bluuebunny/crossref_metadata_2025_split huggingface dataset:
New Device for High-Throughput Viability Screening of Flow Biofilms
Authors: Benoit Michael R. , Conant Carolyn G. , Ionescu-Zanetti Cristian , Schwartz Michael , Matin A. 
Published: May 2010

ABSTRACT

Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well (“static”) biofilms are available, there are no methods for such screening of continuous flow biofilms (“flow biofilms”). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of
Pseudomonas aeruginosa
PAO1 flow biofilms.
“CandidatusAnadelfobacter veles” and “CandidatusCyrtobacter comes,” Two NewRickettsialesSpecies Hosted by the Protist CiliateEuplotes harpa(Ciliophora, Spirotrichea)
Authors: Vannini Claudia , Ferrantini Filippo , Schleifer Karl-Heinz , Ludwig Wolfgang , Verni Franco , Petroni Giulio 
Published: May 2010

ABSTRACTThe orderRickettsiales(Alphaproteobacteria) is a well-known group containing obligate endocellular prokaryotes. The order encompasses three families (Rickettsiaceae,Anaplasmataceae, andHolosporaceae) and a fourth, family-level cluster, which includes only one candidate species, “CandidatusMidichloria mitochondrii,” as well as several unnamed bacterial symbionts. The broad host range exhibited by the members of the “CandidatusMidichloria” clade suggests their eventual relevance for a better understanding of the evolution of symbiosis and host specificity ofRickettsiales. In this paper, two new bacteria belonging to the “CandidatusMidichloria” clade, hosted by two different strains of the ciliate protistEuplotes harpa, are described on the basis of ultrastructural observations, comparative 16S rRNA gene sequence analysis, and an estimation of the percentage of infection. Ultrastructure of these bacteria shows some unusual features: one has an electron-dense cytoplasm, and the other one lacks a symbiosomal membrane. The latter was up to now considered an exclusive feature of bacteria belonging to the familyRickettsiaceae. 16S rRNA gene phylogenetic analysis unambiguously places the new bacteria in the “CandidatusMidichloria” clade, although their phylogenetic relationships with other members of the clade are not clearly resolved. This is the first report of a ciliate-borne bacterium belonging to the “CandidatusMidichloria” clade. On the basis of the data obtained, the two bacteria are proposed as two new candidate genera and species, “CandidatusAnadelfobacter veles” and “CandidatusCyrtobacter comes.”
Rotenone Enhances the Antifungal Properties of Staurosporine
Authors: Castro Ana , Lemos Catarina , Falcão Artur , Fernandes Andreia S. , Glass N. Louise , Videira Arnaldo 
Published: May 2010

ABSTRACT

We studied staurosporine-induced cell death in the filamentous fungus
Neurospora crassa
. The generation of reactive oxygen species during the process appears to be an important signaling event, since addition of the antioxidant glutathione prevents the effects of staurosporine on fungal growth. Selected mutants with mutations in respiratory chain complex I are extremely sensitive to the drug, stressing the involvement of complex I in programmed cell death. Following this finding, we determined that the complex I-specific inhibitor rotenone used in combination with staurosporine results in a synergistic and specific antifungal activity, likely through a concerted action on intracellular glutathione depletion. Paradoxically, the synergistic antifungal activity of rotenone and staurosporine is observed in
N. crassa
complex I mutants and in
Saccharomyces cerevisiae
, which lacks complex I. In addition, it is not observed when other complex I inhibitors are used instead of rotenone. These results indicate that the rotenone effect is independent of complex I inhibition. The combination of rotenone and staurosporine is effective against
N. crassa
as well as against the common pathogens
Aspergillus fumigatus
and
Candida albicans
, pointing to its usefulness as an antifungal agent.
Protein Phosphatase 2B (PP2B, Calcineurin) in Paramecium: Partial Characterization Reveals That Two Members of the Unusually Large Catalytic Subunit Family Have Distinct Roles in Calcium-Dependent Processes
Authors: Fraga D. , Sehring I. M. , Kissmehl R. , Reiss M. , Gaines R. , Hinrichsen R. , Plattner H. 
Published: May 2010

