bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning, Expression, Characterization, and Biocatalytic Investigation of the 4-Hydroxyacetophenone Monooxygenase from
Pseudomonas putida
JD1
Authors: Rehdorf Jessica , Zimmer Christian L. , Bornscheuer Uwe T.
Published: February 2009

While the number of available recombinant Baeyer-Villiger monooxygenases (BVMOs) has grown significantly over the last few years, there is still the demand for other BVMOs to expand the biocatalytic diversity. Most BVMOs that have been described are dedicated to convert efficiently cyclohexanone and related cyclic aliphatic ketones. To cover a broader range of substrate types and enantio- and/or regioselectivities, new BVMOs have to be discovered. The gene encoding a BVMO identified in
Pseudomonas putida
JD1 converting aromatic ketones (HAPMO; 4-hydroxyacetophenone monooxygenase) was amplified from genomic DNA using SiteFinding-PCR, cloned, and functionally expressed in
Escherichia coli
. Furthermore, four other open reading frames could be identified clustered around this HAPMO. It has been suggested that these proteins, including the HAPMO, might be involved in the degradation of 4-hydroxyacetophenone. Substrate specificity studies revealed that a large variety of other arylaliphatic ketones are also converted via Baeyer-Villiger oxidation into the corresponding esters, with preferences for
para
-substitutions at the aromatic ring. In addition, oxidation of aldehydes and some heteroaromatic compounds was observed. Cycloketones and open-chain ketones were not or poorly accepted, respectively. It was also found that this enzyme oxidizes aromatic ketones such as 3-phenyl-2-butanone with excellent enantioselectivity (E ≫100).
Antibody-Mediated Immobilization of
Cryptococcus neoformans
Promotes Biofilm Formation
Authors: Robertson Emma J. , Casadevall Arturo
Published: February 2009

Most microbes, including the fungal pathogen
Cryptococcus neoformans
, can grow as biofilms. Biofilms confer upon microbes a range of characteristics, including an ability to colonize materials such as shunts and catheters and increased resistance to antibiotics. Here, we provide evidence that coating surfaces with a monoclonal antibody to glucuronoxylomannan, the major component of the fungal capsular polysaccharide, immobilizes cryptococcal cells to a surface support and, subsequently, promotes biofilm formation. We used time-lapse microscopy to visualize the growth of cryptococcal biofilms, generating the first movies of fungal biofilm growth. We show that when fungal cells are immobilized using surface-attached specific antibody to the capsule, the initial stages of biofilm formation are significantly faster than those on surfaces with no antibody coating or surfaces coated with unspecific monoclonal antibody. Time-lapse microscopy revealed that biofilm growth was a dynamic process in which cells shuffled position during budding and was accompanied by emergence of planktonic variant cells that left the attached biofilm community. The planktonic variant cells exhibited mobility, presumably by Brownian motion. Our results indicate that microbial immobilization by antibody capture hastens biofilm formation and suggest that antibody coating of medical devices with immunoglobulins must exclude binding to common pathogenic microbes and the possibility that this effect could be exploited in industrial microbiology.
Four-Antigen Mixture Containing V-Cyclin for Serological Screening of Human Herpesvirus 8 Infection
Authors: Burbelo Peter D. , Leahy Hannah P. , Groot Sandra , Bishop Lisa R. , Miley Wendell , Iadarola Michael J. , Whitby Denise , Kovacs Joseph A.
Published: March 2009

Improved diagnostic reagents and testing are currently needed for the serological detection of human herpesvirus 8 (HHV-8) infections. We evaluated the luciferase immunoprecipitation systems (LIPS) for profiling antibody responses to a panel of HHV-8 proteins for diagnosis of Kaposi sarcoma (KS)-infected individuals. Using a pilot serum set, LIPS detected robust antibody responses to several known antigens, and a screen of 14 additional HHV-8 proteins identified v-cyclin as a potentially new diagnostic antigen. In evaluating a training-serum set, a four-antigen panel (K8.1, v-cyclin, ORF65, and a LANA fragment) was found to provide sufficient information for diagnosis. Analysis of a validation serum set using the combined results from these four separate antigen tests showed 100% sensitivity and 100% specificity. Furthermore, a LIPS format using a mixture of the four antigens, which simplifies data collection and analysis, closely matched the diagnostic performance of the combined separate tests (
R
= 0.95). This four-antigen mixture format analyzed with the validation serum set also showed 100% sensitivity and 100% specificity but was not statistically different from two separate enzyme-linked immunosorbent assays (94% sensitivity and 100% specificity) using baculovirus-produced LANA and bacterially produced K8.1. Heat map analysis of KS patient antibody titers revealed marked heterogeneity in humoral responses to this four-antigen panel. Overall, the LIPS assay showed 97% sensitivity, and positive anti-v-cyclin antibodies were detected in approximately 75% of the KS sera. These results suggest that LIPS screening using an antigen mixture is a sensitive and high-throughput method for serological screening of HHV-8 infection in individuals with KS.
Iron Activates In Vivo DNA Binding of
Schizosaccharomyces pombe
Transcription Factor Fep1 through Its Amino-Terminal Region
Authors: Jbel Mehdi , Mercier Alexandre , Pelletier Benoit , Beaudoin Jude , Labbé Simon
Published: February 2009

