bluuebunny/crossref_metadata_2025_split huggingface dataset:
Simple, fast and high‐efficiency transformation system for directed evolution of cellulase in Bacillus subtilis
Authors: Zhang Xiao‐Zhou , Zhang Y.‐H. Percival 
Published: November 2010

Summary Bacillus subtilis can serve as a powerful platform for directed evolution, especially for secretory enzymes. However, cloning and transformation of a DNA mutant library in B. subtilis are not as easy as they are in Escherichia coli. For direct transformation of B. subtilis, here we developed a new protocol based on supercompetent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids. This new protocol is simple (restriction enzyme‐, phosphatase‐ and ligase‐free), fast (i.e. 1 day) and of high efficiency (i.e. ∼107 or ∼104 transformants per µg of multimeric plasmid or ligated plasmid DNA respectively). Supercompetent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose. The DNA mutant library was generated through a two‐round PCR: (i) the mutagenized DNA fragments were generated by error‐prone PCR and linearized plasmids were made using high‐fidelity PCR, and (ii) the multimeric plasmids were generated based on these two DNA templates by using overlap PCR. Both protein expression level and specific activity of glycoside hydrolase family 5 endoglucanse on regenerated amorphous cellulose were improved through this new system. To our limited knowledge, this study is the first report for enhancing secretory cellulase performance on insoluble cellulose.
Adherence with brimonidine in patients with glaucoma aware and not aware of electronic monitoring
Authors: Hermann Manuel Marcel , Papaconstantinou Dimitris , Muether Philipp Sebastian , Georgopoulos Gerasimos , Diestelhorst Michael 
Published: November 2010

Abstract.Purpose:  To assess the impact of open versus masked adherence monitoring on adherence with topical brimonidine using two different dosing schedules.Methods:  Thirty‐seven patients with glaucoma or ocular hypertension were enroled in a prospective, observational cohort study. Patients were randomly assigned to open or masked adherence monitoring and to brimonidine twice daily (BID) or three times daily (TID). Patients received conventional brimonidine eye drops with attached electronic monitoring devices for 4 weeks with weekly intraocular pressure measurements. Adherence calculations comprised dosing intervals, adherence rate and time covered. Subgroup analysis with anova included the factors masking, regimen, diagnosis and age.Results:  Among 36 individually analysed patients, 12 (33%) had adherence rates above 75%, therein two (5%)>90%. The mean adherence rate in 19 patients aware of adherence measurements was 70 ± 17% for brimonidine BID and 65 ± 14% for TID, not significantly different to the rates of 17 patients not informed about adherence measurements (77 ± 6% BID, 62 ± 9% TID, p = 0.24). On average, patients with brimonidine TID achieved significantly lower adherence rates (64 ± 12%) than patients on BID (73 ± 13%, p = 0.02). Still, patients on TID applied brimonidine more often (TID 1.9 ± 0.3, BID 1.5 ± 0.1 mean applications per day). The median coverage was 70% and showed no statistically significant difference between patients on BID and TID (p = 0.36).Conclusion:  The study findings suggest that adherence measurements are not significantly altered by open adherence monitoring, which may simplify future adherence studies. Adherence with brimonidine eye drops was insufficient for most patients. These results demonstrate the necessity to develop new strategies to improve compliance in glaucoma therapy.
Real orthopaedic subluxations versus imaginary chiropractic subluxations
Authors: Homola Samuel 
Published: November 2010

AbstractThe chiropractic vertebral subluxation theory has confused the definition of the word ‘subluxation’; a common medical term. Unlike the mysterious, undetectable chiropractic ‘vertebral subluxation complex’ alleged to be a cause of disease, a real vertebral subluxation can be a cause of mechanical and neuromusculoskeletal symptoms, but has never been shown to be a cause of organic disease. The views offered in this paper may be helpful in clarifying this issue.
Complete Biodegradation of 4-Fluorocinnamic Acid by a Consortium Comprising
Arthrobacter
sp. Strain G1 and
Ralstonia
sp. Strain H1
Authors: Hasan Syed A. , Ferreira Maria Isabel M. , Koetsier Martijn J. , Arif Muhammad I. , Janssen Dick B. 
Published: November 2010