ABSTRACT

We characterized the calcineurin (
CaN
) gene family, including the subunits
CaNA
and
CaNB
, based upon sequence information obtained from the
Paramecium
genome project.
Paramecium tetraurelia
has seven subfamilies of the catalytic CaNA subunit and one subfamily of the regulatory CaNB subunit, with each subfamily having two members of considerable identity on the amino acid level (≥55% between subfamilies, ≥94% within CaNA subfamilies, and full identity in the CaNB subfamily). Within CaNA subfamily members, the catalytic domain and the CaNB binding region are highly conserved and molecular modeling revealed a three-dimensional structure almost identical to a human ortholog. At 14 members, the size of the CaNA family is unprecedented, and we hypothesized that the different CaNA subfamily members were not strictly redundant and that at least some fulfill different roles in the cell. This was tested by selecting two phylogenetically distinct members of this large family for posttranscriptional silencing by RNA interference. The two targets resulted in differing effects in exocytosis, calcium dynamics, and backward swimming behavior that supported our hypothesis that the large, highly conserved CaNA family members are not strictly redundant and that at least two members have evolved diverse but overlapping functions. In sum, the occurrence of CaN in
Paramecium
spp., although disputed in the past, has been established on a molecular level. Its role in exocytosis and ciliary beat regulation in a protozoan, as well as in more complex organisms, suggests that these roles for CaN were acquired early in the evolution of this protein family.
ABacillus anthracisS-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC
Authors: Tarlovsky Yael , Fabian Marian , Solomaha Elena , Honsa Erin , Olson John S. , Maresso Anthony W. 
Published: May 2010

ABSTRACTThe sequestration of iron by mammalian hosts represents a significant obstacle to the establishment of a bacterial infection. In response, pathogenic bacteria have evolved mechanisms to acquire iron from host heme.Bacillus anthracis, the causative agent of anthrax, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, includingB. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Here, we present evidence that aB. anthracisS-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation ofB. anthracisto changing iron reservoirs during an infection. Understanding the mechanism of heme uptake in pathogenic bacteria is important for the development of novel therapeutics to prevent and treat bacterial infections.
Single-Stranded DNA Binding by F TraI Relaxase and Helicase Domains Is Coordinately Regulated
Authors: Dostál Lubomír , Schildbach Joel F. 
Published: May 2010

ABSTRACT
Transfer of conjugative plasmids requires relaxases, proteins that cleave one plasmid strand sequence specifically. The F plasmid relaxase TraI (1,756 amino acids) is also a highly processive DNA helicase. The TraI relaxase activity is located within the N-terminal ∼300 amino acids, while helicase motifs are located in the region comprising positions 990 to 1450. For efficient F transfer, the two activities must be physically linked. The two TraI activities are likely used in different stages of transfer; how the protein regulates the transition between activities is unknown. We examined TraI helicase single-stranded DNA (ssDNA) recognition to complement previous explorations of relaxase ssDNA binding. Here, we show that TraI helicase-associated ssDNA binding is independent of and located N-terminal to all helicase motifs. The helicase-associated site binds ssDNA oligonucleotides with nM-range equilibrium dissociation constants and some sequence specificity. Significantly, we observe an apparent strong negative cooperativity in ssDNA binding between relaxase and helicase-associated sites. We examined three TraI variants having 31-amino-acid insertions in or near the helicase-associated ssDNA binding site. B. A. Traxler and colleagues (J. Bacteriol. 188:6346-6353) showed that under certain conditions, these variants are released from a form of negative regulation, allowing them to facilitate transfer more efficiently than wild-type TraI. We find that these variants display both moderately reduced affinity for ssDNA by their helicase-associated binding sites and a significant reduction in the apparent negative cooperativity of binding, relative to wild-type TraI. These results suggest that the apparent negative cooperativity of binding to the two ssDNA binding sites of TraI serves a major regulatory function in F transfer.
Subcellular Localization and Characterization of the ParAB System fromCorynebacterium glutamicum
Authors: Donovan Catriona , Schwaiger Astrid , Krämer Reinhard , Bramkamp Marc 
Published: May 2010