In
Schizosaccharomyces pombe
, the iron sensor Fep1 mediates the transcriptional repression of iron transport genes in response to high concentrations of iron. On the other hand,
fep1

+

expression is downregulated under conditions of iron starvation by the CCAAT-binding factor Php4. In this study, we created a
fep1
Δ
php4
Δ double mutant strain where expression of
fep1

+

was disengaged from its iron limitation-dependent repression by Php4 to examine the effects of iron on constitutively expressed functional
fep1

+

-
GFP
and
TAP-fep1

+

alleles and their gene products. In these cells, Fep1-green fluorescent protein was invariably localized in the nucleus under both iron-limiting and iron-replete conditions. Using chromatin immunoprecipitation assays, we found that Fep1 is associated with iron-responsive promoters in vivo. Chromatin binding was iron dependent, with a loss of binding observed in the presence of low iron. Functional dissection of the protein revealed that the N-terminal 241-residue segment that includes two consensus Cys
2
/Cys
2
-type zinc finger motifs and a Cys-rich region is required for optimal promoter occupancy by Fep1. Within this segment, a minimal module encompassing amino acids 60 to 241 is sufficient for iron-dependent chromatin binding. Using yeast one-hybrid analysis, we showed that the replacement of the repression domain of Fep1 by fusing the activation domain of VP16 to the chromatin-binding fragment of amino acids 1 to 241 of Fep1 converts the protein from an iron-dependent repressor into an iron-dependent transcriptional activator. Thus, the repression function of Fep1 can be replaced with that of a transcriptional activation function without the loss of its iron-dependent DNA-binding activity.
Yap1-Regulated Glutathione Redox System Curtails Accumulation of Formaldehyde and Reactive Oxygen Species in Methanol Metabolism of
Pichia pastoris
Authors: Yano Taisuke , Takigami Emiko , Yurimoto Hiroya , Sakai Yasuyoshi
Published: February 2009

The glutathione redox system, including the glutathione biosynthesis and glutathione regeneration reaction, has been found to play a critical role in the yeast
Pichia pastoris
during growth on methanol, and this regulation was at least partly executed by the transcription factor PpYap1. During adaptation to methanol medium, PpYap1 transiently localized to the nucleus and activated the expression of the glutathione redox system and upregulated glutathione reductase 1 (Glr1). Glr1 activates the regeneration of the reduced form of glutathione (GSH). Depletion of Glr1 caused a severe growth defect on methanol and hypersensitivity to formaldehyde (HCHO), which could be complemented by addition of GSH to the medium. Disruption of the genes for the HCHO-oxidizing enzymes PpFld1 and PpFgh1 caused a comparable phenotype, but disruption of the downstream gene Pp
FDH1
did not, demonstrating the importance of maintaining intracellular GSH levels. Absence of the peroxisomal glutathione peroxidase Pmp20 also triggered nuclear localization of PpYap1, and although cells were not sensitive to HCHO, growth on methanol was again severely impaired due to oxidative stress. Thus, the PpYap1-regulated glutathione redox system has two important roles, i.e., HCHO metabolism and detoxification of reactive oxygen species.
Candida albicans RFX2
Encodes a DNA Binding Protein Involved in DNA Damage Responses, Morphogenesis, and Virulence
Authors: Hao Binghua , Clancy Cornelius J. , Cheng Shaoji , Raman Suresh B. , Iczkowski Kenneth A. , Nguyen M. Hong
Published: February 2009

We previously showed that
Candida albicans
orf19.4590, which we have renamed
RFX2
, expresses a protein that is reactive with antibodies in persons with candidiasis. In this study, we demonstrate that
C. albicans RFX2
shares some functional redundancy with
Saccharomyces cerevisiae RFX1
. Complementation of an
S. cerevisiae rfx1
mutant with
C. albicans RFX2
partially restored UV susceptibility and the repression of DNA damage response genes. DNA damage- and UV-induced genes
RAD6
and
DDR48
were derepressed in a
C. albicans rfx2
null mutant strain under basal conditions, and the mutant was significantly more resistant to UV irradiation, heat shock, and ethanol than wild-type strain SC5314. The
rfx2
mutant was hyperfilamentous on solid media and constitutively expressed hypha-specific genes
HWP1
,
ALS3
,
HYR1
,
ECE1
, and
CEK1
. The mutant also demonstrated increased invasion of solid agar and significantly increased adherence to human buccal epithelial cells. During hematogenously disseminated candidiasis, mice infected with the mutant had a significantly delayed time to death compared to the wild type. During oropharyngeal candidiasis, mice infected with the mutant had significantly lower tissue burdens in the oral cavity and esophagus at 7 days and they were less likely to develop disseminated infections because of mucosal translocation. The data demonstrate that
C. albicans
Rfx2p regulates DNA damage responses, morphogenesis, and virulence.
Aspergillus fumigatus
Calcipressin CbpA Is Involved in Hyphal Growth and Calcium Homeostasis
Authors: Pinchai Nadthanan , Perfect B. Zachary , Juvvadi Praveen Rao , Fortwendel Jarrod R. , Cramer Robert A. , Asfaw Yohannes G. , Heitman Joseph , Perfect John R. , Steinbach William J.
Published: February 2009