ABSTRACT

A consortium of the newly isolated bacterial strains
Arthrobacter
sp. strain G1 and
Ralstonia
sp. strain H1 utilized 4-fluorocinnamic acid for growth under aerobic conditions. Strain G1 converted 4-fluorocinnamic acid into 4-fluorobenzoic acid and used the two-carbon side chain for growth, with some formation of 4-fluoroacetophenone as a dead-end side product. In the presence of strain H1, complete mineralization of 4-fluorocinnamic acid and release of fluoride were obtained. Degradation of 4-fluorocinnamic acid by strain G1 occurred through a β-oxidation mechanism and started with the formation of 4-fluorocinnamoyl-coenzyme A (CoA), as indicated by the presence of 4-fluorocinnamoyl-CoA ligase. Enzymes for further transformation were detected in cell extract, i.e., 4-fluorocinnamoyl-CoA hydratase, 4-fluorophenyl-β-hydroxy propionyl-CoA dehydrogenase, and 4-fluorophenyl-β-keto propionyl-CoA thiolase. Degradation of 4-fluorobenzoic acid by strain H1 proceeded via 4-fluorocatechol, which was converted by an
ortho
-cleavage pathway.
Mycolic Acid-Containing Bacteria Induce Natural-Product Biosynthesis in
Streptomyces
Species
Authors: Onaka Hiroyasu , Mori Yukiko , Igarashi Yasuhiro , Furumai Tamotsu 
Published: November 2010

ABSTRACT

Natural products produced by microorganisms are important starting compounds for drug discovery. Secondary metabolites, including antibiotics, have been isolated from different
Streptomyces
species. The production of these metabolites depends on the culture conditions. Therefore, the development of a new culture method can facilitate the discovery of new natural products. Here, we show that mycolic acid-containing bacteria can influence the biosynthesis of cryptic natural products in
Streptomyces
species. The production of red pigment by
Streptomyces lividans
TK23 was induced by coculture with
Tsukamurella pulmonis
TP-B0596, which is a mycolic acid-containing bacterium. Only living cells induced this pigment production, which was not mediated by any substances.
T
.
pulmonis
could induce natural-product synthesis in other
Streptomyces
strains too: it altered natural-product biosynthesis in 88.4% of the
Streptomyces
strains isolated from soil. The other mycolic acid-containing bacteria,
Rhodococcus erythropolis
and
Corynebacterium glutamicum
, altered biosynthesis in 87.5 and 90.2% of the
Streptomyces
strains, respectively. The coculture broth of
T
.
pulmonis
and
Streptomyces endus
S-522 contained a novel antibiotic, which we named alchivemycin A. We concluded that the mycolic acid localized in the outer cell layer of the inducer bacterium influences secondary metabolism in
Streptomyces
, and this activity is a result of the direct interaction between the mycolic acid-containing bacteria and
Streptomyces
. We used these results to develop a new coculture method, called the combined-culture method, which facilitates the screening of natural products.
Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression in
Clostridium perfringens
Authors: Hartman Andrea H. , Liu Hualan , Melville Stephen B. 
Published: November 2010

