Bioproduction of Lyxose and Glucose by Two-Step Isomerization
Authors: Patel Darshan H. , Wi Seung Gon , Lee Seong-Gene , Lee Dae-Seok , Song Youn-ho , Bae Hyeun-Jong
Published: March 2011

Enzymatic processes are useful for industrially important sugar production, and
in vitro
two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by
ydaE
of
Bacillus licheniformis
was found to have broad substrate specificities and has shown high catalytic efficiency on
d
-lyxose, suggesting that the enzyme is
d
-lyxose isomerase.
Escherichia coli
BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn
2+
. The apparent

K
m

values for
d
-lyxose and
d
-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (
k
cat
/

K
m

) for lyxose (3.2 ± 0.1 mM
−1
s
−1
) was higher than that for
d
-mannose (1.6 mM
−1
s
−1
). The purified protein was applied to the bioproduction of
d
-lyxose and
d
-glucose from
d
-xylose and
d
-mannose, respectively, along with the thermostable xylose isomerase of
Thermus thermophilus
HB08. From an initial concentration of 10 mM
d
-lyxose and
d
-mannose, 3.7 mM and 3.8 mM
d
-lyxose and
d
-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for
in vitro
catalysis and can be applied to industrial production.
Assessing and Improving Methods Used in Operational Taxonomic Unit-Based Approaches for 16S rRNA Gene Sequence Analysis
Authors: Schloss Patrick D. , Westcott Sarah L.
Published: March 2011

In spite of technical advances that have provided increases in orders of magnitude in sequencing coverage, microbial ecologists still grapple with how to interpret the genetic diversity represented by the 16S rRNA gene. Two widely used approaches put sequences into bins based on either their similarity to reference sequences (i.e., phylotyping) or their similarity to other sequences in the community (i.e., operational taxonomic units [OTUs]). In the present study, we investigate three issues related to the interpretation and implementation of OTU-based methods. First, we confirm the conventional wisdom that it is impossible to create an accurate distance-based threshold for defining taxonomic levels and instead advocate for a consensus-based method of classifying OTUs. Second, using a taxonomic-independent approach, we show that the average neighbor clustering algorithm produces more robust OTUs than other hierarchical and heuristic clustering algorithms. Third, we demonstrate several steps to reduce the computational burden of forming OTUs without sacrificing the robustness of the OTU assignment. Finally, by blending these solutions, we propose a new heuristic that has a minimal effect on the robustness of OTUs and significantly reduces the necessary time and memory requirements. The ability to quickly and accurately assign sequences to OTUs and then obtain taxonomic information for those OTUs will greatly improve OTU-based analyses and overcome many of the challenges encountered with phylotype-based methods.
Genetic Screening Strategy for Rapid Access to Polyether Ionophore Producers and Products in Actinomycetes
Authors: Wang Hao , Liu Ning , Xi Lijun , Rong Xiaoying , Ruan Jisheng , Huang Ying
Published: March 2011

Polyether ionophores are a unique class of polyketides with broad-spectrum activity and outstanding potency for the control of drug-resistant bacteria and parasites, and they are produced exclusively by actinomycetes. A special epoxidase gene encoding a critical tailoring enzyme involved in the biosynthesis of these compounds has been found in all five of the complete gene clusters of polyether ionophores published so far. To detect potential producer strains of these antibiotics, a pair of degenerate primers was designed according to the conserved regions of the five known polyether epoxidases. A total of 44 putative polyether epoxidase gene-positive strains were obtained by the PCR-based screening of 1,068 actinomycetes isolated from eight different habitats and 236 reference strains encompassing eight major families of
Actinomycetales
. The isolates spanned a wide taxonomic diversity based on 16S rRNA gene analysis, and actinomycetes isolated from acidic soils seemed to be a promising source of polyether ionophores. Four genera were detected to contain putative polyether epoxidases, including
Micromonospora
, which has not previously been reported to produce polyether ionophores. The designed primers also detected putative epoxidase genes from diverse known producer strains that produce polyether ionophores unrelated to the five published gene clusters. Moreover, phylogenetic and chemical analyses showed a strong correlation between the sequence of polyether epoxidases and the structure of encoded polyethers. Thirteen positive isolates were proven to be polyether ionophore producers as expected, and two new analogues were found. These results demonstrate the feasibility of using this epoxidase gene screening strategy to aid the rapid identification of known products and the discovery of unknown polyethers in actinomycetes.
Comparative Genomics of Escherichia coli Strains Causing Urinary Tract Infections
Authors: Vejborg Rebecca Munk , Hancock Viktoria , Schembri Mark A. , Klemm Per
Published: March 2011