Calcineurin is a conserved protein phosphatase that plays a critical role in Ca
2+
signaling and stress responses. Previously, a new class of conserved calcineurin-binding proteins, the calcipressins, was identified. However, the role of these proteins remains controversial, and both inhibitory and stimulatory effects on calcineurin were observed. In this study, we investigate the role of CbpA, the
Aspergillus fumigatus
member of the calcipressin family, and report that deletion of the
cbpA
gene resulted in reduced hyphal growth and limited attenuated virulence. Interestingly, under high-calcium-level conditions, the Δ
cbpA
strain displayed improved Ca
2+
tolerance compared to the wild-type strain and revealed increased expression of
vcxA
,
chsA
, and
cnaA
, which encode the vacuolar Ca
2+
/H
+
exchanger VcxA, chitin synthase A, and the calcineurin catalytic subunit CnaA, respectively. The increased transcript levels of these three genes were reversed in the presence of the calcineurin inhibitor FK506, indicating a calcineurin-dependent mechanism. Overexpression of
cbpA
resulted in decreased transcription of
vcxA
,
chsA
, and
cnaA
, associated with wild-type sensitivity to Ca
2+
. Taken together, our study highlights the importance of CbpA in the regulation of hyphal growth and calcium adaptation of
A. fumigatus
and provides evidence that CbpA may serve as a feedback inhibitor in some aspects of calcineurin functions.
Deregulation of
DSE1
Gene Expression Results in Aberrant Budding within the Birth Scar and Cell Wall Integrity Pathway Activation in
Saccharomyces cerevisiae
Authors: Frýdlová Ivana , Malcová Ivana , Vašicová Pavla , Hašek Jiří
Published: February 2009

Strains of
Saccharomyces cerevisiae
lacking Isw2, the catalytic subunit of the Isw2 chromatin remodeling complex, show the mating type-independent activation of the cell wall integrity (CWI) signaling pathway. Since the CWI pathway activation usually reflects cell wall defects, we searched for the cell wall-related genes changed in expression. The genes
DSE1
,
CTS1
, and
CHS1
were upregulated as a result of the absence of Isw2, according to previously published gene expression profiles (I. Frydlova, M. Basler, P. Vasicova, I. Malcova, and J. Hasek, Curr. Genet. 52:87-95, 2007). Western blot analyses of double deletion mutants, however, did not indicate the contribution of the chitin metabolism-related genes
CTS1
and
CHS1
to the CWI pathway activation. Nevertheless, the deletion of the
DSE1
gene encoding a daughter cell-specific protein with unknown function suppressed CWI pathway activation in
isw2
Δ cells. In addition, the deletion of
DSE1
also abolished the budding-within-the-birth-scar phenotype of
isw2
Δ cells. The plasmid-driven overexpression proved that the deregulation of Dse1 synthesis was also responsible for CWI pathway activation and manifestation of the budding-within-the-birth-scar phenotype in wild-type cells. The overproduced Dse1-green fluorescent protein localized to both sides of the septum and persisted in unbudded cells. Although the exact cellular role of this daughter cell-specific protein has to be elucidated, our data point to the involvement of Dse1 in bud site selection in haploid cells.
The Stationary-Phase Sigma Factor σ
S
Is Responsible for the Resistance of
Escherichia coli
Stationary-Phase Cells to
mazEF
-Mediated Cell Death
Authors: Kolodkin-Gal Ilana , Engelberg-Kulka Hanna
Published: February 2009

Escherichia coli mazEF
is a toxin-antitoxin gene module that mediates cell death during exponential-phase cellular growth through either reactive oxygen species (ROS)-dependent or ROS-independent pathways. Here, we found that the stationary-phase sigma factor σ
S
was responsible for the resistance to
mazEF
-mediated cell death during stationary growth phase. Deletion of
rpoS
, the gene encoding σ
S
from the bacterial chromosome, permitted
mazEF
-mediated cell death during stationary growth phase.
The Putative Coupling Protein TcpA Interacts with Other pCW3-Encoded Proteins To Form an Essential Part of the Conjugation Complex
Authors: Steen Jennifer A. , Bannam Trudi L. , Teng Wee Lin , Devenish Rodney J. , Rood Julian I.
Published: February 2009

Conjugative plasmids encode antibiotic resistance determinants or toxin genes in the anaerobic pathogen
Clostridium perfringens
. The paradigm conjugative plasmid in this bacterium is pCW3, a 47-kb tetracycline resistance plasmid that encodes the unique
tcp
transfer locus. The
tcp
locus consists of 11 genes,
intP
and
tcpA
-
tcpJ
, at least three of which,
tcpA
,
tcpF
, and
tcpH
, are essential for the conjugative transfer of pCW3. In this study we examined protein-protein interactions involving TcpA, the putative coupling protein. Use of a bacterial two-hybrid system identified interactions between TcpA and TcpC, TcpG, and TcpH. This analysis also demonstrated TcpA, TcpC, and TcpG self-interactions, which were confirmed by chemical cross-linking studies. Examination of a series of deletion and site-directed derivatives of TcpA identified the domains and motifs required for these interactions. Based on these results, we have constructed a model for this unique conjugative transfer apparatus.
Activation and Repression at the
Escherichia coli ynfEFGHI
Operon Promoter
Authors: Xu Meng , Busby Stephen J. W. , Browning Douglas F.
Published: February 2009

