bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biosynthesis and Degradation of H
2
O
2
by Vaginal Lactobacilli
Authors: Martín Rebeca , Suárez Juan E. 
Published: December 2009

ABSTRACT

Hydrogen peroxide production by vaginal lactobacilli represents one of the most important defense mechanisms against vaginal colonization by undesirable microorganisms. To quantify the ability of a collection of 45 vaginal
Lactobacillus
strains to generate H
2
O
2
, we first compared three published colorimetric methods. It was found that the use of DA-64 as a substrate rendered the highest sensitivity, while tetramethyl-benzidine (TMB) maintained its linearity from nanomolar to millimolar H
2
O
2
concentrations. Generation of H
2
O
2
was found to be especially common and strong for
L. jensenii
strains, while it was variable among
L. crispatus
and
L. gasseri
strains. Biosynthesis of H
2
O
2
only occurred upon agitation of the cultures, but the H
2
O
2
-producing machinery was already present in them before aeration started. Calcium, magnesium, manganese, and zinc ions did not affect H
2
O
2
production, while Cu
2+
inhibited the growth of
Lactobacillus jensenii
CECT 4306, which was chosen as a model strain. Cultures with Fe
3+
, hemin, and hemoglobin did not accumulate H
2
O
2
. Fe
3+
activated an extracellular peroxidase that destroyed the H
2
O
2
being produced by the cultures. This protected the lactobacilli against its antimicrobial effect. The production of the enzyme appears to be constitutive, the Fe
3+
ions being a necessary cofactor of the reaction.
Contributions of the Pre- and Pro-Regions of a
Staphylococcus hyicus
Lipase to Secretion of a Heterologous Protein by
Bacillus subtilis
Authors: Kouwen Thijs R. H. M. , Nielsen Allan K. , Denham Emma L. , Dubois Jean-Yves F. , Dorenbos Ronald , Rasmussen Michael D. , Quax Wim J. , Freudl Roland , van Dijl Jan Maarten 
Published: December 2009

ABSTRACT

Bacillus subtilis
is a well-established cell factory for efficient secretion of many biotechnologically relevant enzymes that are naturally produced by it or related organisms. However, the use of
B. subtilis
as a host for production of heterologous secretory proteins can be complicated by problems related to inefficient translocation of the foreign proteins across the plasma membrane or to inefficient release of the exported proteins from the cell surface into the surrounding medium. Therefore, there is a clear need for tools that allow more efficient membrane targeting, translocation, and release during the production of these proteins. In the present study, we investigated the contributions of the pre (pre
lip
) and pro (pro
lip
) sequences of a
Staphylococcus hyicus
lipase to secretion of a heterologous protein, the alkaline phosphatase PhoA of
Escherichia coli
, by
B. subtilis
. The results indicate that the presence of the pro
lip
-peptide, in combination with the lipase signal peptide (pre
lip
), contributes significantly to the efficient secretion of PhoA by
B. subtilis
and that pre
lip
directs PhoA secretion more efficiently than the authentic signal peptide of PhoA. Genome-wide transcriptional analyses of the host cell responses indicate that, under the conditions tested, no known secretion or membrane-cell wall stress responses were provoked by the production of PhoA with any of the pre- and pro-region sequences used. Our data underscore the view that the pre-pro signals of the
S. hyicus
lipase are very useful tools for secretion of heterologous proteins in
B. subtilis
.
Contributions ofFrancisella tularensissubsp.novicidaChitinases and Sec Secretion System to Biofilm Formation on Chitin
Authors: Margolis Jeffrey J. , El-Etr Sahar , Joubert Lydia-Marie , Moore Emily , Robison Richard , Rasley Amy , Spormann Alfred M. , Monack Denise M. 
Published: December 2009