ABSTRACT

Clostridium perfringens
is a Gram-positive anaerobic pathogen which causes many diseases in humans and animals. While some genetic tools exist for working with
C. perfringens
, a tightly regulated, inducible promoter system is currently lacking. Therefore, we constructed a plasmid-based promoter system that provided regulated expression when lactose was added. This plasmid (pKRAH1) is an
Escherichia coli-C. perfringens
shuttle vector containing the gene encoding a transcriptional regulator, BgaR, and a divergent promoter upstream of gene
bgaL
(
bgaR
-P

bgaL

). To measure transcription at the
bgaL
promoter in pKRAH1, the
E. coli
reporter gene
gusA
, encoding β-glucuronidase, was placed downstream of the P

bgaL

promoter to make plasmid pAH2. When transformed into three strains of
C. perfringens
, pAH2 exhibited lactose-inducible expression.
C. perfringens
strain 13, a commonly studied strain, has endogenous β-glucuronidase activity. We mutated gene
bglR
, encoding a putative β-glucuronidase, and observed an 89% decrease in endogenous activity with no lactose. This combination of a system for regulated gene expression and a mutant of strain 13 with low β-glucuronidase activity are useful tools for studying gene regulation and protein expression in an important pathogenic bacterium. We used this system to express the
yfp
-
pilB
gene, comprised of a yellow fluorescent protein (YFP)-encoding gene fused to an assembly ATPase gene involved in type IV pilus-dependent gliding motility in
C. perfringens
. Expression in the wild-type strain showed that YFP-PilB localized mostly to the poles of cells, but in a
pilC
mutant it localized throughout the cell, demonstrating that the membrane protein PilC is required for polar localization of PilB.
Utilization of Natural Fucosylated Oligosaccharides by Three Novel α-
l
-Fucosidases from a Probiotic
Lactobacillus casei
Strain
Authors: Rodríguez-Díaz Jesús , Monedero Vicente , Yebra María J. 
Published: November 2010

ABSTRACT

Three putative α-
l
-fucosidases encoded in the
Lactobacillus casei
BL23 genome were cloned and purified. The proteins displayed different abilities to hydrolyze natural fucosyloligosaccharides like 2′-fucosyllactose, H antigen disaccharide, H antigen type II trisaccharide, and 3′-, 4′-, and 6′-fucosyl-GlcNAc. This indicated a possible role in the utilization of oligosaccharides present in human milk and intestinal mucosa.
Acute Parvovirus B19 Infection Causes Nonspecificity Frequently in
Borrelia
and Less Often in
Salmonella
and
Campylobacter
Serology, Posing a Problem in Diagnosis of Infectious Arthropathy
Authors: Tuuminen Tamara , Hedman Klaus , Söderlund-Venermo Maria , Seppälä Ilkka 
Published: November 2010

ABSTRACT

Several infectious agents may cause arthritis or arthropathy. For example, infection with
Borrelia burgdorferi
, the etiologic agent of Lyme disease, may in the late phase manifest as arthropathy. Infections with
Campylobacter
,
Salmonella
, or
Yersinia
may result in a postinfectious reactive arthritis. Acute infection with parvovirus B19 (B19V) may likewise initiate transient or chronic arthropathy. All these conditions may be clinically indistinguishable from rheumatoid arthritis. Here, we present evidence that acute B19V infection may elicit IgM antibodies that are polyspecific or cross-reactive with a variety of bacterial antigens. Their presence may lead to misdiagnosis and improper clinical management, exemplified here by two case descriptions. Further, among 33 subjects with proven recent B19V infection we found IgM enzyme immunoassay (EIA) positivity for
Borrelia
only; for
Borrelia
and
Salmonella
; for
Borrelia
and
Campylobacter
; and for
Borrelia
,
Campylobacter
, and
Salmonella
in 26 (78.7%), 1 (3%), 2 (6%), and 1 (3%), respectively; however, when examined by
Borrelia
LineBlot, all samples were negative. These antibodies persisted over 3 months in 4/13 (38%) patients tested. Likewise, in a retrospective comparison of the results of a diagnostic laboratory, 9/11 (82%) patients with confirmed acute B19V infection showed IgM antibody to
Borrelia
. However, none of 12 patients with confirmed borreliosis showed any serological evidence of acute B19V infection. Our study demonstrates that recent B19V infection can be misinterpreted as secondary borreliosis or enteropathogen-induced reactive arthritis. To obtain the correct diagnosis, we emphasize caution in interpretation of polyreactive IgM and exclusion of recent B19V infection in patients examined for infectious arthritis or arthropathy.
Differential Responses of
Bacillus subtilis
rRNA Promoters to Nutritional Stress
Authors: Samarrai Walied , Liu David X. , White Ann-Marie , Studamire Barbara , Edelstein Jacob , Srivastava Anita , Widom Russell L. , Rudner Rivka 
Published: November 2010

