bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Inner Membrane Retention Signals inPseudomonas aeruginosaLipoproteins
Authors: Lewenza Shawn , Mhlanga Musa M. , Pugsley Anthony P. 
Published: July 2008

ABSTRACTThe ultimate membrane localization and function of most of the 185 predictedPseudomonas aeruginosaPAO1 lipoproteins remain unknown. We constructed a fluorescent lipoprotein, CSFPOmlA-ChFP, by fusing the signal peptide and the first four amino acids of theP. aeruginosaouter membrane lipoprotein OmlA to the monomeric red fluorescent protein mCherry (ChFP). When cells were plasmolyzed with 0.5 M NaCl, the inner membrane separated from the outer membrane and formed plasmolysis bays. This permits the direct observation of fluorescence in either the outer or inner membrane. CSFPOmlA-ChFP was shown to localize in the outer membrane by fluorescence microscopy and immunoblotting analysis of inner and outer membrane fractions. The site-directed substitution of the amino acids at positions +2, +3, and +4 in CSFPOmlA-ChFP was performed to test the effects on lipoprotein localization of a series of amino acid sequences selected from a panel of predicted lipoproteins. We confirmed Asp+2and Lys+3Ser+4function as inner membrane retention signals and identified four novel inner membrane retention signals: CK+2V+3E+4, CG+2G+3G+4, CG+2D+3D+4, and CQ+2G+3S+4. These inner membrane retention signals are found in 5% of the 185 predictedP. aeruginosalipoproteins. Full-length chimeras of predicted lipoproteins PA4370 and PA3262 fused to mCherry were shown to reside in the inner membrane and showed a nonuniform or patchy distribution in the membrane. The optical sectioning of cells producing PA4370CGDD-ChFP and PA3262CDSQ-ChFP by confocal microscopy improved the resolution and indicated a helix-like localization pattern in the inner membrane. The method described here permits the in situ visualization of lipoprotein localization and should work equally well for other membrane-associated proteins.
Genomic Analysis of the Role of RNase R in the Turnover ofPseudomonas putidamRNAs
Authors: Fonseca Pilar , Moreno Renata , Rojo Fernando 
Published: July 2008

ABSTRACTRNase R is a 3′-5′ highly processive exoribonuclease that can digest RNAs with extensive secondary structure. We analyzed the global effect of eliminating RNase R on thePseudomonas putidatranscriptome and the expression of thernrgene under diverse conditions. The absence of RNase R led to increased levels of many mRNAs, indicating that it plays an important role in mRNA turnover.
SoxAX Binding Protein, a Novel Component of the Thiosulfate-Oxidizing Multienzyme System in the Green Sulfur Bacterium
Chlorobium tepidum
Authors: Ogawa Takuro , Furusawa Toshinari , Nomura Ryohei , Seo Daisuke , Hosoya-Matsuda Naomi , Sakurai Hidehiro , Inoue Kazuhito 
Published: July 2008

ABSTRACT

From the photosynthetic green sulfur bacterium
Chlorobium tepidum
(pro synon.
Chlorobaculum tepidum
), we have purified three factors indispensable for the thiosulfate-dependent reduction of the small, monoheme cytochrome
c
554
. These are homologues of sulfur-oxidizing (Sox) system factors found in various thiosulfate-oxidizing bacteria. The first factor is SoxYZ that serves as the acceptor for the reaction intermediates. The second factor is monomeric SoxB that is proposed to catalyze the hydrolytic cleavage of sulfate from the SoxYZ-bound oxidized product of thiosulfate. The third factor is the trimeric cytochrome
c
551
, composed of the monoheme cytochrome SoxA, the monoheme cytochrome SoxX, and the product of the hypothetical open reading frame CT1020. The last three components were expressed separately in
Escherichia coli
cells and purified to homogeneity. In the presence of the other two Sox factors, the recombinant SoxA and SoxX showed a low but discernible thiosulfate-dependent cytochrome
c
554
reduction activity. The further addition of the recombinant CT1020 protein greatly increased the activity, and the total activity was as high as that of the native SoxAX-CT1020 protein complex. The recombinant CT1020 protein participated in the formation of a tight complex with SoxA and SoxX and will be referred to as SAXB (SoxAX binding protein). Homologues of the SAXB gene are found in many strains, comprising roughly about one-third of the thiosulfate-oxidizing bacteria whose
sox
gene cluster sequences have been deposited so far and ranging over the
Chlorobiaciae, Chromatiaceae, Hydrogenophilaceae, Oceanospirillaceae
, etc. Each of the deduced SoxA and SoxX proteins of these bacteria constitute groups that are distinct from those found in bacteria that apparently lack SAXB gene homologues.
CDP-Alcohol Hydrolase, a Very Efficient Activity of the 5′-Nucleotidase/UDP-Sugar Hydrolase Encoded by the
ushA
Gene of
Yersinia intermedia
and
Escherichia coli
Authors: Alves-Pereira Isabel , Canales José , Cabezas Alicia , Martín Cordero Paloma , Costas María Jesús , Cameselle José Carlos 
Published: July 2008

