bluuebunny/crossref_metadata_2025 huggingface dataset:
Adaptation and Antibiotic Tolerance of Anaerobic Burkholderia pseudomallei
Authors: Hamad Mohamad A. , Austin Chad R. , Stewart Amanda L. , Higgins Mike , Vázquez-Torres Andrés , Voskuil Martin I.
Published: May 2011

The Gram-negative bacterium
Burkholderia pseudomallei
is the etiological agent of melioidosis and is remarkably resistant to most classes of antibacterials. Even after months of treatment with antibacterials that are relatively effective
in vitro
, there is a high rate of treatment failure, indicating that this pathogen alters its patterns of antibacterial susceptibility in response to cues encountered in the host. The pathology of melioidosis indicates that
B. pseudomallei
encounters host microenvironments that limit aerobic respiration, including the lack of oxygen found in abscesses and in the presence of nitric oxide produced by macrophages. We investigated whether
B. pseudomallei
could survive in a nonreplicating, oxygen-deprived state and determined if this physiological state was tolerant of conventional antibacterials.
B. pseudomallei
survived initial anaerobiosis, especially under moderately acidic conditions similar to those found in abscesses. Microarray expression profiling indicated a major shift in the physiological state of hypoxic
B. pseudomallei
, including induction of a variety of typical anaerobic-environment-responsive genes and genes that appear specific to anaerobic
B. pseudomallei
. Interestingly, anaerobic
B. pseudomallei
was unaffected by antibacterials typically used in therapy. However, it was exquisitely sensitive to drugs used against anaerobic pathogens. After several weeks of anaerobic culture, a significant loss of viability was observed. However, a stable subpopulation that maintained complete viability for at least 1 year was established. Thus, during the course of human infection, if a minor subpopulation of bacteria inhabited an oxygen-restricted environment, it might be indifferent to traditional therapy but susceptible to antibiotics frequently used to treat anaerobic infections.
Novel Diamidines with Activity against Babesia divergens
In Vitro
and Babesia microti
In Vivo
Authors: Nehrbass-Stuedli Angela , Boykin David , Tidwell Richard R. , Brun Reto
Published: May 2011

Dicationic diamidines, such as diminazene and pentamidine, are well-studied chemotherapeutic agents with significant activity against parasitic diseases. The
in vitro
activities of novel diamidine compounds against the
Babesia divergens
strains 1903B and 4201 were investigated. The most potent compound, a diphenyl furan, had a 50% inhibitory concentration (IC
50
) of 1.5 ng/ml. In a murine model, several test compounds were effective enough to cure mice infected with
Babesia microti
at a dose of 12.5 and/or 25 mg/kg of body weight given by the subcutaneous route for 4 days. The best antibabesial properties were exhibited by terphenyls, benzimidazoles, diphenyl furans, pentamidine, and pentamidine analogues.
Inducible
l
-Alanine Exporter Encoded by the Novel Gene
ygaW
(
alaE
) in Escherichia coli
Authors: Hori Hatsuhiro , Yoneyama Hiroshi , Tobe Ryuta , Ando Tasuke , Isogai Emiko , Katsumata Ryoichi
Published: April 2011

We previously isolated a mutant hypersensitive to
l
-alanyl-
l
-alanine from a non-
l
-alanine-metabolizing
Escherichia coli
strain and found that it lacked an inducible
l
-alanine export system. Consequently, this mutant showed a significant accumulation of intracellular
l
-alanine and a reduction in the
l
-alanine export rate compared to the parent strain. When the mutant was used as a host to clone a gene(s) that complements the dipeptide-hypersensitive phenotype, two uncharacterized genes,
ygaW
and
ytfF
, and two characterized genes,
yddG
and
yeaS
, were identified. Overexpression of each gene in the mutant resulted in a decrease in the intracellular
l
-alanine level and enhancement of the
l
-alanine export rate in the presence of the dipeptide, suggesting that their products function as exporters of
l
-alanine. Since
ygaW
exhibited the most striking impact on both the intra- and the extracellular
l
-alanine levels among the four genes identified, we disrupted the
ygaW
gene in the non-
l
-alanine-metabolizing strain. The resulting isogenic mutant showed the same intra- and extracellular
l
-alanine levels as observed in the dipeptide-hypersensitive mutant obtained by chemical mutagenesis. When each gene was overexpressed in the wild-type strain, which does not intrinsically excrete alanine, only the
ygaW
gene conferred on the cells the ability to excrete alanine. In addition, expression of the
ygaW
gene was induced in the presence of the dipeptide. On the basis of these results, we concluded that YgaW is likely to be the physiologically most relevant exporter for
l
-alanine in
E. coli
and proposed that the gene be redesignated
alaE
for
ala
nine
e
xport.
Identification of Mycoparasitism-Related Genes in Trichoderma atroviride
Authors: Reithner Barbara , Ibarra-Laclette Enrique , Mach Robert L. , Herrera-Estrella Alfredo
Published: April 2011