ABSTRACT

The
in vivo
expression levels of four rRNA promoter pairs (
rrn
p
1
p
2
) of
Bacillus subtilis
were determined by employing single-copy
lacZ
fusions integrated at the
amyE
locus. The
rrnO
,
rrnJ
,
rrnD
, and
rrnB
promoters displayed unique growth rate regulation and stringent responses. Both
lacZ
activity and mRNA levels were highest for
rrnO
under all growth conditions tested, while
rrnJ
,
rrnB
,
and rrnD
showed decreasing levels of activity. During amino acid starvation induced by serine hydroxamate (SHX), only the strong
rrnO
and
rrnJ
promoters demonstrated stringent responses. Under the growth conditions used, the
rrn
promoters showed responses similar to the responses to carbon source limitation induced by α-methyl glucoside (α-MG). The ratio of P2 to P1 transcripts, determined by primer extension analysis, was high for the strong
rrnO
and
rrnJ
promoters, while only P2 transcripts were detected for the weak
rrnD
and
rrnB
promoters. Cloned P1 or P2 promoter fragments of
rrnO
or
rrnJ
were differentially regulated. In wild-type (

relA
+

) and suppressor [
relA
(S)] strains under the conditions tested, only P2 responded to carbon source limitation by a decrease in RNA synthesis, correlating with an increase in (p)ppGpp levels and a decrease in the GTP concentration. The weak P1 promoter elements remain relaxed in the three genetic backgrounds [

relA
+

,
relA
,
relA
(S)] in the presence of α-MG. During amino acid starvation, P2 was stringently regulated in

relA
+

and
relA
(S) cells, while only
rrnJ
p
1
was also regulated, but to a lesser extent. Both the
relA
+
and
relA
(S) strains showed (p)ppGpp accumulation after α-MG treatment but not after SHX treatment. These data reveal the complex nature of
B. subtilis rrn
promoter regulation in response to stress, and they suggest that the P2 promoters may play a more prominent role in the stringent response.
The Peptidoglycan-Binding Protein FimV Promotes Assembly of the
Pseudomonas aeruginosa
Type IV Pilus Secretin
Authors: Wehbi Hania , Portillo Eder , Harvey Hanjeong , Shimkoff Anthony E. , Scheurwater Edie M. , Howell P. Lynne , Burrows Lori L. 
Published: November 2010

ABSTRACT

The
Pseudomonas aeruginosa
inner membrane protein FimV is among several proteins of unknown function required for type IV pilus-mediated twitching motility, arising from extension and retraction of pili from their site of assembly in the inner membrane. The pili transit the periplasm and peptidoglycan (PG) layer, ultimately exiting the cell through the PilQ secretin. Although
fimV
mutants are nonmotile, they are susceptible to killing by pilus-specific bacteriophage, a hallmark of retractable surface pili. Here we show that levels of recoverable surface pili were markedly decreased in
fimV pilT
retraction-deficient mutants compared with levels in the
pilT
control, demonstrating that FimV acts at the level of pilus assembly. Levels of inner membrane assembly subcomplex proteins PilM/N/O/P were decreased in
fimV
mutants, but supplementation of these components in
trans
did not restore pilus assembly or motility. Loss of FimV dramatically reduced the levels of the PilQ secretin multimer through which pili exit the cell, in part due to decreased levels of PilQ monomers, while PilF pilotin levels were unchanged. Expression of
pilQ
in
trans
in the wild type or
fimV
mutants increased total PilQ monomer levels but did not alter secretin multimer levels or motility. PG pulldown assays showed that the N terminus of FimV bound PG in a LysM motif-dependent manner, and a mutant with an in-frame chromosomal deletion of the LysM motif had reduced motility, secretin levels, and surface piliation. Together, our data show that FimV's role in pilus assembly is to promote secretin formation and that this function depends upon its PG-binding domain.
DNA-Binding Properties of the
Bacillus subtilis
and
Aeribacillus pallidus
AC6 σ
D
Proteins
Authors: Sevim Elif , Gaballa Ahmed , Beldüz A. Osman , Helmann John D. 
Published: November 2010