ABSTRACTFrancisella tularensis, the zoonotic cause of tularemia, can infect numerous mammals and other eukaryotes. Although studyingF. tularensispathogenesis is essential to comprehending disease, mammalian infection is just one step in the ecology ofFrancisellaspecies.F. tularensishas been isolated from aquatic environments and arthropod vectors, environments in which chitin could serve as a potential carbon source and as a surface for attachment and growth. We show thatF. tularensissubsp.novicidaforms biofilms during the colonization of chitin surfaces. The ability ofF. tularensisto persist using chitin as a sole carbon source is dependent on chitinases, since mutants lackingchiAorchiBare attenuated for chitin colonization and biofilm formation in the absence of exogenous sugar. A genetic screen for biofilm mutants identified the Sec translocon export pathway and 14 secreted proteins. We show that these genes are important for initial attachment during biofilm formation. We generated defined deletion mutants by targeting two chaperone genes (secB1andsecB2) involved in Sec-dependent secretion and four genes that encode putative secreted proteins. All of the mutants were deficient in attachment to polystyrene and chitin surfaces and for biofilm formation compared to wild-typeF. novicida. In contrast, mutations in the Sec translocon and secreted factors did not affect virulence. Our data suggest that biofilm formation byF. tularensispromotes persistence on chitin surfaces. Further study of the interaction ofF. tularensiswith the chitin microenvironment may provide insight into the environmental survival and transmission mechanisms of this pathogen.
Fates of Acid-Resistant and Non-Acid-Resistant Shiga Toxin-Producing
Escherichia coli
Strains in Ruminant Digestive Contents in the Absence and Presence of Probiotics
Authors: Chaucheyras-Durand Frédérique , Faqir Fahima , Ameilbonne Aurélie , Rozand Christine , Martin Christine 
Published: December 2009

ABSTRACT

Healthy ruminants are the main reservoir of Shiga toxin-producing
Escherichia coli
(STEC). During their transit through the ruminant gastrointestinal tract, STEC encounters a number of acidic environments. As all STEC strains are not equally resistant to acidic conditions, the purpose of this study was to investigate whether acid resistance confers an ecological advantage to STEC strains in ruminant digestive contents and whether acid resistance mechanisms are induced in the rumen compartment. We found that acid-resistant STEC survived at higher rates during prolonged incubation in rumen fluid than acid-sensitive STEC and that they resisted the highly acidic conditions of the abomasum fluid, whereas acid-sensitive strains were killed. However, transit through the rumen contents allowed acid-sensitive strains to survive in the abomasum fluid at levels similar to those of acid-resistant STEC. The acid resistance status of the strains had little influence on STEC growth in jejunal and cecal contents. Supplementation with the probiotic
Saccharomyces cerevisiae
CNCM I-1077 or
Lactobacillus acidophilus
BT-1386 led to killing of all of the strains tested during prolonged incubation in the rumen contents, but it did not have any influence in the other digestive compartments. In addition,
S. cerevisiae
did not limit the induction of acid resistance in the rumen fluid. Our results indicate that the rumen compartment could be a relevant target for intervention strategies that could both limit STEC survival and eliminate induction of acid resistance mechanisms in order to decrease the number of viable STEC cells reaching the hindgut and thus STEC shedding and food contamination.
Alanine Scanning Analyses of the Three Major Loops in Domain II of
Bacillus thuringiensis
Mosquitocidal Toxin Cry4Aa
Authors: Howlader Mohammad Tofazzal Hossain , Kagawa Yasuhiro , Miyakawa Ai , Yamamoto Ayaka , Taniguchi Tetsuya , Hayakawa Tohru , Sakai Hiroshi 
Published: December 2009

ABSTRACT

Cry4Aa produced by
Bacillus thuringiensis
is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using
Culex pipiens
larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.
Enhanced Display of Lipase on the
Escherichia coli
Cell Surface, Based on Transcriptome Analysis
Authors: Baek Jong Hwan , Han Mee-Jung , Lee Seung Hwan , Lee Sang Yup 
Published: December 2009

ABSTRACT

A cell surface display system was developed using
Escherichia coli
OmpC as an anchoring motif. The fused
Pseudomonas fluorescens
SIK W1 lipase was successfully displayed on the surface of
E. coli
cells, and the lipase activity could be enhanced by the coexpression of the
gadBC
genes identified by transcriptome analysis.
A Putative P-Type ATPase, Apt1, Is Involved in Stress Tolerance and Virulence in
Cryptococcus neoformans
Authors: Hu Guanggan , Kronstad James W. 
Published: December 2009