ABSTRACTFaithful segregation of chromosomes and plasmids is a vital prerequisite to produce viable and genetically identical progeny. Bacteria use a specialized segregation system composed of the partitioning proteins ParA and ParB to segregate certain plasmids. Strikingly, homologues of ParA and ParB are found to be encoded in many chromosomes. Although mutations in the chromosomal Par system have effects on segregation efficiency, the exact mechanism by which the chromosomes are segregated into the daughter cells is not fully understood. We describe the polar localization of the ParB origin nucleoprotein complex in the actinomyceteCorynebacterium glutamicum. ParB and the origin of replication were found to be stably localized to the cell poles. After replication, the origins move toward the opposite pole. Purified ParB was able to bind to theparSconsensus sequencein vitro.C. glutamicumpossesses two ParA-like partitioning ATPase proteins. Both proteins interact with ParB but show a slightly different subcellular localization and phenotype. While ParA might be part of a conventional partitioning system, PldP seems to play a role in division site selection.
Regulatory System of the Protocatechuate 4,5-Cleavage Pathway Genes Essential for Lignin Downstream Catabolism
Authors: Kamimura Naofumi , Takamura Kazuhiro , Hara Hirofumi , Kasai Daisuke , Natsume Ryo , Senda Toshiya , Katayama Yoshihiro , Fukuda Masao , Masai Eiji 
Published: May 2010

ABSTRACT

Sphingobium
sp. strain SYK-6 converts various lignin-derived biaryls with guaiacyl (4-hydroxy-3-methoxyphenyl) and syringyl (4-hydroxy-3,5-dimethoxyphenyl) moieties to vanillate and syringate. These compounds are further catabolized through the protocatechuate (PCA) 4,5-cleavage (PCA45) pathway. In this article, the regulatory system of the PCA45 pathway is described. A LysR-type transcriptional regulator (LTTR), LigR, activated the transcription of the
ligK-orf1-ligI-lsdA
and
ligJABC
operons in the presence of PCA or gallate (GA), which is an intermediate metabolite of vanillate or syringate, respectively, and repressed transcription of its own gene. LigR bound to the positions −77 to −51 and −80 to −48 of the
ligK
and
ligJ
promoters, respectively, and induced DNA bending. In the presence of PCA or GA, DNA bending on both promoters was enhanced. The LigR-binding regions of the
ligK
and
ligJ
promoters in the presence of inducer molecules were extended and shortened, respectively. The LTTR consensus sequences (Box-K and Box-J) in the
ligK
and
ligJ
promoters were essential for the binding of LigR and transcriptional activation of both operons. In addition, the regions between the LigR binding boxes and the −35 regions were required for the enhancement of DNA bending, although the binding of LigR to the −35 region of the
ligJ
promoter was not observed in DNase I footprinting experiments. This study shows the binding features of LigR on the
ligK
and
ligJ
promoters and explains how the PCA45 pathway genes are expressed during degradation of lignin-derived biaryls by this bacterium.
Functional Characterization of
Synechocystis
sp. Strain PCC 6803
pst1
and
pst2
Gene Clusters Reveals a Novel Strategy for Phosphate Uptake in a Freshwater Cyanobacterium
Authors: Pitt Frances D. , Mazard Sophie , Humphreys Lee , Scanlan David J. 
Published: May 2010

ABSTRACT

Synechocystis
sp. strain PCC 6803 possesses two putative ABC-type inorganic phosphate (P
i
) transporters with three associated P
i
-binding proteins (PBPs), SphX (encoded by
sll0679
), PstS1 (encoded by
sll0680
), and PstS2 (encoded by
slr1247
), organized in two spatially discrete gene clusters,
pst1
and
pst2
. We used a combination of mutagenesis, gene expression, and radiotracer uptake analyses to functionally characterize the role of these PBPs and associated gene clusters. Quantitative PCR (qPCR) demonstrated that
pstS1
was expressed at a high level in P
i
-replete conditions compared to
sphX
or
pstS2
. However, a P
i
stress shift increased expression of
pstS2
318-fold after 48 h, compared to 43-fold for
pstS1
and 37-fold for
sphX
. A shift to high-light conditions caused a transient increase of all PBPs, whereas N stress primarily increased expression of
sphX
. Interposon mutagenesis of each PBP demonstrated that disruption of
pstS1
alone caused constitutive expression of
pho
regulon genes, implicating PstS1 as a major component of the P
i
sensing machinery. The
pstS1
mutant was also transformation incompetent.
32
P
i
radiotracer uptake experiments using
pst1
and
pst2
deletion mutants showed that Pst1 acts as a low-affinity, high-velocity transporter (