Induction of the
Escherichia coli
K-12
ynfEFGHI
operon in response to anaerobiosis is repressed by nitrate ions. In this study, we show that the global transcription factor FNR is a class II activator at the
ynfEFGHI
promoter and that NarL represses activation by binding to a single target that overlaps the promoter −10 element. Electromobility shift assays show that NarL does not prevent RNA polymerase binding and suggest that repression may involve a quaternary NarL-FNR-RNA polymerase-promoter complex.
Regulation of
Bacillus subtilis aprE
Expression by
glnA
through Inhibition of
scoC
and σ
D
-Dependent
degR
Expression
Authors: Abe Sadanobu , Yasumura Ayako , Tanaka Teruo
Published: February 2009

Expression of the gene for the extracellular alkaline protease (
aprE
) of
Bacillus subtilis
is subject to regulation by many positive and negative regulators. We have found that
aprE
expression was increased by disruption of the glutamine synthetase gene
glnA
. The increase in
aprE
expression was attributed to a decreased in expression of
scoC
, which encodes a negative regulator of
aprE
expression. The
glnA
effect on
scoC
expression was abolished by further disruption of
tnrA
, indicating that
aprE
expression is under global regulation through TnrA. In the
scoC
background, however,
aprE
expression was decreased by
glnA
deletion, and it was shown that the decrease was due to a defect in positive regulation by DegU. Among the genes that affect
aprE
expression through DegU, the expression of
degR
, encoding a protein that stabilizes phosphorylated DegU, was inhibited by
glnA
deletion. It was further shown that the decrease in
degR
expression by
glnA
deletion was caused by inhibition of the expression of
sigD
, encoding the σ
D
factor, which is required for
degR
expression. In accordance with these findings, the expression levels of
aprE-lacZ
in
glnA scoC degR
and
scoC degR
strains were identical. These results led us to conclude that
glnA
deletion brings about two effects on
aprE
expression, i.e., a positive effect through inhibition of
scoC
expression and a negative effect through inhibition of
degR
expression, with the former predominating over the latter.
AStenotrophomonas maltophiliaMultilocus Sequence Typing Scheme for Inferring Population Structure
Authors: Kaiser Sabine , Biehler Klaus , Jonas Daniel
Published: February 2009

Stenotrophomonas maltophiliais an opportunistic, highly resistant, and ubiquitous pathogen. Strains have been assigned to genogroups using amplified fragment length polymorphism. Hence, isolates of environmental and clinical origin predominate in different groups. A multilocus sequence typing (MLST) scheme was developed using a highly diverse selection of 70 strains of various ecological origins from seven countries on all continents including strains of the 10 previously defined genogroups. Sequence data were assigned to 54 sequence types (ST) based on seven loci. Indices of association for all isolates and clinical isolates of 2.498 and 2.562 indicated a significant linkage disequilibrium, as well as high congruence of tree topologies from different loci. Potential recombination events were detected in one-sixth of all ST. Calculation of the mean divergence between and within predicted clusters confirmed previously defined groups and revealed five additional groups. Consideration of the different ecological origins showed that 18 out of 31 respiratory tract isolates, including 12 out of 19 isolates from cystic fibrosis (CF) patients, belonged to genogroup 6. In contrast, 16 invasive strains isolated from blood cultures were distributed among nine different genogroups. Three genogroups contained isolates of strictly environmental origin that also featured high sequence distances to other genogroups, including theS. maltophiliatype strain. On the basis of this MLST scheme, isolates can be assigned to the genogroups of this species in order to further scrutinize the population structure of this species and to unravel the uneven distribution of environmental and clinical isolates obtained from infected, colonized, or CF patients.
The Dienes Phenomenon: Competition and Territoriality in Swarming
Proteus mirabilis
Authors: Budding A. E. , Ingham C. J. , Bitter W. , Vandenbroucke-Grauls C. M. , Schneeberger P. M.
Published: February 2009

When two different strains of swarming
Proteus mirabilis
encounter one another on an agar plate, swarming ceases and a visible line of demarcation forms. This boundary region is known as the Dienes line and is associated with the formation of rounded cells. While the Dienes line appears to be the product of distinction between self and nonself, many aspects of its formation and function are unclear. In this work, we studied Dienes line formation using clinical isolates labeled with fluorescent proteins. We show that round cells in the Dienes line originate exclusively from one of the swarms involved and that these round cells have decreased viability. In this sense one of the swarms involved is dominant over the other. Close cell proximity is required for Dienes line formation, and when strains initiate swarming in close proximity, the dominant Dienes type has a significant competitive advantage. When one strain is killed by UV irradiation, a Dienes line does not form. Killing of the dominant strain limits the induction of round cells. We suggest that both strains are actively involved in boundary formation and that round cell formation is the result of a short-range killing mechanism that mediates a competitive advantage, an advantage highly specific to the swarming state. Dienes line formation has implications for the physiology of swarming and social recognition in bacteria.
Spatial Arrangement of the β-Glucoside Transporter fromEscherichia coli
Authors: Yagur-Kroll Sharon , Ido Ayelet , Amster-Choder Orna
Published: February 2009