A high-throughput sequencing approach was utilized to carry out a comparative transcriptome analysis of
Trichoderma atroviride
IMI206040 during mycoparasitic interactions with the plant-pathogenic fungus
Rhizoctonia solani
. In this study, transcript fragments of 7,797
Trichoderma
genes were sequenced, 175 of which were host responsive. According to the functional annotation of these genes by KOG (eukaryotic orthologous groups), the most abundant group during direct contact was “metabolism.” Quantitative reverse transcription (RT)-PCR confirmed the differential transcription of 13 genes (including
swo1
, encoding an expansin-like protein;
axe1
, coding for an acetyl xylan esterase; and homologs of genes encoding the aspartyl protease
papA
and a trypsin-like protease,
pra1
) in the presence of
R. solani
. An additional relative gene expression analysis of these genes, conducted at different stages of mycoparasitism against
Botrytis cinerea
and
Phytophthora capsici
, revealed a synergistic transcription of various genes involved in cell wall degradation. The similarities in expression patterns and the occurrence of regulatory binding sites in the corresponding promoter regions suggest a possible analog regulation of these genes during the mycoparasitism of
T. atroviride
. Furthermore, a chitin- and distance-dependent induction of
pra1
was demonstrated.
Molecular Characterization of Cronobacter Lipopolysaccharide O-Antigen Gene Clusters and Development of Serotype-Specific PCR Assays
Authors: Jarvis K. G. , Grim C. J. , Franco A. A. , Gopinath G. , Sathyamoorthy V. , Hu L. , Sadowski J. A. , Lee C. S. , Tall B. D.
Published: April 2011

Cronobacter
(formerly
Enterobacter sakazakii
) is a recently defined genus consisting of six species,
C. sakazakii
,
C. malonaticus
,
C. dublinensis
,
C. muytjensii
,
C. turicensis
, and
Cronobacter
genomospecies 1. In this study, MboII restriction fragment length polymorphism (RFLP) patterns of O-antigen gene clusters, located between
galF
and
gnd
, were used to identify serotypes in
Cronobacter
spp. Seven O-antigen RFLP clusters were generated, including three
C. sakazakii
clusters, previously identified as serotypes O1, O2, and O3. The O-antigen regions of six strains with unique RFLP patterns, including two
C. sakazakii
strains, two
C. malonaticus
strains, one
C. turicensis
strain, and one
C. muytjensii
strain, revealed three O-antigen gene clusters shared among
Cronobacter
species. PCR assays were developed, targeting the
wzx
O-antigen polymerase gene, and used to screen 231
Cronobacter
strains to determine the frequency of these newly identified serotypes.
Bacterial Subfamily of LuxR Regulators That Respond to Plant Compounds
Authors: Subramoni Sujatha , Gonzalez Juan F. , Johnson Aaron , Péchy-Tarr Maria , Rochat Laurène , Paulsen Ian , Loper Joyce E. , Keel Christoph , Venturi Vittorio
Published: April 2011

Pseudomonas fluorescens
are rhizobacteria known for their biocontrol properties. Several antimicrobial functions are crucial for this process, and the experiments described here investigate the modulation of their expression during the plant-bacterium interaction. The role of a LuxR family regulator in interkingdom signaling has been investigated using genome-scale transcriptome analysis, gene promoter studies
in vivo
and
in vitro
, biocontrol assays, and response to plant compounds. PsoR, a LuxR solo or orphan regulator of
P. fluorescens
, was identified. PsoR is solubilized and activates a
lux
-box-containing promoter only in the presence of macerated plants, suggesting the presence of a plant molecule(s) that most likely binds to PsoR. Gene expression profiles revealed that genes involved in the inhibition of plant pathogens were affected by PsoR, including a chitinase gene, iron metabolism genes, and biosynthetic genes of antifungal compounds. 2,4-Diacetylphloroglucinol production is PsoR dependent both
in vitro
and
in vivo
.
psoR
mutants were significantly reduced for their ability to protect wheat plants from root rot, and damping-off caused by
Pythium ultimum
infection. PsoR most likely senses a molecule(s) in the plant and modulates expression of genes that have a role in biocontrol. PsoR and related proteins form a subfamily of LuxR family regulators in plant-associated bacteria.
Squalene-Hopene Cyclases
Authors: Siedenburg Gabriele , Jendrossek Dieter
Published: April 2011

Hopanoids and sterols are members of a large group of cyclic triterpenoic compounds that have important functions in many prokaryotic and eukaryotic organisms. They are biochemically synthesized from linear precursors (squalene, 2,3-oxidosqualene) in only one enzymatic step that is catalyzed by squalene-hopene cyclase (SHC) or oxidosqualene cyclase (OSC). SHCs and OSCs are related in amino acid sequences and probably are derived from a common ancestor. The SHC reaction requires the formation of five ring structures, 13 covalent bonds, and nine stereo centers and therefore is one of the most complex one-step enzymatic reactions. We summarize the knowledge of the properties of triterpene cyclases and details of the reaction mechanism ofAlicyclobacillus acidocaldariusSHC. Properties of other SHCs are included.
Increased Fitness and Alteration of Metabolic Pathways during Bacillus subtilis Evolution in the Laboratory
Authors: Maughan Heather , Nicholson Wayne L.
Published: April 2011