ABSTRACT

σ
D
proteins from
Aeribacillus pallidus
AC6 and
Bacillus subtilis
bound specifically, albeit weakly, to promoter DNA even in the absence of core RNA polymerase. Binding required a conserved CG motif within the −10 element, and this motif is known to be recognized by σ region 2.4 and critical for promoter activity.
Enteric Campylobacteria and RNA Viruses Associated with Healthy and Diarrheic Humans in the Chinook Health Region of Southwestern Alberta, Canada
Authors: Inglis G. Douglas , Boras Valerie F. , Houde Alain 
Published: November 2010

ABSTRACT

The presence of
Campylobacter
species and enteric RNA viruses in stools from diarrheic (
n
= 442) and healthy (
n
= 58) humans living in southwestern Alberta was examined (May to October 2005). A large number of diarrheic individuals who were culture negative for
C. jejuni
(
n
= 54) or
C. coli
(
n
= 19) were PCR positive for these taxa. Overall detection rates for
C. jejuni
and
C. coli
in diarrheic stools were 29% and 5%, respectively. In contrast, 3% and 0% of stools from healthy humans were positive for these taxa, respectively. Infection with
C. jejuni
was endemic over the study period. However, there was no difference in infection rates between individuals living in urban or rural locations. Stools from a large number of diarrheic (74%) and healthy (88%) individuals were positive for
Campylobacter
DNA. The prevalence rates of
C. concisus
,
C. curvus
,
C. fetus
,
C. gracilis
,
C. helveticus
,
C. hominis
,
C. hyointestinalis
,
C. mucosalis
,
C. showae
,
C. sputorum
, and
C. upsaliensis
DNA were either not significantly different or were significantly lower in stools from diarrheic than from healthy individuals. No
C. lanienae
or
C. lari
DNA was detected. Stools from 4% and 0% of diarrheic and healthy humans, respectively, were positive for rotavirus, sapovirus, or norovirus (GI/GII). Our results showed a high prevalence of diarrheic individuals living in southwestern Alberta who were infected by
C. jejuni
and, to a lesser extent, by
C. coli
. However, other
Campylobacter
species, norovirus, rotavirus, sapovirus, and bovine enteric calicivirus were either inconsequential pathogens during the study period or are not pathogens at all.
Multilocus Sequence Analysis and
rpoB
Sequencing of Mycobacterium abscessus (Sensu Lato) Strains
Authors: Macheras Edouard , Roux Anne-Laure , Bastian Sylvaine , Leão Sylvia Cardoso , Palaci Moises , Sivadon-Tardy Valérie , Gutierrez Cristina , Richter Elvira , Rüsch-Gerdes Sabine , Pfyffer Gaby , Bodmer Thomas , Cambau Emmanuelle , Gaillard Jean-Louis , Heym Beate 
Published: November 2010