K
s

, 3.7 ± 0.7 μM;
V
max
, 31.18 ± 3.96 fmol cell
−1
min
−1
) and Pst2 acts as a high-affinity, low-velocity system (

K
s

, 0.07 ± 0.01 μM;
V
max
, 0.88 ± 0.11 fmol cell
−1
min
−1
). These P
i
ABC transporters thus exhibit differences in both kinetic and regulatory properties, the former trait potentially dramatically increasing the dynamic range of P
i
transport into the cell, which has potential implications for our understanding of the ecological success of this key microbial group.
Treponema denticola
PrcB Is Required for Expression and Activity of the PrcA-PrtP (Dentilisin) Complex
Authors: Godovikova Valentina , Wang Hong-Tao , Goetting-Minesky M. Paula , Ning Yu , Capone Ricardo F. , Slater Claudia K. , Fenno J. Christopher 
Published: May 2010

ABSTRACT

The
Treponema denticola
surface protease complex, consisting of PrtP protease (dentilisin) and two auxiliary polypeptides (PrcA1 and PrcA2), is believed to contribute to periodontal disease by degrading extracellular matrix components and disrupting host intercellular signaling. Previously, we showed that transcription of the protease operon initiates upstream of TDE0760 (herein designated
prcB
), the open reading frame immediately 5′ of
prcA-prtP
. The
prcB
gene is conserved in
T. denticola
strains. PrcB localizes to the detergent phase of Triton X-114 cell surface extracts and migrates as a 22-kDa polypeptide, in contrast to the predicted 17-kDa cytoplasmic protein encoded in the annotated
T. denticola
genome. Consistent with this observation, the PrcB N terminus is unavailable for Edman sequencing, suggesting that it is acylated. Nonpolar deletion of
prcB
in
T. denticola
showed that PrcB is required for production of PrtP protease activity, including native PrtP cleavage of PrcA to PrcA1 and PrcA2. A 6×His-tagged PrcB protein coimmunoprecipitates with native PrtP, using either anti-PrtP or anti-His-tag antibodies, and recombinant PrtP copurifies with PrcB-6×His in nickel affinity chromatography. Taken together, these data are consistent with identification of PrcB as a PrtP-binding lipoprotein that likely stabilizes the PrtP polypeptide during localization to the outer membrane.
RNA Processing of Nitrogenase Transcripts in the CyanobacteriumAnabaena variabilis
Authors: Ungerer Justin L. , Pratte Brenda S. , Thiel Teresa 
Published: May 2010

ABSTRACTLittle is known about the regulation of nitrogenase genes in cyanobacteria. Transcription of thenifH1andvnfHgenes, encoding dinitrogenase reductases for the heterocyst-specific Mo-nitrogenase and the alternative V-nitrogenase, respectively, was studied by using alacZreporter. Despite evidence for a transcription start site just upstream ofnifH1andvnfH, promoter fragments that included these start sites did not drive the transcription oflacZand, fornifH1, did not drive the expression ofnifHDK1. Further analysis using larger regions upstream ofnifH1indicated that a promoter withinnifU1and a promoter upstream ofnifB1both contributed to expression ofnifHDK1, with thenifB1promoter contributing to most of the expression. Similarly, while the region upstream ofvnfH, containing the putative transcription start site, did not drive expression oflacZ, the region that included the promoter for the upstream gene,ava4055, did. Characterization of the previously reportednifH1andvnfHtranscriptional start sites by 5′RACE (5′ rapid amplification of cDNA ends) revealed that these 5′ ends resulted from processing of larger transcripts rather than byde novotranscription initiation. The 5′ positions of both thevnfHandnifH1transcripts lie at the base of a stem-loop structure that may serve to stabilize thenifHDK1andvnfHspecific transcripts compared to the transcripts for other genes in the operons providing the proper stoichiometry for the Nif proteins for nitrogenase synthesis.
Direct and Indirect Control of Late Sporulation Genes by GerR of
Bacillus subtilis
Authors: Cangiano Giuseppina , Mazzone Antonio , Baccigalupi Loredana , Isticato Rachele , Eichenberger Patrick , De Felice Maurilio , Ricca Ezio 
Published: May 2010