Five batch cultures of
Bacillus subtilis
were subjected to evolution in the laboratory for 6,000 generations under conditions repressing sporulation in complex liquid medium containing glucose. Between generations 1,000 and 2,000, variants with a distinct small-colony morphology arose and swept through four of the five populations that had been previously noted for their loss of sporulation (H. Maughan et al., Genetics 177:937-948, 2007). To better understand the nature of adaptation in these variants, individual strains were isolated from one population before (WN715) and after (WN716) the sweep. In addition to colony morphology, strains WN715 and WN716 differed in their motility, aerotaxis, and cell morphology. Competition experiments showed that strain WN716 had evolved a distinct fitness advantage over the ancestral strain and strain WN715 during growth and the transition to the postexponential growth phase, which was more pronounced when WN715 was present in the coculture. Microarray analyses revealed candidate genes in which mutations may have produced some of the observed phenotypes. For example, loss of motility in WN716 was accompanied by decreased transcription of all flagellar, motility, and chemotaxis genes on the microarray. Transcription of
alsS
and
alsD
was also lower in strain WN716, and the predicted loss of acetoin production and enhanced acetate production was confirmed by high-performance liquid chromatography (HPLC) analysis. The results suggested that the derived colony morphology of strain WN716 was associated with increased fitness, the alteration of several metabolic pathways, and the loss of a typical postexponential-phase response.
Modular Synthase-Encoding Gene Involved in α-Olefin Biosynthesis in Synechococcus sp. Strain PCC 7002
Authors: Mendez-Perez Daniel , Begemann Matthew B. , Pfleger Brian F.
Published: April 2011

A gene involved in the production of medium-chain α-olefins was identified in the cyanobacterium
Synechococcus
sp. strain PCC 7002. The gene encodes a large multidomain protein with homology to type I polyketide synthases, suggesting a route for hydrocarbon biosynthesis from fatty acids via an elongation decarboxylation mechanism.
BTI1, an Azoreductase with pH-Dependent Substrate Specificity
Authors: Johansson Hans E. , Johansson Mary K. , Wong Albert C. , Armstrong Eliana S. , Peterson Erik J. , Grant Richard E. , Roy Margaret A. , Reddington Mark V. , Cook Ronald M.
Published: April 2011

The group II azoreductase BTI1 utilizes NADPH to directly cleave azo bonds in water-soluble azo dyes, including quenchers of fluorescence. Unexpectedly, optimal reduction was dye specific, ranging from a pH of <5.5 for Janus green B, to pH 6.0 for methyl red, methyl orange, and BHQ-10, to pH >8.3 for flame orange.
Effects of Bacterial Microflora of the Lower Digestive Tract of Free-Range Waterfowl on Influenza Virus Activation
Authors: King Marcus D. , Guentzel M. Neal , Arulanandam Bernard P. , Bodour Adria A. , Brahmakshatriya Vinayak , Lupiani Blanca , Chambers James P.
Published: April 2011

Proteolytic cleavage activation of influenza virus hemagglutinin (HA0) is required for cell entry via receptor-mediated endocytosis. Despite numerous studies describing bacterial protease-mediated influenza A viral activation in mammals, very little is known about the role of intestinal bacterial flora of birds in hemagglutinin cleavage/activation. Therefore, the cloaca of wild waterfowl was examined for (i) representative bacterial types and (ii) their ability to cleave in a “trypsin-like” manner the precursor viral hemagglutinin molecule (HA0). Using radiolabeled HA0, bacterial secretion-mediated trypsin-like conversion of HA0 to HA1 and HA2 peptide products was observed to various degrees in 42 of 44 bacterial isolates suggestive of influenza virus activation in the cloaca of wild waterfowl. However, treatment of uncleaved virus with all bacterial isolates gave rise to substantially reduced emergent virus progeny compared with what was expected. Examination of two isolates exhibiting pronounced trypsin-like conversion of HA0 to HA1 and HA2 peptide products and low infectivity revealed lipase activity to be present. Because influenza virus possesses a complex lipid envelope, the presence of lipid hydrolase activity could in part account for the observed less-than-expected level of viable progeny. A thorough characterization of respective isolate protease HA0 hydrolysis products as well as other resident activities (i.e., lipase) is ongoing such that the role of these respective contributors in virus activation/inactivation can be firmly established.
Diversity of Somatic Coliphages in Coastal Regions with Different Levels of Anthropogenic Activity in São Paulo State, Brazil
Authors: Burbano-Rosero E. M. , Ueda-Ito M. , Kisielius J. J. , Nagasse-Sugahara T. K. , Almeida B. C. , Souza C. P. , Markman C. , Martins G. G. , Albertini L. , Rivera I. N. G.
Published: April 2011

Bacteriophages are the most abundant and genetically diverse viruses on Earth, with complex ecology in both quantitative and qualitative terms. Somatic coliphages (SC) have been reported to be good indicators of fecal pollution in seawater. This study focused on determining the concentration of SC and their diversity by electron microscopy of seawater, plankton, and bivalve samples collected at three coastal regions in São Paulo, Brazil. The SC counts varied from <1 to 3.4 × 103PFU/100 ml in seawater (73 samples tested), from <1 to 4.7 × 102PFU/g in plankton (46 samples tested), and from <1 to 2.2 × 101PFU/g in bivalves (11 samples tested). In seawater samples, a relationship between the thermotolerant coliforms andEscherichia coliand SC was observed at the three regions (P= 0.0001) according to the anthropogenic activities present at each region. However, SC were found in plankton samples from three regions: Baixada Santista (17/20), Canal de São Sebastião (6/14), and Ubatuba (3/12). In seawater samples collected from Baixada Santista, four morphotypes were observed: A1 (4.5%), B1 (50%), C1 (36.4%), and D1 (9.1%). One coliphage,Siphoviridaetype T1, had the longest tail: between 939 and 995 nm. In plankton samples,Siphoviridae(65.8%),Podoviridae(15.8%),Microviridae(15.8%), andMyoviridae(2.6%) were found. In bivalves, only the morphotype B1 was observed. These SC were associated with enteric hosts: enterobacteria,E. coli,Proteus,Salmonella, andYersinia. Baixada Santista is an area containing a high level of fecal pollution compared to those in the Canal de São Sebastião and Ubatuba. This is the first report of coliphage diversity in seawater, plankton, and bivalve samples collected from São Paulo coastal regions. A better characterization of SC diversity in coastal environments will help with the management and evaluation of the microbiological risks for recreation, seafood cultivation, and consumption.
Bacterial Diversity across Individual Lichens
Authors: Mushegian Alexandra A. , Peterson Celeste N. , Baker Christopher C. M. , Pringle Anne
Published: April 2011