TheEscherichia coliBglF protein, a sugar permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), catalyzes concomitant transport and phosphorylation of β-glucosides across the cytoplasmic membrane. Despite intensive studies of PTS permeases, the mechanism that couples sugar translocation to phosphorylation and the nature of the translocation apparatus are poorly understood. Like many PTS permeases, BglF consists of a transmembrane domain, which in addition to transmembrane helices (TMs) contains a big cytoplasmic loop and two hydrophilic domains, one containing a conserved cysteine that phosphorylates the incoming sugar. We previously reported that the big hydrophilic loop, which connects TM VI to TM VII, contains regions that alternate between facing-in and facing-out states and speculated that it is involved in creating the sugar translocation channel. In the current study we used [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET), a membrane-impermeative thiol-specific reagent, to identify sites that are involved in sugar transport. These sites map to the regions that border the big loop. Using cross-linking reagents that penetrate the cell, we could demonstrate spatial proximity between positions at the center of the big loop and the phosphorylation site, suggesting that the two regions come together to execute sugar phosphotransfer. Additionally, positions on opposite ends of the big loop were found to be spatially close. Cys accessibility analyses suggested that the sugar induces a change in this region. Taken together, our results demonstrate that the big loop participates in creating the sugar pathway and explain the observed coupling between translocation of PTS sugars from the periplasm to the cytoplasm and their phosphorylation.
Similar and Divergent Effects of ppGpp and DksA Deficiencies on Transcription in
Escherichia coli
Authors: Åberg Anna , Fernández-Vázquez Jorge , Cabrer-Panes Juan David , Sánchez Alex , Balsalobre Carlos
Published: February 2009

The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in
Escherichia coli
. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp
0
) and/or DksA (Δ
dksA
). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksA-deficient strains, respectively, increasing to 13% of all genes in the ppGpp
0
Δ
dksA
strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in
E. coli
.
Pyruvate Kinase-Deficient
Escherichia coli
Exhibits Increased Plasmid Copy Number and Cyclic AMP Levels
Authors: Cunningham Drew S. , Liu Zhu , Domagalski Nathan , Koepsel Richard R. , Ataai Mohammad M. , Domach Michael M.
Published: February 2009

Previously established consequences of abolishing pyruvate kinase (Pyk) activity in
Escherichia coli
during aerobic growth on glucose include reduced acetate production, elevated hexose monophosphate (HMP) pathway flux, elevated phosphoenolpyruvate carboxylase (Ppc) flux, and an increased ratio of phosphoenolpyruvate (PEP) to pyruvate. These traits inspired two hypotheses. First, the mutant (PB25) may maintain more plasmid than the wild type (JM101) by combining traits reported to facilitate plasmid DNA synthesis (i.e., decreased Pyk flux and increased HMP pathway and Ppc fluxes). Second, PB25 likely possesses a higher level of cyclic AMP (cAMP) than JM101. This is based on reports that connect elevated PEP/pyruvate ratios to phosphotransferase system signaling and adenylate cyclase activation. To test the first hypothesis, the strains were transformed with a pUC-based, high-copy-number plasmid (pGFPuv), and copy numbers were measured. PB25 exhibited a fourfold-higher copy number than JM101 when grown at 37°C. At 42°C, its plasmid content was ninefold higher than JM101 at 37°C. To test the second hypothesis, cAMP was measured, and the results confirmed it to be higher in PB25 than JM101. This elevation was not enough to elicit a strong regulatory effect, however, as indicated by the comparative expression of the pGFPuv-based reporter gene,
gfp

uv

, under the control of the cAMP-responsive
lac
promoter. The elevated cAMP in PB25 suggests that Pyk may participate in glucose catabolite repression by serving among all of the factors that tighten gene expression.
Interactions betweenBrucella suisVirB8 and Its Homolog TraJ from the Plasmid pSB102 Underline the Dynamic Nature of Type IV Secretion Systems
Authors: Bourg Gisèle , Sube Romain , O'Callaghan David , Patey Gilles
Published: February 2009

The proteinVirB8 plays a critical role in the assembly and function of theAgrobacterium tumefaciens virBtype IV secretion system (T4SS). The structure of the periplasmic domain of bothA.tumefaciensandBrucella suisVirB8 has been determined, and site-directed mutagenesis has revealed amino acids involved in the dimerization of VirB8 and interactions with VirB4 and VirB10. We have shown previously that TraJ, the VirB8 homologue from pSB102, and the chimeric protein TraJB8, encompassing the cytoplasmic and transmembrane (TM) domains of TraJ and the periplasmic domain of VirB8, were unable to complement aB.suismutant containing an in-frame deletion of thevirB8gene. This suggested that the presence of the TraJ cytoplasmic and TM domains could block VirB8 dimerization or assembly in the inner membrane. By bacterial two-hybrid analysis, we found that VirB8, TraJ, and the chimeras can all interact to form both homo- and heterodimers. However, the presence of the TM domain of TraJ resulted in much stronger interactions in both the homo- and heterodimers. We expressed the wild-type and chimeric proteins in wild-typeB.suis. The presence of proteins carrying the TM domain of TraJ had a dominant negative effect, leading to complete loss of virulence. This suggests that the T4SS is a dynamic structure and that strong interactions block the spatial flexibility required for correct assembly and function.
Docking and Assembly of the Type II Secretion Complex of
Vibrio cholerae
Authors: Lybarger Suzanne R. , Johnson Tanya L. , Gray Miranda D. , Sikora Aleksandra E. , Sandkvist Maria
Published: February 2009