The virulence determinants of uropathogenic
Escherichia coli
have been studied extensively over the years, but relatively little is known about what differentiates isolates causing various types of urinary tract infections. In this study, we compared the genomic profiles of 45 strains from a range of different clinical backgrounds, i.e., urosepsis, pyelonephritis, cystitis, and asymptomatic bacteriuria (ABU), using comparative genomic hybridization analysis. A microarray based on 31 complete
E. coli
sequences was used. It emerged that there is little correlation between the genotypes of the strains and their disease categories but strong correlation between the genotype and the phylogenetic group association. Also, very few genetic differences may exist between isolates causing symptomatic and asymptomatic infections. Only relatively few genes that could potentially differentiate between the individual disease categories were identified. Among these were two genomic islands, namely, pathogenicity island (PAI)-CFT073-
serU
and PAI-CFT073-
pheU
, which were significantly more associated with the pyelonephritis and urosepsis isolates than with the ABU and cystitis isolates. These two islands harbor genes encoding virulence factors, such as P fimbriae (pyelonephritis-associated fimbriae) and an important immunomodulatory protein, TcpC. It seems that both urovirulence and growth fitness can be attributed to an assortment of genes rather than to a specific gene set. Taken together, urovirulence and fitness are the results of the interplay of a mixture of factors taken from a rich menu of genes.
Characterization of Putative Virulence Genes on the Related RepFIB Plasmids Harbored by Cronobacter spp
Authors: Franco A. A. , Hu L. , Grim C. J. , Gopinath G. , Sathyamoorthy V. , Jarvis K. G. , Lee C. , Sadowski J. , Kim J. , Kothary M. H. , McCardell B. A. , Tall B. D.
Published: March 2011

Cronobacter
spp. are emerging neonatal pathogens that cause meningitis, sepsis, and necrotizing enterocolitis. The genus
Chronobacter
consists of six species:
C. sakazakii
,
C. malonaticus
,
C. muytjensii
,
C. turicensis
,
C. dublinensis
, and
Cronobacter
genomospecies group 1. Whole-genome sequencing of
C. sakazakii
BAA-894 and
C. turicensis
z3032 revealed that they harbor similarly sized plasmids identified as pESA3 (131 kb) and pCTU1 (138 kb), respectively.
In silico
analysis showed that both plasmids encode a single RepFIB-like origin of replication gene,
repA
, as well as two iron acquisition systems (
eitCBAD
and
iucABCD/iutA
). In a chrome azurol S agar diffusion assay, it was demonstrated that siderophore activity was associated with the presence of pESA3 or pCTU1. Additionally, pESA3 contains a
cpa
(
Cronobacter
plasminogen activator) gene and a 17-kb type 6 secretion system (T6SS) locus, while pCTU1 contains a 27-kb region encoding a filamentous hemagglutinin gene (
fhaB
), its specifc transporter gene (
fhaC
), and associated putative adhesins (FHA locus), suggesting that these are virulence plasmids. In a
repA
-targeted PCR assay, 97% of 229
Cronobacter
species isolates were found to possess a homologous RepFIB plasmid. All
repA
PCR-positive strains were also positive for the
eitCBAD
and
iucABCD/iutA
iron acquisition systems. However, the presence of
cpa
, T6SS, and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type, and species. These results support the hypothesis that these plasmids have evolved from a single archetypical plasmid backbone through the cointegration, or deletion, of specific virulence traits in each species.
Efficient Extension of Slipped DNA Intermediates by DinB Is Required To Escape Primer Template Realignment by DnaQ
Authors: Foti James J. , Walker Graham C.
Published: March 2011

We show that
Escherichia coli
DinB polymerase, which creates single-base deletions, prefers to extend slipped DNA substrates with the skipped base at the −4 position. A DinB(Y79L) variant, which extends these substrates less efficiently
in vitro
, allows the proofreading function of polymerase III to reverse their formation
in vivo
.
The Y-Family DNA Polymerase Dpo4 Uses a Template Slippage Mechanism To Create Single-Base Deletions
Authors: Wu Yifeng , Wilson Ryan C. , Pata Janice D.
Published: March 2011