ABSTRACT

The export of virulence factors, such as the capsule polysaccharide, to the cell surface is a critical aspect of the pathogenicity of
Cryptococcus neoformans.
A view of capsule export via exocytosis and extracellular vesicles is emerging, but the molecular mechanisms underlying virulence factor transport pathways remain to be established. In this study, we characterized the
APT1
gene, which encodes a predicted integral membrane P-type ATPase belonging to the type IV, Drs2 family of aminophospholipid translocases (flippases) (APTs). APTs maintain the phospholipid asymmetry that is critical in membrane fusion events for trafficking and in establishing cell polarity. Deletion of the
APT1
gene resulted in phenotypes consistent with similar roles in
C. neoformans
. These included altered actin distribution, increased sensitivity to stress conditions (oxidative and nitrosative stress) and to trafficking inhibitors, such as brefeldin A and monensin, a reduction in exported acid phosphatase activity, and hypersensitivity to the antifungal drugs amphotericin B, fluconazole, and cinnamycin. However, there was no difference in growth, capsule size, or melanin production between the wild type and the
apt1
mutant strains at either 30°C or 37°C. Despite the absence of an influence on these major virulence factors, Apt1 was required for survival during interactions with macrophages, and
apt1
mutants exhibited attenuated virulence in a mouse inhalation model of cryptococcosis. Therefore, Apt1 contributes to virulence and the stress response in
C. neoformans
through apparent functions in membrane fusion and trafficking that do not influence the deposition of major virulence factors, such as capsule and melanin, outside the cell.
Synthetic β-(1→6)-Linked N-Acetylated and Nonacetylated Oligoglucosamines Used To Produce Conjugate Vaccines for Bacterial Pathogens
Authors: Gening Marina L. , Maira-Litrán Tomás , Kropec Andrea , Skurnik David , Grout Martha , Tsvetkov Yury E. , Nifantiev Nikolay E. , Pier Gerald B. 
Published: December 2009

ABSTRACT

Vaccines for pathogens usually target strain-specific surface antigens or toxins, and rarely is there broad antigenic specificity extending across multiple species. Protective antibodies for bacteria are usually specific for surface or capsular antigens. β-(1→6)-Poly-
N
-acetyl-
d
-glucosamine (PNAG) is a surface polysaccharide produced by many pathogens, including
Staphylococcus aureus
,
Escherichia coli
,
Yersinia pestis
,
Bordetella pertussis
,
Acinetobacter baumannii
, and others. Protective antibodies to PNAG are elicited when a deacetylated glycoform (deacetylated PNAG [dPNAG]; <30% acetate) is used in conjugate vaccines, whereas highly acetylated PNAG does not induce such antibodies. Chemical derivation of dPNAG from native PNAG is imprecise, so we synthesized both β-(1→6)-
d
-glucosamine (GlcNH
2
) and β-(1→6)-
d
-
N
-acetylglucosamine (GlcNAc) oligosaccharides with linkers on the reducing termini that could be activated to produce sulfhydryl groups for conjugation to bromoacetyl groups introduced onto carrier proteins. Synthetic 5-mer GlcNH
2
(5GlcNH
2
) or 9GlcNH
2
conjugated to tetanus toxoid (TT) elicited mouse antibodies that mediated opsonic killing of multiple
S. aureus
strains, while the antibodies that were produced in response to 5GlcNAc- or 9GlcNAc-TT did not mediate opsonic killing. Rabbit antibodies to 9GlcNH
2
-TT bound to PNAG and dPNAG antigens, mediated killing of
S. aureus
and
E. coli
, and protected against
S. aureus
skin abscesses and lethal
E. coli
peritonitis. Chemical synthesis of a series of oligoglucosamine ligands with defined differences in N acetylation allowed us to identify a conjugate vaccine formulation that generated protective immune responses to two of the most challenging bacterial pathogens. This vaccine could potentially be used to engender protective immunity to the broad range of pathogens that produce surface PNAG.
The
Mycobacterium tuberculosis
High-Affinity Iron Importer, IrtA, Contains an FAD-Binding Domain
Authors: Ryndak Michelle B. , Wang Shuishu , Smith Issar , Rodriguez G. Marcela 
Published: December 2009