ABSTRACT

Nucleoside 5′-diphosphate-X hydrolases are interesting enzymes to study due to their varied activities and structure-function relationships and the roles they play in the disposal, assimilation, and modulation of the effects of their substrates. Few of these enzymes with a preference for CDP-alcohols are known. In
Yersinia intermedia
suspensions prepared from cultures on Columbia agar with 5% sheep blood, we found a CDP-alcohol hydrolase liberated to Triton X-100-containing medium. Growth at 25°C was deemed optimum in terms of the enzyme-activity yield. The purified enzyme also displayed 5′-nucleotidase, UDP-sugar hydrolase, and dinucleoside-polyphosphate hydrolase activities. It was identified as the protein product (UshA
Yi
) of the
Y. intermedia ushA
gene (
ushA
Yi
) by its peptide mass fingerprint and by PCR cloning and expression to yield active enzyme. All those activities, except CDP-alcohol hydrolase, have been shown to be the properties of UshA of
Escherichia coli
(UshA
Ec
). Therefore, UshA
Ec
was expressed from an appropriate plasmid and tested for CDP-alcohol hydrolase activity. UshA
Ec
and UshA
Yi
behaved similarly. Besides being the first study of a UshA enzyme in the genus
Yersinia
, this work adds CDP-alcohol hydrolase to the spectrum of UshA activities and offers a novel perspective on these proteins, which are viewed here for the first time as highly efficient enzymes with
k
cat
/K

m

ratios near the theoretical maximum level of catalytic activities. The results are discussed in the light of the known structures of UshA
Ec
conformers and the respective homology models constructed for UshA
Yi
, and also in relation to possible biological functions. Interestingly, every
Yersinia
species with a sequenced genome contains an intact
ushA
gene, except
Y. pestis
, which in all its sequenced biovars contains a
ushA
gene inactivated by frameshift mutations.
Translational Regulation by an Intramolecular Stem-Loop Is Required for Intermolecular RNA Regulation of theparAddiction Module
Authors: Shokeen Sonia , Patel Smita , Greenfield Tony J. , Brinkman Cassandra , Weaver Keith E. 
Published: July 2008

ABSTRACTTheparstability determinant ofEnterococcus faecalisplasmid pAD1 is the only antisense RNA-regulated addiction module identified to date in gram-positive bacteria.parencodes two small, convergently transcribed RNAs, designated RNA I and RNA II, that function as the toxin (Fst)-encoding and antitoxin components, respectively. Previous work showed that structures at the 5′ end of RNA I are important in regulating its translation. The work presented here reveals that a stem-loop sequestering the Fst ribosome binding site is required for translational repression but a helix sequestering the 5′ end of RNA I is not. Furthermore, disruption of the stem-loop prevented RNA II-mediated repression of Fst translation in vivo. Finally, although Fst-encoding wild-type RNA I is not toxic inEscherichia coli, mutations affecting stem-loop stability resulted in toxicity in this host, presumably due to increased translation.
Two Members of a Network of Putative Na
+
/H
+
Antiporters Are Involved in Salt and pH Tolerance of the Freshwater Cyanobacterium
Synechococcus elongatus
Authors: Billini Maria , Stamatakis Kostas , Sophianopoulou Vicky 
Published: July 2008