Secretion of cholera toxin and other virulence factors from
Vibrio cholerae
is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (
eps
) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC- and GFP-EpsM-expressing strains disperse upon deletion of
epsD
, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.
A Conserved Gene Cluster Rules Anaerobic Oxidative Degradation of
l
-Ornithine
Authors: Fonknechten Nuria , Perret Alain , Perchat Nadia , Tricot Sabine , Lechaplais Christophe , Vallenet David , Vergne Carine , Zaparucha Anne , Le Paslier Denis , Weissenbach Jean , Salanoubat Marcel
Published: February 2009

For the ornithine fermentation pathway, described more than 70 years ago, genetic and biochemical information are still incomplete. We present here the experimental identification of the last four missing genes of this metabolic pathway. They encode
l
-ornithine racemase, (2
R
,4
S
)-2,4-diaminopentanoate dehydrogenase, and the two subunits of 2-amino-4-ketopentanoate thiolase. While described only for the
Clostridiaceae
to date, this pathway is shown to be more widespread.
Genome Sequences of ThreeAgrobacteriumBiovars Help Elucidate the Evolution of Multichromosome Genomes in Bacteria
Authors: Slater Steven C. , Goldman Barry S. , Goodner Brad , Setubal João C. , Farrand Stephen K. , Nester Eugene W. , Burr Thomas J. , Banta Lois , Dickerman Allan W. , Paulsen Ian , Otten Leon , Suen Garret , Welch Roy , Almeida Nalvo F. , Arnold Frank , Burton Oliver T. , Du Zijin , Ewing Adam , Godsy Eric , Heisel Sara , Houmiel Kathryn L. , Jhaveri Jinal , Lu Jing , Miller Nancy M. , Norton Stacie , Chen Qiang , Phoolcharoen Waranyoo , Ohlin Victoria , Ondrusek Dan , Pride Nicole , Stricklin Shawn L. , Sun Jian , Wheeler Cathy , Wilson Lindsey , Zhu Huijun , Wood Derek W.
Published: February 2009

The familyRhizobiaceaecontains plant-associated bacteria with critical roles in ecology and agriculture. Within this family, manyRhizobiumandSinorhizobiumstrains are nitrogen-fixing plant mutualists, while many strains designated asAgrobacteriumare plant pathogens. These contrasting lifestyles are primarily dependent on the transmissible plasmids each strain harbors. Members of theRhizobiaceaealso have diverse genome architectures that include single chromosomes, multiple chromosomes, and plasmids of various sizes.Agrobacteriumstrains have been divided into three biovars, based on physiological and biochemical properties. The genome of a biovar I strain,A. tumefaciensC58, has been previously sequenced. In this study, the genomes of the biovar II strainA. radiobacterK84, a commercially available biological control strain that inhibits certain pathogenic agrobacteria, and the biovar III strainA. vitisS4, a narrow-host-range strain that infects grapes and invokes a hypersensitive response on nonhost plants, were fully sequenced and annotated. Comparison with other sequenced members of theAlphaproteobacteriaprovides new data on the evolution of multipartite bacterial genomes. Primary chromosomes show extensive conservation of both gene content and order. In contrast, secondary chromosomes share smaller percentages of genes, and conserved gene order is restricted to short blocks. We propose that secondary chromosomes originated from an ancestral plasmid to which genes have been transferred from a progenitor primary chromosome. Similar patterns are observed in selectBeta- andGammaproteobacteriaspecies. Together, these results define the evolution of chromosome architecture and gene content among theRhizobiaceaeand support a generalized mechanism for second-chromosome formation among bacteria.
Staphylococcus aureus
MazF Specifically Cleaves a Pentad Sequence, UACAU, Which Is Unusually Abundant in the mRNA for Pathogenic Adhesive Factor SraP
Authors: Zhu Ling , Inoue Koichi , Yoshizumi Satoshi , Kobayashi Hiroshi , Zhang Yonglong , Ouyang Ming , Kato Fuminori , Sugai Motoyuki , Inouye Masayori
Published: February 2009

Escherichia coli
mRNA interferases, such as MazF and ChpBK, are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems present in its genome. A MazF homologue in
Staphylococcus aureus
(MazF
Sa
) has been shown to inhibit cell growth when induced in
E. coli
. Here, we determined the cleavage site for MazF
Sa
with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. MazF
Sa
specifically cleaves the RNA at a pentad sequence, U↓ACAU. Bioinformatics analysis revealed that this pentad sequence is significantly abundant in several genes, including the
sraP
gene in the
S. aureus
N315 strain. This gene encodes a serine-rich protein, which is known to play an important role in adhesion of the pathogen to human tissues and thus in endovascular infection. We demonstrated that the
sraP
mRNA became extremely unstable in comparison with the
ompA
mRNA only when MazF
Sa
was induced in
E. coli
. Further bioinformatics analysis indicated that the pentad sequence is also significantly abundant in the mRNAs for all the pathogenic factors in
S. aureus
. This observation suggests a possible regulatory relationship between the MazEF
Sa
TA module and the pathogenicity in
S. aureus
.
A one-pot synthesis of some novel tetrakisazo disperse dyes bearing twenty-membered macrocyclic poly(azomethine)
Authors: Lagvilava Irma , Matitaishvili Tea , Iardalashvili Ia , Elizbarashvili Elizbar
Published: February 2009