ABSTRACT

Mycobacterium abscessus
,
Mycobacterium bolletii
, and
Mycobacterium massiliense
(
Mycobacterium abscessus
sensu lato) are closely related species that currently are identified by the sequencing of the
rpoB
gene. However, recent studies show that
rpoB
sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of
M. abscessus
(sensu lato) strains by partial
rpoB
sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes:
argH
,
cya
,
glpK
,
gnd
,
murC
,
pgm
,
pta
, and
purH
. The strains studied included the three type strains (
M. abscessus
CIP 104536
T
,
M. massiliense
CIP 108297
T
, and
M. bolletii
CIP 108541
T
) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The
rpoB
phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the
M. massiliense
and
M. bolletii
clusters all were below 3% and between the
M. abscessus
and
M. massiliense
clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The
M. abscessus
cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the
M. massiliense
and
M. bolletii
branches were much lower (71 and 61%, respectively), with the
M. massiliense
cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the
M. abscessus
and
M. massiliense
clusters, 2.37% (1.5 to 2.85%) between the
M. abscessus
and
M. bolletii
clusters, and 2.28% (0.86 to 2.68%) between the
M. massiliense
and
M. bolletii
clusters. Adding the
rpoB
sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and
rpoB
sequence were discordant (e.g.,
M. massiliense
MLSA sequence and
M. abscessus rpoB
sequence), suggesting the intergroup lateral transfers of
rpoB
. In conclusion, our study strongly supports the recent proposal that
M. abscessus
,
M. massiliense
, and
M. bolletii
should constitute a single species. Our findings also indicate that there has been a horizontal transfer of
rpoB
sequences between these subgroups, precluding the use of
rpoB
sequencing alone for the accurate identification of the two proposed
M. abscessus
subspecies.
Hepatitis B and C Virus Infections in the Three Pygmy Groups in Cameroon
Authors: Foupouapouognigni Yacouba , Sadeuh Mba Serge Alain , à Betsem Edouard Betsem , Rousset Dominique , Froment Alain , Gessain Antoine , Njouom Richard 
Published: November 2010

ABSTRACT
A high (11.8%) level of hepatitis B virus (HBV) infection was found among 524 Pygmies in Cameroon, whereas the extent of hepatitis C virus (HCV) infection in the same population was low (0.6%). Phylogenetic analyses showed cocirculation of two HBV genotypes, HBV-A3 and -E. Taken together, our results suggest different epidemiological scenarios concerning HBV and HCV infections in this population.
Role of the CM2 Protein in the Influenza C Virus Replication Cycle
Authors: Furukawa Takatoshi , Muraki Yasushi , Noda Takeshi , Takashita Emi , Sho Ri , Sugawara Kanetsu , Matsuzaki Yoko , Shimotai Yoshitaka , Hongo Seiji 
Published: November 2010

ABSTRACT
CM2 is the second membrane protein of influenza C virus. Although its biochemical characteristics, coding strategy, and properties as an ion channel have been extensively studied, the role(s) of CM2 in the virus replication cycle remains to be clarified. In order to elucidate this role, in the present study we generated CM2-deficient influenza C virus-like particles (VLPs) and examined the VLP-producing 293T cells, VLPs, and VLP-infected HMV-II cells. Quantification of viral RNA (vRNA) in the VLPs by real-time PCR revealed that the CM2-deficient VLPs contain approximately one-third of the vRNA found in wild-type VLPs although no significant differences were detected in the expression levels of viral components in VLP-producing cells or in the number and morphology of the generated VLPs. This finding suggests that CM2 is involved in the genome packaging process into VLPs. Furthermore, HMV-II cells infected with CM2-deficient VLPs exhibited significantly reduced reporter gene expression. Although CM2-deficient VLPs could be internalized into HMV-II cells as efficiently as wild-type VLPs, a smaller amount of vRNA was detected in the nuclear fraction of CM2-deficient VLP-infected cells than in that of wild-type VLP-infected cells, suggesting that the uncoating process of the CM2-deficient VLPs in the infected cells did not proceed in an appropriate manner. Taken together, the data obtained in the present study indicate that CM2 has a potential role in the genome packaging and uncoating processes of the virus replication cycle.