ABSTRACT

Synechococcus elongatus
strain PCC 7942 is an alkaliphilic cyanobacterium that tolerates a relatively high salt concentration as a freshwater microorganism. Its genome sequence revealed seven genes,
nha1
to
nha7
(syn_pcc79420811, syn_pcc79421264, syn_pcc7942359, syn_pcc79420546, syn_pcc79420307, syn_pcc79422394, and syn_pcc79422186), and the deduced amino acid sequences encoded by these genes are similar to those of Na
+
/H
+
antiporters. The present work focused on molecular and functional characterization of these
nha
genes encoding Na
+
/H
+
antiporters. Our results show that of the
nha
genes expressed in
Escherichia coli
, only
nha3
complemented the deficient Na
+
/H
+
antiporter activity of the Na
+
-sensitive TO114 recipient strain. Moreover, two of the cyanobacterial strains with separate disruptions in the
nha
genes (Δ
nha1
, Δ
nha2
, Δ
nha3
, Δ
nha4
, Δ
nha5
, and Δ
nha7
) had a phenotype different from that of the wild type. In particular, Δ
nhA3
cells showed a high-salt- and alkaline-pH-sensitive phenotype, while Δ
nha2
cells showed low salt and alkaline pH sensitivity. Finally, the transcriptional profile of the
nha1
to
nha7
genes, monitored using the real-time PCR technique, revealed that the
nha6
gene is upregulated and the
nha1
gene is downregulated under certain environmental conditions.
Function of a Chemotaxis-Like Signal Transduction Pathway in Modulating Motility, Cell Clumping, and Cell Length in the Alphaproteobacterium
Azospirillum brasilense
Authors: Bible Amber N. , Stephens Bonnie B. , Ortega Davi R. , Xie Zhihong , Alexandre Gladys 
Published: July 2008

ABSTRACT

A chemotaxis signal transduction pathway (hereafter called Che1) has been previously identified in the alphaproteobacterium
Azospirillum brasilens
e. Previous experiments have demonstrated that although mutants lacking CheB and/or CheR homologs from this pathway are defective in chemotaxis, a mutant in which the entire chemotaxis pathway has been mutated displayed a chemotaxis phenotype mostly similar to that of the parent strain, suggesting that the primary function of this Che1 pathway is not the control of motility behavior. Here, we report that mutants carrying defined mutations in the
cheA1
(strain AB101) and the
cheY1
(strain AB102) genes and a newly constructed mutant lacking the entire operon [Δ(
cheA1-cheR1
)::Cm] (strain AB103) were defective, but not null, for chemotaxis and aerotaxis and had a minor defect in swimming pattern. We found that mutations in genes of the Che1 pathway affected the cell length of actively growing cells but not their growth rate. Cells of a mutant lacking functional
cheB1
and
cheR1
genes (strain BS104) were significantly longer than wild-type cells, whereas cells of mutants impaired in the
cheA1
or
cheY1
genes, as well as a mutant lacking a functional Che1 pathway, were significantly shorter than wild-type cells. Both the modest chemotaxis defects and the observed differences in cell length could be complemented by expressing the wild-type genes from a plasmid. In addition, under conditions of high aeration, cells of mutants lacking functional
cheA1
or
cheY1
genes or the Che1 operon formed clumps due to cell-to-cell aggregation, whereas the mutant lacking functional CheB1 and CheR1 (BS104) clumped poorly, if at all. Further analysis suggested that the nature of the exopolysaccharide produced, rather than the amount, may be involved in this behavior. Interestingly, mutants that displayed clumping behavior (lacking
cheA1
or
cheY1
genes or the Che1 operon) also flocculated earlier and quantitatively more than the wild-type cells, whereas the mutant lacking both CheB1 and CheR1 was delayed in flocculation. We propose that the Che1 chemotaxis-like pathway modulates the cell length as well as clumping behavior, suggesting a link between these two processes. Our data are consistent with a model in which the function of the Che1 pathway in regulating these cellular functions directly affects flocculation, a cellular differentiation process initiated under conditions of nutritional imbalance.
The Metal Dependence of Pyoverdine Interactions with Its Outer Membrane Receptor FpvA
Authors: Greenwald Jason , Zeder-Lutz Gabrielle , Hagege Agnès , Celia Hervé , Pattus Franc 
Published: July 2008