Symbioses are unique habitats for bacteria. We surveyed the spatial diversity of bacterial communities across multiple individuals of closely related lichens using terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing. Centers of lichens house richer, more consistent assemblages than species-poor and compositionally disparate lichen edges, suggesting that ecological succession plays a role in structuring these communities.
Simple Enrichment System for Hydrogen Producers
Authors: Tolvanen Katariina E. S. , Mangayil Rahul K. , Karp Matti T. , Santala Ville P.
Published: April 2011

This study presents a simple enrichment system where gas pressure produced by microbes performs functions that are normally done by labor. The system was tested with
Escherichia coli
strains with different hydrogen production and growth capabilities. The results show that the system can enrich the best hydrogen producer.
Type II Toxoplasma gondiiKU80Knockout Strains Enable Functional Analysis of Genes Required for Cyst Development and Latent Infection
Authors: Fox Barbara A. , Falla Alejandra , Rommereim Leah M. , Tomita Tadakimi , Gigley Jason P. , Mercier Corinne , Cesbron-Delauw Marie-France , Weiss Louis M. , Bzik David J.
Published: April 2011

Type IIToxoplasma gondiiKU80knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80Δhxgprtstrain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80Δhxgprtstrain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4,GRA6,ROP7, andtgd057) that encode characterized CD8+T cell epitopes that elicit corresponding antigen-specific CD8+T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8+T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains withGRA4and/orGRA6deleted. Complementation of the Δgra4and Δgra6mutant strains using a functional allele of the deletedGRAcoding region placed under the control of the endogenousUPRTlocus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80Δhxgprtgenetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.
Three Mitochondrial DNA Polymerases Are Essential for Kinetoplast DNA Replication and Survival of Bloodstream Form Trypanosoma brucei
Authors: Bruhn David F. , Sammartino Mark P. , Klingbeil Michele M.
Published: April 2011

Trypanosoma brucei
, the causative agent of human African trypanosomiasis, has a complex life cycle that includes multiple life cycle stages and metabolic changes as the parasite switches between insect vector and mammalian host. The parasite's single mitochondrion contains a unique catenated mitochondrial DNA network called kinetoplast DNA (kDNA) that is composed of minicircles and maxicircles. Long-standing uncertainty about the requirement of kDNA in bloodstream form (BF)
T. brucei
has recently eroded, with reports of posttranscriptional editing and subsequent translation of kDNA-encoded transcripts as essential processes for BF parasites. These studies suggest that kDNA and its faithful replication are indispensable for this life cycle stage. Here we demonstrate that three kDNA replication proteins (mitochondrial DNA polymerases IB, IC, and ID) are required for BF parasite viability. Silencing of each polymerase was lethal, resulting in kDNA loss, persistence of prereplication DNA monomers, and collapse of the mitochondrial membrane potential. These data demonstrate that kDNA replication is indeed crucial for BF
T. brucei
. The contributions of mitochondrial DNA polymerases IB, IC, and ID to BF parasite viability suggest that these and other kDNA replication proteins warrant further investigation as a new class of targets for the development of antitrypanosomal drugs.
Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate
N
-Acetyltransferase from Trypanosoma brucei
Authors: Mariño Karina , Güther M. Lucia Sampaio , Wernimont Amy K. , Qiu Wei , Hui Raymond , Ferguson Michael A. J.
Published: April 2011

A gene predicted to encode
Trypanosoma brucei
glucosamine 6-phosphate
N
-acetyltransferase (
TbGNA1
; EC 2.3.1.4) was cloned and expressed in
Escherichia coli
. The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form
T. brucei
GNA1
conditional null mutant was constructed and shown to be unable to sustain growth
in vitro
under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the
TbGNA1
mutant under nonpermissive conditions revealed that poly-
N
-acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed.
Burkholderia cenocepacia Phenotypic Clonal Variation during a 3.5-Year Colonization in the Lungs of a Cystic Fibrosis Patient
Authors: Coutinho Carla P. , de Carvalho Carla C. C. R. , Madeira Andreia , Pinto-de-Oliveira Ana , Sá-Correia Isabel
Published: May 2011