ABSTRACT

Iron is an essential nutrient not freely available to microorganisms infecting mammals. To overcome iron deficiency, bacteria have evolved various strategies including the synthesis and secretion of high-affinity iron chelators known as siderophores. The siderophores produced and secreted by
Mycobacterium tuberculosis
, exomycobactins, compete for iron with host iron-binding proteins and, together with the iron-regulated ABC transporter IrtAB, are required for the survival of
M. tuberculosis
in iron deficient conditions and for normal replication in macrophages and in mice. This study further characterizes the role of IrtAB in
M. tuberculosis
iron acquisition. Our results demonstrate a role for IrtAB in iron import and show that the amino terminus domain of IrtA is a flavin-adenine dinucleotide-binding domain essential for iron acquisition. These results suggest a model in which the amino terminus of IrtA functions to couple iron transport and assimilation.
Genetic Basis for
Rhizobium etli
CE3 O-Antigen O-Methylated Residues That Vary According to Growth Conditions
Authors: Ojeda Kristylea J. , Box Jodie M. , Noel K. Dale 
Published: December 2009

ABSTRACT

The
Rhizobium etli
CE3 O antigen is a fixed-length heteropolymer with O methylation being the predominant type of sugar modification. There are two O-methylated residues that occur, on average, once per complete O antigen: a multiply O-methylated terminal fucose and 2-O methylation of a fucose residue within a repeating unit. The amount of the methylated terminal fucose decreases and the amount of 2-
O
-methylfucose increases when bacteria are grown in the presence of the host plant,
Phaseolus vulgaris
, or its seed exudates. Insertion mutagenesis was used to identify open reading frames required for the presence of these O-methylated residues. The presence of the methylated terminal fucose required genes
wreA
,
wreB
,
wreC
,
wreD
, and
wreF
, whereas 2-O methylation of internal fucoses required the methyltransferase domain of bifunctional gene
wreM
. Mutants lacking only the methylated terminal fucose, lacking only 2-O methylation, or lacking both the methylated terminal fucose and 2-O methylation exhibited no other lipopolysaccharide structural defects. Thus, neither of these decorations is required for normal O-antigen length, transport, or assembly into the final lipopolysaccharide. This is in contrast to certain enteric bacteria in which the absence of a terminal decoration severely affects O-antigen length and transport.
R. etli
mutants lacking only the methylated terminal fucose were not altered in symbiosis with host
Phaseolus vulgaris
, whereas mutants lacking only 2-
O
-methylfucose exhibited a delay in nodule development during symbiosis. These results support previous conclusions that the methylated terminal fucose is dispensable for symbiosis, whereas 2-O methylation of internal fucoses somehow facilitates early events in symbiosis.
Genome Sequence of a Recently Emerged, Highly Transmissible, Multi-Antibiotic- and Antiseptic-Resistant Variant of Methicillin-Resistant
Staphylococcus aureus
, Sequence Type 239 (TW)
Authors: Holden Matthew T. G. , Lindsay Jodi A. , Corton Craig , Quail Michael A. , Cockfield Joshua D. , Pathak Smriti , Batra Rahul , Parkhill Julian , Bentley Stephen D. , Edgeworth Jonathan D. 
Published: December 2009

ABSTRACT

The 3.1-Mb genome of an outbreak methicillin-resistant
Staphylococcus aureus
(MRSA) strain (TW20) contains evidence of recently acquired DNA, including two large regions (635 kb and 127 kb). The strain is resistant to a wide range of antibiotics, antiseptics, and heavy metals due to resistance genes encoded on mobile genetic elements and also mutations in housekeeping genes.
Expression of Factor H Binding Protein of Meningococcus Responds to Oxygen Limitation through a Dedicated FNR-Regulated Promoter
Authors: Oriente Francesca , Scarlato Vincenzo , Delany Isabel 
Published: December 2009

ABSTRACT

Factor H binding protein (fHBP) is a surface-exposed lipoprotein in
Neisseria meningitidis
, which is a component of several investigational vaccines against serogroup B meningococcus (MenB) currently in development. fHBP enables the bacterium to evade complement-mediated killing by binding factor H, a key downregulator of the complement alternative pathway, and, in addition, fHBP is important for meningococcal survival in the presence of the antimicrobial peptide LL-37. In this study, we investigate the molecular mechanisms involved in transcription and regulation of the fHBP-encoding gene,
fhbp
. We show that the fHBP protein is expressed from two independent transcripts: one bicistronic transcript that includes the upstream gene and a second shorter monocistronic transcript from its own dedicated promoter, P