Multiple-chromophore-containing dyes have been synthesized by azo coupling of twenty-membered poly(azomethine) dye 1 and 3-nitro- (2a) or 2,4-dinitrobenzene-1-diazonium tetrafluoroborate (2b) in solvents of various polarity (water, chloroform) in the presence or absence of dibenzo-18-crown-6 as phase transfer catalyst. The dyeing ability of the dyes was investigated as well as spectral and kinetic study of azo coupling was performed. The obtained disperse dyes are suitable for dyeing Nylon and polyester fibers from light reddish-brown to dark brown colors with good uniformity of dyeing as well as light, washing and sublimation fastness.
Improved synthesis of [closo-1-CB9H10]– anion and new C-substituted derivatives
Authors: Ringstrand Bryan , Bateman Devon , Shoemaker Richard K. , Janoušek Zbyněk
Published: February 2009

The Brellochs method was utilized to gain access to the [closo-1-CB9H10]–anion (1). Previous work describing the details of the synthesis of1via the [closo-2-CB9H10]–anion (2) was brief and insufficient. Therefore, we report an optimized procedure for the synthesis of1, the preparation of the unknown C-halogenated series usingN-halosuccinimides and bis(benzenesulfonyl)fluoroamine, and the unknown C-methylated product using CH3I. NMR properties and regularities within the series were also investigated.
Rh- and Ru-complex-catalyzed dimerization of arylethynes in aqueous environment
Authors: Novák Petr , Kotora Martin
Published: February 2009

Complexes [RhCl(PPh3)3] and [Ru(CHPh)Cl2(PCy3)2] efficiently catalyzed the dimerization of arylethynes to the corresponding 1,4-substituted enynes in aqueous environment in the presence of sodium dodecyl sulfate. The Rh catalyst exhibited almost exclusive preference for the formation of E-isomers, the Ru one exhibits strong preference for the formation of Z-isomers.
Growth and sexual size dimorphism in Alberta populations of the eastern short-horned lizard, Phrynosoma douglassi brevirostre
Authors: Powell G. Lawrence , Russell Anthony P.
Published: March 2009

Alberta populations of Phrynosoma douglassi brevirostre display marked sexual size dimorphism, adult females being considerably larger than adult males. Discriminant analyses of whole mensural characters and of scaled mensural characters indicate that this dimorphism is present from birth, although it is more strongly expressed after sexual maturity. Recapture data were used to generate modified logistic by weight growth models for snout–vent length (SVL), and allometric models for each sex were generated for growth in tail length, head length, and head width. The SVL growth model for females indicates delayed maturity leading to greater adult size, an expected feature of a female viviparine. The SVL growth model for males indicates that growth ceases sooner than in females, resulting in a smaller adult size. This is possibly a result of male dispersal competition, an hypothesis further borne out by the results of a preliminary analysis of mobility in the two sexes, and may also be influenced by intersexual dietary competition. Differences in head dimensions between the sexes are a function of the differences in SVL at adulthood, but there is a significant sexual difference in the allometric relationship of tail length to SVL. No difference in the growth patterns and adult size of either sex was found to exist over the range in Alberta.
Female spacing patterns in brown-headed cowbirds
Authors: Teather Kevin L. , Robertson Raleigh J.
Published: March 2009

Breeding female cowbirds at the Queen's University Biological Station are often aggressive when encountering other females within their ranges, but they do not defend exclusive territories. The likelihood of aggression appears to be a function of two factors. First, females are more aggressive near the centres of their ranges than around its periphery, suggesting a gradient of aggression based on location within the female's range. Second, females are most aggressive in woodland and swampy areas and least aggressive in grassy areas and at artificial feeding stations. We suggest this is related to the availability of host nests.
Comparative morphology, seasonality, and a key to planktonic fish eggs from the Nova Scotian shelf
Authors: Markle Douglas F. , Frost Lee-Anne
Published: March 2009

The pelagic fish egg fauna of the Nova Scotian shelf consists of 16 species plus 5 predominantly slope species that occasionally drift onto the shelf. In 9 of these 21 species there were no morphological characters that could be used to identify early, pre-embryonic eggs. In the remainder, chorion structure, egg diameter, or egg versus oil globule diameter scattergrams could be used to completely or partially diagnose species identities. A key to the 21 species of eggs is presented. On the shelf proper pronounced seasonal variation was observed in presence or absence of oil globules with species having oil globules restricted to the period April to October. Seasonal variation was also observed in egg sizes and adult sizes, with species attaining a large body size producing large eggs in colder months, while smaller species producing smaller eggs (usually with an oil globule) spawned in warmer months. These seasonal patterns are not resolved but appear related to buoyancy requirements, metabolic rates, first feeding requirements, and phylogenetic constraints.
Rythmes circannuels chez les mammifères hibernants sauvages
Authors: Canguilhem B.
Published: March 2009

Circannual rhythms have been studied for 25 years. Most information has come from work on hibernators. Like circadian rhythms, circannuals rhythms are endogenous, innate, self sustained, and their spontaneous frequencies (biological period) are always different from astronomical time. They are insensitive to temperature changes. Their mechanisms are not yet known. Circannual rhythms are synchronized by external factors. There is, however, a great variability among these rhythms, some species being better synchronized than others. Differences in species ecology could be involved in this variability. The adaptative value of these rhythms is discussed.
Do Northern Harriers (Circus cyaneus) choose nest sites adaptively?
Authors: Simmons Robert , Smith P. C.
Published: March 2009