Chronic lung infection is the major cause of morbidity and premature mortality in cystic fibrosis (CF) patients. Bacteria of the
Burkholderia cepacia
complex are the most threatening pathogens in CF, and a better understanding of how these bacteria adapt to the CF airway environment and resist the host defense mechanisms and therapeutically administered antibiotics is crucial. To provide clues to the adaptive strategies adopted by
Burkholderia cenocepacia
during long-term colonization, we carried out a phenotypic assessment of 11 clonal variants obtained at the major Portuguese CF Center in Lisbon from sputa of the same CF patient during 3.5 years of colonization of the lungs, until the patient's death with cepacia syndrome. Phenotypic characterization included susceptibility assays against different classes of antimicrobials and characterization of cell motility, cell hydrophobicity and zeta potential, colony and cell morphology, fatty acid composition, growth under iron limitation/load conditions, exopolysaccharide production, and size of the biofilms formed. The results suggest the occurrence of clonal expansion during long-term colonization. For a number of the characteristics tested, no isolation time-dependent consistent alteration pattern could be identified. However, the values for antimicrobial susceptibility and swarming motility for the first
B. cenocepacia
isolate, thought to have initiated the infection, were consistently above those for the clonal variants obtained during the course of infection, and the opposite was found for the zeta potential. The adaptive strategy for long-term colonization, described here for the first time, involved the alteration of membrane fatty acid composition, in particular a reduction of the degree of fatty acid saturation, in the
B. cenocepacia
variants retrieved, along with the deterioration of pulmonary function and severe oxygen limitation.
Tumor Necrosis Factor Alpha Production from CD8+T Cells Mediates Oviduct Pathological Sequelae following Primary Genital Chlamydia muridarum Infection
Authors: Murthy Ashlesh K. , Li Weidang , Chaganty Bharat K. R. , Kamalakaran Sangamithra , Guentzel M. Neal , Seshu J. , Forsthuber Thomas G. , Zhong Guangming , Arulanandam Bernard P.
Published: May 2011

The immunopathogenesis ofChlamydia trachomatis-induced oviduct pathological sequelae is not well understood. Mice genetically deficient in perforin (perforin−/−mice) or tumor necrosis factor alpha (TNF-α) production (TNF-α−/−mice) displayed comparable vaginal chlamydial clearance rates but significantly reduced oviduct pathology (hydrosalpinx) compared to that of wild-type mice. Since both perforin and TNF-α are effector mechanisms of CD8+T cells, we evaluated the role of CD8+T cells during genitalChlamydia muridaruminfection and oviduct sequelae. Following vaginal chlamydial challenge, (i) mice deficient in TAP I (and therefore the major histocompatibility complex [MHC] I pathway and CD8+T cells), (ii) wild-type mice depleted of CD8+T cells, and (iii) mice genetically deficient in CD8 (CD8−/−mice) all displayed similar levels of vaginal chlamydial clearance but significantly reduced hydrosalpinx, compared to those of wild-type C57BL/6 mice, suggesting a role for CD8+T cells in chlamydial pathogenesis. Repletion of CD8−/−mice with wild-type or perforin−/−, but not TNF-α−/−, CD8+T cells at the time of challenge restored hydrosalpinx to levels observed in wild-type C57BL/6 mice, suggesting that TNF-α production from CD8+T cells is important for pathogenesis. Additionally, repletion of TNF-α−/−mice with TNF-α+/+CD8+T cells significantly enhanced the incidence of hydrosalpinx and oviduct dilatation compared to those of TNF-α−/−mice but not to the levels found in wild-type mice, suggesting that TNF-α production from CD8+T cells and non-CD8+cells cooperates to induce optimal oviduct pathology following genital chlamydial infection. These results provide compelling new evidence supporting the contribution of CD8+T cells and TNF-α production toChlamydia-induced reproductive tract sequelae.
The Anaerobe-Specific Orange Protein Complex of Desulfovibrio vulgaris Hildenborough Is Encoded by Two Divergent Operons Coregulated by σ
54
and a Cognate Transcriptional Regulator
Authors: Fiévet Anouchka , My Laetitia , Cascales Eric , Ansaldi Mireille , Pauleta Sofia R. , Moura Isabel , Dermoun Zorah , Bernard Christophe S. , Dolla Alain , Aubert Corinne
Published: April 2011

Analysis of sequenced bacterial genomes revealed that the genomes encode more than 30% hypothetical and conserved hypothetical proteins of unknown function. Among proteins of unknown function that are conserved in anaerobes, some might be determinants of the anaerobic way of life. This study focuses on two divergent clusters specifically found in anaerobic microorganisms and mainly composed of genes encoding conserved hypothetical proteins. We show that the two gene clusters
DVU2103-DVU2104-DVU2105
(
orp2
) and
DVU2107-DVU2108-DVU2109
(
orp1
) form two divergent operons transcribed by the σ
54
-RNA polymerase. We further demonstrate that the σ
54
-dependent transcriptional regulator DVU2106, located between
orp1
and
orp2
, collaborates with σ
54
-RNA polymerase to orchestrate the simultaneous expression of the divergent
orp
operons. DVU2106, whose structural gene is transcribed by the σ
70
-RNA polymerase, negatively retrocontrols its own expression. By using an endogenous pulldown strategy, we identify a physiological complex composed of DVU2103, DVU2104, DVU2105, DVU2108, and DVU2109. Interestingly, inactivation of DVU2106, which is required for
orp
operon transcription, induces morphological defects that are likely linked to the absence of the ORP complex. A putative role of the ORP proteins in positioning the septum during cell division is discussed.
Asp2 and Asp3 Interact Directly with GspB, the Export Substrate of the Streptococcus gordonii Accessory Sec System
Authors: Yen Yihfen T. , Seepersaud Ravin , Bensing Barbara A. , Sullam Paul M.
Published: April 2011