The Y-family polymerases help cells tolerate DNA damage by performing translesion synthesis, yet they also can be highly error prone. One distinctive feature of the DinB class of Y-family polymerases is that they make single-base deletion errors at high frequencies in repetitive sequences, especially those that contain two or more identical pyrimidines with a 5′ flanking guanosine. Intriguingly, different deletion mechanisms have been proposed, even for two archaeal DinB polymerases that share 54% sequence identity and originate from two strains of
Sulfolobus
. To reconcile these apparent differences, we have characterized Dpo4 from
Sulfolobus solfataricus
using the same biochemical and crystallographic approaches that we have used previously to characterize Dbh from
Sulfolobus acidocaldarius
. In contrast to previous suggestions that Dpo4 uses a deoxynucleoside triphosphate (dNTP)-stabilized misalignment mechanism when creating single-base deletions, we find that Dpo4 predominantly uses a template slippage deletion mechanism when replicating repetitive DNA sequences, as was previously shown for Dbh. Dpo4 stabilizes the skipped template base in an extrahelical conformation between the polymerase and the little-finger domains of the enzyme. This contrasts with Dbh, in which the extrahelical base is stabilized against the surface of the little-finger domain alone. Thus, despite sharing a common deletion mechanism, these closely related polymerases use different contacts with the substrate to accomplish the same result.
An Agrobacterium VirB10 Mutation Conferring a Type IV Secretion System Gating Defect
Authors: Banta Lois M. , Kerr Jennifer E. , Cascales Eric , Giuliano Meghan E. , Bailey Megan E. , McKay Cedar , Chandran Vidya , Waksman Gabriel , Christie Peter J.
Published: March 2011

Agrobacterium
VirB7, VirB9, and VirB10 form a “core complex” during biogenesis of the VirB/VirD4 type IV secretion system (T4SS). VirB10 spans the cell envelope and, in response to sensing of ATP energy consumption by the VirB/D4 ATPases, undergoes a conformational change required for DNA transfer across the outer membrane (OM). Here, we tested a model in which VirB10 regulates substrate passage by screening for mutations that allow for unregulated release of the VirE2 secretion substrate to the cell surface independently of target cell contact. One mutation, G272R, conferred VirE2 release and also rendered VirB10 conformationally insensitive to cellular ATP depletion. Strikingly, G272R did not affect substrate transfer to target cells (Tra
+
) but did block pilus production (Pil
−
). The G272R mutant strain displayed enhanced sensitivity to vancomycin and SDS but did not nonspecifically release periplasmic proteins or VirE2 truncated of its secretion signal. G272 is highly conserved among VirB10 homologs, including pKM101 TraF, and in the TraF X-ray structure the corresponding Gly residue is positioned near an α-helical domain termed the antenna projection (AP), which is implicated in formation of the OM pore. A partial AP deletion mutation (ΔAP) also confers a Tra
+
Pil
−
phenotype; however, this mutation did not allow VirE2 surface exposure but instead allowed the release of pilin monomers or short oligomers to the milieu. We propose that (i) G272R disrupts a gating mechanism in the core chamber that regulates substrate passage across the OM and (ii) the G272R and ΔAP mutations block pilus production at distinct steps of the pilus biogenesis pathway.
Inactivation of σ
F
in Clostridium acetobutylicum ATCC 824 Blocks Sporulation Prior to Asymmetric Division and Abolishes σ
E
and σ
G
Protein Expression but Does Not Block Solvent Formation
Authors: Jones Shawn W. , Tracy Bryan P. , Gaida Stefan M. , Papoutsakis Eleftherios T.
Published: March 2011