fhbp

. Transcription from the promoter P

fhbp

responds to oxygen limitation in an FNR-dependent manner, and, accordingly, the FNR protein binds to a P

fhbp

probe
in vitro
. Furthermore, expression in meningococci of a constitutively active FNR mutant results in the overexpression of the fHBP protein. Finally, the analysis of fHBP regulation was extended to a panel of strains expressing different fHBP allelic variants at different levels, and we demonstrate that FNR is involved in the regulation of this antigen in all but one of the strains tested. Our data suggest that oxygen limitation may play an important role in inducing the expression of fHBP from a dedicated FNR-regulated promoter. This implies a role for this protein in microenvironments lacking oxygen, for instance in the submucosa or intracellularly, in addition to its demonstrated role in serum resistance in the blood.
Emerging impact of CTA/perfusion CT on acute stroke thrombolysis in a community hospital
Authors: Brant-Zawadzki M N , Brown D M , Whitaker L A , Peck W W 
Published: December 2009

Our objective was to retrospectively review the emerging role of CT, CTA, and perfusion CT (pCT) in the hyperacute stroke population of a community hospital. We reviewed 50 consecutive patients' records and imaging studies, who were treated with thrombolytic therapy within 6 h of symptom onset. Multidetector CT, CTA, and pCT studies were evaluated. Subsequent CT, magnetic resonance, or angiographic studies when available were correlated. Patients' clinical data at admission and outcomes at discharge were evaluated. Complications were tabulated. Of the 50 patients treated with thrombolytics, 37 had CT/CTA/pCT, the others non-contrast CT only. CT blood volume defect was present in a total of 14 patients, presaging permanent infarct in all. Arterial clot was seen in 28/37 CTAs (carotid “T” 6, MCA 16, vertebrobasilar 6). Viable penumbra was shown in 20/37; rescued penumbra was depicted after treatment in 14.39 patients were treated with intravenous, nine with intra-arterial, two with both forms of thrombolysis. Modified Rankin score showed clinical improvement in 58%, three patients had complete recovery. Subsequent bleed was shown in two (4%), symptomatic in one (2%). Two patients died. Our experience suggests advanced CT is more sensitive to ischemia than routine CT, that salvageable penumbra can be identified, and that triage of patients with acute stroke for thrombolysis with CT/CTA/pCT is more robust than routine CT alone, and may improve outcomes in the community hospital setting.
Molecular cloning, sequence characteristics, and polymorphism analyses of the tyrosinase-related protein 2 / DOPAchrome tautomerase gene of black-boned sheep (Ovis aries)
Authors: Deng Weidong , Tan Yuwen , Wang Xinyu , Xi Dongmei , He Yiduo , Yang Shuli , Mao Huaming , Gao Shizheng 
Published: December 2009

Tyrosinase-related protein 2 (TYRP2) plays a pivotal role in the biosynthesis of eumelanin. Black-boned sheep have excessive melanin and eumelanin, resulting in dark (black) muscles and organs. This study was designed to investigate the effects of variants of the TYRP2 gene on black traits and coat colour of black-boned sheep. Melanin traits were measured in three populations of sheep (Nanping black-boned, Nanping normal, and Romney Marsh) and compared in this study. From the TYRP2 cDNA, all 8 exons and their flanking regions were amplified and characterized. Fifteen single nucleotide polymorphisms (SNPs) were identified in the exons and their flanking regions. Five exonic polymorphic sites, including two synonymous (c.93T>G and c.1140C>T) and three non-synonymous mutations (c.163C>T (p.R55W), c.605G>A (p.R202H), and c.1141A>G (p.T381A)), were retrieved. PCR-RFLP analysis of c.605G>A showed that the frequencies of allele G in the Nanping black-boned, Nanping normal, and Romney Marsh sheep were 0.632, 0.603, and 0.886, respectively. Sheep with the GG genotype had significantly (P < 0.05) lower tyrosinase activity, alkali-soluble melanin content, and ratio of eumelanin : total melanin than sheep with GA and AA genotypes when measured across all investigated samples but not when samples within each population of sheep were compared. However, there was no association of TYRP2 genotype at a single SNP position with coat colour across populations. Nonetheless, the two breeds with higher overall tyrosinase activity did produce darker and more varied coat colours than the breed with lower tyrosinase activity.