GspB is a serine-rich glycoprotein adhesin of
Streptococcus gordonii
that is exported to the bacterial surface by the accessory Sec system. This dedicated export pathway is comprised of seven components (SecA2, SecY2, and five accessory Sec proteins [Asp1 to Asp5]). The latter proteins have no known homologs beyond the Asps of other species. Asp1 to Asp3 are absolutely required for export of the substrate GspB, but their roles in this process are unknown. Using copurification analysis and far-Western blotting, we found that Asp2 and Asp3 could individually bind the serine-rich repeat (SRR) domains of GspB. Deletion of both SRR regions of GspB led to a decrease in its export, suggesting that binding of the Asps to the SRR regions is important for GspB transport by the accessory Sec system. The Asps also bound a heterologous substrate for the accessory Sec system containing a slow-folding MalE variant, but they did not bind wild-type MalE. The combined results indicate that the Asps may recognize the export substrate through preferential interactions with its unstructured or unfolded regions. Glycosylation of the SRR domains on GspB prevented Asp binding, suggesting that binding of the Asps to the preprotein occurs prior to its full glycosylation. Together, these findings suggest that Asp2 and Asp3 are likely to function in part as chaperones in the early phase of GspB transport.
The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities
Authors: Duong-Ly Krisna C. , Gabelli Sandra B. , Xu WenLian , Dunn Christopher A. , Schoeffield Andrew J. , Bessman Maurice J. , Amzel L. Mario
Published: April 2011

A Nudix enzyme from
Bacillus cereus
(NCBI RefSeq accession no.
NP_831800
) catalyzes the hydrolysis of CDP-choline to produce CMP and phosphocholine. Here, we show that in addition, the enzyme has a 3′→5′ RNA exonuclease activity. The structure of the free enzyme, determined to a 1.8-Å resolution, shows that the enzyme is an asymmetric dimer. Each monomer consists of two domains, an N-terminal helical domain and a C-terminal Nudix domain. The N-terminal domain is placed relative to the C-terminal domain such as to result in an overall asymmetric arrangement with two distinct catalytic sites: one with an “enclosed” Nudix pyrophosphatase site and the other with a more open, less-defined cavity. Residues that may be important for determining the asymmetry are conserved among a group of uncharacterized Nudix enzymes from Gram-positive bacteria. Our data support a model where CDP-choline hydrolysis is catalyzed by the enclosed Nudix site and RNA exonuclease activity is catalyzed by the open site. CDP-Chase is the first identified member of a novel Nudix family in which structural asymmetry has a profound effect on the recognition of substrates.
Dual Function of a Tip Fimbrillin of Actinomyces in Fimbrial Assembly and Receptor Binding
Authors: Wu Chenggang , Mishra Arunima , Yang Jinghua , Cisar John O. , Das Asis , Ton-That Hung
Published: April 2011

Interaction ofActinomyces oriswith salivary proline-rich proteins (PRPs), which serve as fimbrial receptors, involves type 1 fimbriae. Encoded by the gene locusfimQ-fimP-srtC1, the type 1 fimbria is comprised of the fimbrial shaft FimP and the tip fimbrillin FimQ. Fimbrial polymerization requires the fimbria-specific sortase SrtC1, which catalyzes covalent linkage of fimbrial subunits. Using genetics, biochemical methods, and electron microscopy, we provide evidence that the tip fimbrillin, FimQ, is involved in fimbrial assembly and interaction with PRPs. Specifically, while deletion offimPcompletely abolished the type 1 fimbrial structures, surface display of monomeric FimQ was not affected by this mutation. Surprisingly, deletion offimQsignificantly reduced surface assembly of the type 1 fimbriae. This defect was rescued by recombinant FimQ ectopically expressed from a plasmid. In agreement with the role of type 1 fimbriae in binding to PRPs, aggregation ofA. oriswith PRP-coated beads was abrogated in cells lackingsrtC1orfimP. This aggregation defect of the ΔfimPmutant was mainly due to significant reduction of FimQ on the bacterial surface, as the aggregation was not observed in a strain lackingfimQ. Increasing expression of FimQ in the ΔfimPmutant enhanced aggregation, while overexpression of FimP in the ΔfimQmutant did not. Furthermore, recombinant FimQ, not FimP, bound surface-associated PRPs in a dose-dependent manner. Thus, not only does FimQ function as the major adhesin of the type 1 fimbriae, it also plays an important role in fimbrial assembly.
Binding and Spreading of ParB on DNA Determine Its Biological Function in Pseudomonas aeruginosa
Authors: Kusiak Magdalena , Gapczyńska Anna , Płochocka Danuta , Thomas Christopher M. , Jagura-Burdzy Grażyna
Published: April 2011

ParB protein of
Pseudomonas aeruginosa
belongs to a widely represented ParB family of chromosomally and plasmid-encoded partitioning type IA proteins. Ten putative
parS
sites are dispersed in the
P. aeruginosa
chromosome, with eight of them localizing in the
oriC
domain. After binding to
parS
, ParB spreads on the DNA, causing transcriptional silencing of nearby genes (A. A. Bartosik et al., J. Bacteriol. 186:6983–6998, 2004). We have studied ParB derivatives impaired in spreading either due to loss of DNA-binding ability or oligomerization. We defined specific determinants outside of the helix-turn-helix motif responsible for DNA binding. Analysis confirmed the localization of the main dimerization domain in the C terminus of ParB but also mapped another self-interactive domain in the N-terminal domain. Reverse genetics were used to introduce five
parB
alleles impaired in spreading into the
P. aeruginosa
chromosome. The single amino acid substitutions in ParB causing a defect in oligomerization but not in DNA binding caused a chromosome segregation defect, slowed the growth rate, and impaired motilities, similarly to the pleiotropic phenotype of
parB
-null mutants, indicating that the ability to spread is vital for ParB function in the cell. The toxicity of ParB overproduction in
Pseudomonas
spp. is not due to the spreading since several ParB derivatives defective in oligomerization were still toxic for
P. aeruginosa
when provided in excess.
CheY3 of Borrelia burgdorferi Is the Key Response Regulator Essential for Chemotaxis and Forms a Long-Lived Phosphorylated Intermediate
Authors: Motaleb M. A. , Sultan Syed Z. , Miller Michael R. , Li Chunhao , Charon Nyles W.
Published: April 2011