Clostridium acetobutylicum
is both a model organism for the understanding of sporulation in solventogenic clostridia and its relationship to solvent formation and an industrial organism for anaerobic acetone-butanol-ethanol (ABE) fermentation. How solvent production is coupled to endospore formation—both stationary-phase events—remains incompletely understood at the molecular level. Specifically, it is unclear how sporulation-specific sigma factors affect solvent formation. Here the
sigF
gene in
C. acetobutylicum
was successfully disrupted and silenced. Not only σ
F
but also the sigma factors σ
E
and σ
G
were not detected in the
sigF
mutant (FKO1), and differentiation was stopped prior to asymmetric division. Since plasmid expression of the
spoIIA
operon (
spoIIAA
-
spoIIAB
-
sigF
) failed to complement FKO1, the operon was integrated into the FKO1 chromosome to generate strain FKO1-C. In FKO1-C, σ
F
expression was restored along with sporulation and σ
E
and σ
G
protein expression. Quantitative reverse transcription-PCR (RT-PCR) analysis of a select set of genes (
csfB
,
gpr
,
spoIIP
,
sigG
,
lonB
, and
spoIIR
) that could be controlled by σ
F
, based on the
Bacillus subtilis
model, indicated that
sigG
may be under the control of σ
F
, but
spoIIR
, an important activator of σ
E
in
B. subtilis
, is not, and neither are the rest of the genes investigated. FKO1 produced solvents at a level similar to that of the parent strain, but solvent levels were dependent on the physiological state of the inoculum. Finally, the complementation strain FKO1-C is the first reported instance of purposeful integration of multiple functional genes into a clostridial chromosome—here, the
C. acetobutylicum
chromosome—with the aim of altering cell metabolism and differentiation.
Development and Use of a Gene Deletion Strategy for Flavobacterium johnsoniae To Identify the Redundant Gliding Motility Genes
remF
,
remG
,
remH
, and
remI
Authors: Rhodes Ryan G. , Pucker Halley G. , McBride Mark J.
Published: March 2011

Cells of
Flavobacterium johnsoniae
exhibit rapid gliding motility over surfaces. Cell movement is thought to involve motor complexes comprised of Gld proteins that propel the cell surface adhesin SprB. The four distal genes of the
sprB
operon (
sprC
,
sprD
,
sprB
, and
sprF
) are required for normal motility and for formation of spreading colonies, but the roles of the remaining three genes (
remF
,
remG
, and
fjoh
_
0982
) are unclear. A gene deletion strategy was developed to determine whether these genes are involved in gliding. A spontaneous streptomycin-resistant
rpsL
mutant of
F. johnsoniae
was isolated. Introduction of wild-type
rpsL
on a plasmid restored streptomycin sensitivity, demonstrating that wild-type
rpsL
is dominant to the mutant allele. The gene deletion strategy employed a suicide vector carrying wild-type
rpsL
and used streptomycin for counterselection. This approach was used to delete the region spanning
remF
,
remG
, and
fjoh
_
0982
. The mutant cells formed spreading colonies, demonstrating that these genes are not required for normal motility. Analysis of the genome revealed a paralog of
remF
(
remH
) and a paralog of
remG
(
remI
). Deletion of
remH
and
remI
had no effect on motility of wild-type cells, but cells lacking
remF
and
remH
, or cells lacking
remG
and
remI
, formed nonspreading colonies. The motility defects resulting from the combination of mutations suggest that the paralogous proteins perform redundant functions in motility. The
rpsL
counterselection strategy allows construction of unmarked mutations to determine the functions of individual motility proteins or to analyze other aspects of
F. johnsoniae
physiology.
Overexpression of
pknE
Blocks Heterocyst Development in Anabaena sp. Strain PCC 7120
Authors: Saha Sushanta K. , Golden James W.
Published: March 2011

The upstream intergenic regions for each of four genes encoding Ser/Thr kinases, all2334,
pknE
(alr3732), all4668, and all4838, were fused to a
gfpmut2
reporter gene to determine their expression during heterocyst development in the cyanobacterium
Anabaena
(
Nostoc
) sp. strain PCC 7120. P

pknE

-gfp
was upregulated after nitrogen step-down and showed strong expression in differentiating cells. Developmental regulation of
pknE
required a 118-bp upstream region and was abolished in a
hetR
mutant. A
pknE
mutant strain had shorter filaments with slightly higher heterocyst frequency than did the wild type. Overexpression of
pknE
from its native promoter inhibited heterocyst development in the wild type and in four mutant backgrounds that overproduce heterocysts. Overexpression of
pknE
from the copper-inducible
petE
promoter did not completely inhibit heterocyst development but caused a 24-h delay in heterocyst differentiation and cell bleaching 4 to 5 days after nitrogen step-down. Strains overexpressing
pknE
and containing P

hetR

-gfp
or P

patS

-gfp
reporters failed to show developmental regulation of the reporters and had undetectable levels of HetR protein. Genetic epistasis experiments suggest that overexpression of
pknE
blocks HetR activity or downstream regulation.