ABSTRACT

GerR is a sporulation-specific transcriptional factor of
Bacillus subtilis
that has been identified as a negative regulator of genes transcribed by σ
E
-containing RNA polymerase and as a positive effector of the expression of three late sporulation genes. Here we confirmed that
gerR
transcription is dependent on σ
E
-containing RNA polymerase but also observed that it requires the transcriptional regulator SpoIIID. The study of the role of GerR in regulating the expression of several late sporulation genes allowed us to observe that its effect is strongly positive on
spoVIF
,
cotC
, and
cotG
, weakly positive on
cotB
, and negative on
cotU
. The results of chromatin immunoprecipitation (ChIP) experiments indicated that GerR binds to the promoter regions of some, but not all, of the GerR-controlled genes, leading us to propose that GerR controls late sporulation genes in two ways: (i) directly, by acting on the transcription of
cotB
,
cotU
and
spoVIF
; and (ii) indirectly, through the activation of SpoVIF, which stabilizes the transcriptional activator GerE and consequently induces the expression of the GerE-dependent genes
cotC
and
cotG
.
Genome Sequence of
Lactobacillus crispatus
ST1
Authors: Ojala Teija , Kuparinen Veera , Koskinen J. Patrik , Alatalo Edward , Holm Liisa , Auvinen Petri , Edelman Sanna , Westerlund-Wikström Benita , Korhonen Timo K. , Paulin Lars , Kankainen Matti 
Published: May 2010

ABSTRACT

Lactobacillus crispatus
is a common member of the beneficial microbiota present in the vertebrate gastrointestinal and human genitourinary tracts. Here, we report the genome sequence of
L. crispatus
ST1, a chicken isolate displaying strong adherence to vaginal epithelial cells.
Mutations in the Lipopolysaccharide Biosynthesis Pathway Interfere with Crescentin-Mediated Cell Curvature in
Caulobacter crescentus
Authors: Cabeen Matthew T. , Murolo Michelle A. , Briegel Ariane , Bui N. Khai , Vollmer Waldemar , Ausmees Nora , Jensen Grant J. , Jacobs-Wagner Christine 
Published: May 2010

ABSTRACT

Bacterial cell morphogenesis requires coordination among multiple cellular systems, including the bacterial cytoskeleton and the cell wall. In the vibrioid bacterium
Caulobacter crescentus
, the intermediate filament-like protein crescentin forms a cell envelope-associated cytoskeletal structure that controls cell wall growth to generate cell curvature. We undertook a genetic screen to find other cellular components important for cell curvature. Here we report that deletion of a gene (
wbqL
) involved in the lipopolysaccharide (LPS) biosynthesis pathway abolishes cell curvature. Loss of WbqL function leads to the accumulation of an aberrant O-polysaccharide species and to the release of the S layer in the culture medium. Epistasis and microscopy experiments show that neither S-layer nor O-polysaccharide production is required for curved cell morphology
per se
but that production of the altered O-polysaccharide species abolishes cell curvature by apparently interfering with the ability of the crescentin structure to associate with the cell envelope. Our data suggest that perturbations in a cellular pathway that is itself fully dispensable for cell curvature can cause a disruption of cell morphogenesis, highlighting the delicate harmony among unrelated cellular systems. Using the
wbqL
mutant, we also show that the normal assembly and growth properties of the crescentin structure are independent of its association with the cell envelope. However, this envelope association is important for facilitating the local disruption of the stable crescentin structure at the division site during cytokinesis.
BRAID GROUP REPRESENTATIONS ARISING FROM THE YANG–BAXTER EQUATION
Authors: FRANKO JENNIFER M. 
Published: April 2010

This paper aims to determine the images of the braid group under representations afforded by the Yang–Baxter equation when the solution is a non-trivial 4 × 4 matrix. Making the assumption that all the eigenvalues of the Yang–Baxter solution are roots of unity, leads to the conclusion that all the images are finite. Using results of Turaev, we have also identified cases in which one would get a link invariant. Finally, by observing the group algebra generated by the image of the braid group sometimes factor through known algebras, in certain instances we can identify the invariant as particular specializations of a known invariant.