Spirochetes have a unique cell structure: These bacteria have internal periplasmic flagella subterminally attached at each cell end. How spirochetes coordinate the rotation of the periplasmic flagella for chemotaxis is poorly understood. In other bacteria, modulation of flagellar rotation is essential for chemotaxis, and phosphorylation-dephosphorylation of the response regulator CheY plays a key role in regulating this rotary motion. The genome of the Lyme disease spirochete
Borrelia burgdorferi
contains multiple homologues of chemotaxis genes, including three copies of
cheY
, referred to as
cheY1
,
cheY2
, and
cheY3
. To investigate the function of these genes, we targeted them separately or in combination by allelic exchange mutagenesis. Whereas wild-type cells ran, paused (flexed), and reversed, cells of all single, double, and triple mutants that contained an inactivated
cheY3
gene constantly ran. Capillary tube chemotaxis assays indicated that only those strains with a mutation in
cheY3
were deficient in chemotaxis, and
cheY3
complementation restored chemotactic ability.
In vitro
phosphorylation assays indicated that CheY3 was more efficiently phosphorylated by CheA2 than by CheA1, and the CheY3-P intermediate generated was considerably more stable than the CheY-P proteins found in most other bacteria. The results point toward CheY3 being the key response regulator essential for chemotaxis in
B. burgdorferi
. In addition, the stability of CheY3-P may be critical for coordination of the rotation of the periplasmic flagella.
Do the mercaptocarbene (H–C–S–H) and selenocarbene (H–C–Se–H) congeners of hydroxycarbene (H–C–O–H) undergo 1,2-H-tunneling?
Authors: Sarka János , Császár Attila G. , Schreiner Peter R.
Published: May 2011

The principal purpose of this investigation is the determination of the tunneling half-lives of the trans-HCSH → H2CS and the trans-HCSeH → H2CSe unimolecular isomerization reactions at temperatures close to 0 K. To aid these determinations, accurate electronic structure computations were performed, with electron correlation treatments as extensive as CCSDT(Q) and basis sets as large as aug-cc-pCV5Z, for the isomers of [H,H,C,S] and [H,H,C,Se] on their lowest singlet surfaces and for the appropriate transition states yielding structural data for key stationary points characterizing the isomerization reactions. The computational results were subjected to a focal-point analysis (FPA) that yields accurate relative energies with uncertainty estimates. The tunneling half-lives were determined by a simple Eckart-barrier approach and via the more sophisticated though still one-dimensional Wentzel–Kramers–Brillouin (WKB) approximation. Only stationary-point information is needed for the former while an intrinsic reaction path (IRP) is necessary for the latter approach. Both protocols suggest that, unlike for the parent hydroxymethylene (HCOH), at the low temperatures of matrix isolation experiments no tunneling will be observable for the trans-HCSH and trans-HCSeH systems.
Distribution of 10-year and lifetime predicted risk for cardiovascular disease in the Indian Sentinel Surveillance Study population (cross-sectional survey results)
Authors: Jeemon Panniyammakal , Prabhakaran Dorairaj , Huffman Mark D , Ramakrishnan Lakshmy , Goenka Shifalika , Thankappan K R , Mohan V , Joshi Prashant P , Mohan B V M , Ahmed F , Ramanathan Meera , Ahuja R , Chaturvedi Vivek , Lloyd-Jones Donald M , Reddy K Srinath
Published: April 2011

Introduction

Cardiovascular disease (CVD) prevention guidelines recommend lifetime risk stratification for primary prevention of CVD, but no such risk stratification has been performed in India to date.

Methods

The authors estimated short-term and lifetime predicted CVD risk among 10 054 disease-free, adult Indians in the 20–69-year age group who participated in a nationwide risk factor surveillance study. The study population was then stratified into high short-term (≥10% 10-year risk or diabetes), low short-term (<10%)/high lifetime and low short-term/low lifetime CVD risk groups.

Results

The mean age (SD) of the study population (men=63%) was 40.8±10.9 years. High short-term risk for coronary heart disease was prevalent in more than one-fifth of the population (23.5%, 95% CI 22.7 to 24.4). Nearly half of individuals with low short-term predicted risk (48.2%, 95% CI 47.1 to 49.3) had a high predicted lifetime risk for CVD. While the proportion of individuals with all optimal risk factors was 15.3% (95% CI 14.6% to 16.0%), it was 20.6% (95% CI 18.7% to 22.6%) and 8.8% (95% CI 7.7% to 10.5%) in the highest and lowest educational groups, respectively.