Northern Harrier (Circus cyaneus) nest success was studied in relation to habitat choices and availability from 1980 to 1982 in New Brunswick. Measurements of moisture, vegetation, and visibility at nest sites indicated that harriers most often used wet sites surrounded by cattails, with high visibility. Moisture and vegetation had a significant effect on nest success (p < 0.03), while visibility played no significant role. Discriminant analysis indicated that of the three variables, moisture was consistently the best predictor of nest success over 3 years (≥ 64%). Paradoxically, the order of settling by females was weakly correlated with the quality of nest sites in these years, yet choice was not limited by availability. We suggest, therefore, that nest site selection was a compromise between a wet nest site, close proximity to optimum foraging habitat, and, for females, access to a mate with a high food provisioning rate.
Reproduction of selected Mollusca in some low alkalinity lakes in south-central Ontario
Authors: Servos M. R. , Rooke J. B. , Mackie G. L.
Published: March 2009

Growth and reproduction of several species of molluscs were examined in six low alkalinity lakes in south-central Ontario. Amnicola limosa (Gastropoda: Amnicolidae) held through the summer of 1982 in cages in acidic lakes (pH 5.78–5.89) grew slower (p < 0.01) than those held in less acidic lakes (pH 6.03–6.84). The development of eggs of A. limosa in the laboratory was impaired at and below pH 5.0 and delayed at pH 5.5 relative to pH 6.0. Hence the recruitment failure and elimination of A. limosa that has been reported in acidifying lakes may be caused by reduced summer growth and (or) impaired development of the eggs. In contrast, Pisidium spp. (Bivalvia: Pisidiidae) do not appear to be adversely affected by low pH in the lakes in this study (pH 5.78–6.84). Caged Pisidium equilaterale did not have reduced growth in the acidic lakes during the summer of 1982. Pisidium casertanum and Pisidium ferrugineum had only slightly reduced (p < 0.05) fecundity (number of extramarsupial larvae per adult) in acidic lakes relative to less acidic lakes sampled in 1980.
Hydroids and medusae of Sarsia apicula (Murbach and Shearer, 1902) and Sarsia princeps (Haeckel, 1879) from British Columbia and Puget Sound with an evaluation of their systematic characters
Authors: Brinckmann-Voss Anita
Published: March 2009

The life cycles of Sarsia apicula (Murbach &Shearer) and Sarsia princeps (Haeckel) are described and figured and a comparison of all developmental stages and their taxonomic significance with related species is given. The value of different taxonomic characters, including colour, of the genus Sarsia is discussed.
Ligictaluridus Beverley-Burton, 1984 (Monogenea: Ancyrocephalidae) from catfishes (Siluriformes: Ictaluridae) in North America with redescriptions of the type species, Ligictaluridus pricei (Mueller, 1936), and three others
Authors: Klassen G. J. , Beverley-Burton M.
Published: March 2009

Ligictaluridus Beverley-Burton, 1984 has been proposed for ancyrocephalid monogeneans found on North American catfishes (Siluriformes: Ictaluridae). The genus is characterized by the following: penis curved, sclerotized, tubular; accessory piece with proximally articulating base and distally projecting limb of varying size and shape; dorsal and ventral hamuli with slender, curving shaft, dorsal hamuli smaller than ventral; dorsal and ventral transverse bars bowed with flange projecting medianly; larval hooks of variable size and shape. The type species, Ligictaluridus pricei (Mueller, 1936) Beverley-Burton, 1984, together with L. monticellii (Cognetti de Martiis, 1924) n. comb., L. floridanus (Mueller, 1936) Beverley-Burton, 1984, and L. mirabilis (Mueller, 1937) n. comb., is redescribed. The finding of L. monticellii on Ictalurus nebulosus taken in Lake Erie, Ontario, is a new record for North America. All the above species, as well as L. bychowskyi (Price and Mura, 1969) n. comb., are transferred from Cleidodiscus Mueller, 1934. The effects of variable cover-slip pressure and different preservation techniques on the morphology of some of the sclerotized structures are discussed.
A comparison of foliage discrimination learning in a specialist and a generalist species of migrant wood warbler (Aves: Parulidae)
Authors: Greenberg Russell
Published: March 2009

The Bay-breasted Warbler (Dendroica castanea) is more generalized in its foraging behavior than the Chestnut-sided Warbler (Dendroica pensylvanica) where the two species winter together in Panama. I tested the hypothesis that the Bay-breasted Warbler would learned to discriminate between foliage types of differing profitability more rapidly than the more specialized Chestnut-sided Warbler. The performance of the two species in a laboratory discrimination experiment involving two foliage types was similar. Both species learned the foliage discrimination rapidly, in an average of 22 trials. Extinction occurred at a similarly rapid rate (17 trials) in both species as well. Differences in apparent foraging plasticity between the two species may be based primarily on their different responses to novelty, rather than differences in learning abilities. None of the warblers showed any tendency to attempt to solve the problem based on the irrelevant spatial cues prior to acquiring the correct response. Visual discrimination problems based on habitat cues are more relevant to insectivorous birds than are the more commonly administered spatial learning problems.