Conclusion

Approximately one in two men and three in four women in India had low short-term predicted risks for CVD in this national study, based on aggregate risk factor burden. However, two in three men and one in two women had high lifetime predicted risks for CVD, highlighting a key limitation of short-term risk stratification.
Wood quality in artificially inclined 1-year-old trees of Eucalyptus regnans — differences in tension wood and opposite wood properties
Authors: Chauhan Shakti S. , Walker John C.F.
Published: May 2011

This paper presents a new approach to assess wood quality in 1-year-old Eucalyptus regnans F. Muell. Twenty-two seedlings were grown tilted to induce tension wood and acoustic velocity, basic density, longitudinal shrinkage, and volumetric shrinkage of both opposite wood and tension wood were assessed subsequently. Longitudinal growth strains were also estimated in the leaning stems by sawing along the length through the pith and measuring the bending of the two halves. The derived longitudinal growth strain, which varied from 708 to 2319 µε, was uncorrelated with stem and wood characteristics. Wood characteristics differed significantly between upper-side wood (predominantly tension wood) and lower-side wood (opposite wood). Tension wood was characterized by a higher acoustic velocity (high stiffness), basic density, and volumetric shrinkage compared with opposite wood. Tension wood also exhibited significant collapse and dimensional distortion such as twisting. Longitudinal shrinkage exhibited a significant negative relationship with acoustic velocity in opposite wood and a positive relationship with the basic density in tension wood. This new approach has potential in early selection of breeding material with superior normal wood properties from 1-year-old material by isolating the influence of tension wood. This approach can also be useful in understanding the variability in propensity of tension wood production in breeding populations.
An efficient single nucleotide polymorphism assay to diagnose the genomic identity of poplar species and hybrids on the Canadian prairies
Authors: Talbot Patricia , Thompson Stacey Lee , Schroeder William , Isabel Nathalie
Published: May 2011

Hybridization frequently occurs among poplars, both naturally and artificially, hindering identification. Over 32 million clonal poplars, predominantly hybrids, have been planted throughout the Canadian prairies over the past century, making confirmation of genomic identity important. We developed a genotyping assay that rapidly diagnoses four compatible Populus species ( Populus balsamifera L. and Populus deltoides Bartr. ex Marsh.: indigenous, Populus laurifolia Ledeb. and Populus nigra L.: exotics) and their hybrids found throughout this ecozone. First, we sequenced 23 genes from representative provenances of the four Populus species to discover single nucleotide polymorphisms (SNPs). Second, we developed and validated a medium-throughput genotyping assay of 26 diagnostic SNPs within these genes. We used this assay to genotype 198 trees from natural populations as well as 30 clones (pure species and hybrids), including those broadly distributed by Agriculture and Agri-Food Canada’s Agroforestry Development Centre since 1910. This suite of SNPs has the resolving power to correctly identify pure species and hybrids of Populus. We confirmed the identity of clones of well-documented origin, complex hybrids with exotic components, and paternity of open-pollinated progenies from breeding programs. This diagnostic tool should prove useful for efficient molecular fingerprinting of breeding material and for further studies of interspecific gene flow on the Canadian prairies.
Comparing selection system and diameter-limit cutting in uneven-aged northern hardwoods using computer simulation
Authors: Bohn Kimberly K. , Nyland Ralph D. , Yanai Ruth D.
Published: May 2011

Comparisons of selection system and diameter-limit cutting based on trials in specific settings have often yielded conflicting results. We used a simulation approach to evaluate sawtimber production over three cutting cycles on 10 sugar maple ( Acer saccharum Marsh.) dominated plots of varying initial forest structure. Treatments on each plot included light, moderate, and heavy intensities of selection system silviculture and diameter-limit cutting. Harvested sawtimber volumes were initially higher on all plots using diameter-limit cutting, but selection system outperformed diameter-limit cutting at later entries on 7 of the 10 plots. Volume differences between cutting types ranged among plots from 0.3 to 26 m3·ha–1, equating to a less than 1% to as much as a twofold difference. Average volumes from selection system at later entries were 20%–40% greater than diameter-limit cutting, due in part to consistent production in large sawtimber (≥46 cm). Yields from real stands could vary from these simulations where mortality losses (not modeled here) differ between treatments as a result of competition or logging damage. Findings suggest that cumulative sawtimber volumes from repeated selection system silviculture could eventually surpass that of diameter-limit cutting, but at a rate depending on initial stand conditions and harvesting intensity.
Pre-fire forest conditions and fire severity as determinants of the quality of burned forests for deadwood-dependent species: the case of the black-backed woodpecker
Authors: Nappi Antoine , Drapeau Pierre
Published: May 2011

Burned forests represent high-quality habitats for many deadwood-dependent species. Yet, post-fire conditions may vary greatly within and among burns and thereby may affect habitat suitability for these species. We studied habitat selection of nesting black-backed woodpeckers ( Picoides arcticus Swainson) in recently burned spruce-dominated boreal forests. Our objectives were to (i) identify factors involved in snag selection for both nesting and foraging and (ii) examine selection of nest sites within the burned landscape. A total of 92 nests and 1612 foraging observations were used to investigate snag selection. Our results show that both pre-fire forest conditions and fire severity are important in determining the quality of burned forests for black-backed woodpeckers. This species selected large snags for both nesting (>20 cm DBH) and foraging (>15 cm DBH). Woodpeckers selected deciduous and degraded “pre-fire” snags for nesting whereas black spruce snags that had been created by fire and that were moderately burned were preferred for foraging. Nest sites were concentrated in burned mature stands and supported higher densities of large snags (e.g., >15 cm DBH). Our results suggest that burned forest patches of at least 20 ha and composed mainly of burned mature and old-growth forests should be maintained during post-fire harvesting. The decrease in the amount of late seral stands in managed forest landscapes raises concerns about the future availability of high-quality burned forests for this species.