ABSTRACTTo acquire iron,Pseudomonas aeruginosasecretes the fluorescent siderophore pyoverdine (Pvd), which chelates iron and shuttles it into the cells via the specific outer membrane transporter FpvA. We studied the role of iron and other metals in the binding and transport of Pvd by FpvA and conclude that there is no significant affinity between FpvA and metal-free Pvd. We found that the fluorescent in vivo complex of iron-free FpvA-Pvd is in fact a complex with aluminum (FpvA-Pvd-Al) formed from trace aluminum in the growth medium. WhenPseudomonas aeruginosawas cultured in a medium that had been treated with a metal affinity resin, the in vivo formation of the FpvA-Pvd complex and the recycling of Pvd on FpvA were nearly abolished. The accumulation of Pvd in the periplasm ofPseudomonas aeruginosawas also reduced in the treated growth medium, while the addition of 1 μM AlCl3to the treated medium restored the effects of trace metals observed in standard growth medium. Using fluorescent resonance energy transfer and surface plasmon resonance techniques, the in vitro interactions between Pvd and detergent-solubilized FpvA were also shown to be metal dependent. We demonstrated that FpvA binds Pvd-Fe but not Pvd and that Pvd did not compete with Pvd-Fe for FpvA binding. In light of our finding that the Pvd-Al complex is transported across the outer membrane ofPseudomonas aeruginosa, a model for siderophore recognition based on a metal-induced conformation followed by redox selectivity for iron is discussed.
The
Bacillus cereus
GerN and GerT Protein Homologs Have Distinct Roles in Spore Germination and Outgrowth, Respectively
Authors: Senior Adam , Moir Anne 
Published: July 2008

ABSTRACT

The GerT protein of
Bacillus cereus
shares 74% amino acid identity with its homolog GerN. The latter is a Na
+
/H
+
-K
+
antiporter that is required for normal spore germination in inosine. The germination properties of single and double mutants of
B. cereus
ATCC 10876 reveal that unlike GerN, which is required for all germination responses that involve the GerI germinant receptor, the GerT protein does not have a significant role in germination, although it is required for the residual GerI-mediated inosine germination response of a
gerN
mutant. In contrast, GerT has a significant role in outgrowth;
gerT
mutant spores do not outgrow efficiently under alkaline conditions and outgrow more slowly than the wild type in the presence of high NaCl concentrations. The GerT protein in
B. cereus
therefore contributes to the success of spore outgrowth from the germinated state during alkaline or Na
+
stress.
Characterization of Sec-Translocon-Dependent Extracytoplasmic Proteins of
Rickettsia typhi
Authors: Ammerman Nicole C. , Rahman M. Sayeedur , Azad Abdu F. 
Published: July 2008

ABSTRACT

As obligate intracellular, vector-borne bacteria, rickettsiae must adapt to both mammalian and arthropod host cell environments. Deciphering the molecular mechanisms of the interactions between rickettsiae and their host cells has largely been hindered by the genetic intractability of these organisms; however, research in other gram-negative pathogens has demonstrated that many bacterial determinants of attachment, entry, and pathogenesis are extracytoplasmic proteins. The annotations of several rickettsial genomes indicate the presence of homologs of the Sec translocon, the major route for bacterial protein secretion from the cytoplasm. For
Rickettsia typhi
, the etiologic agent of murine typhus, homologs of the Sec-translocon-associated proteins LepB, SecA, and LspA have been functionally characterized; therefore, the
R. typhi
Sec apparatus represents a mechanism for the secretion of rickettsial proteins, including virulence factors, into the extracytoplasmic environment. Our objective was to characterize such Sec-dependent
R. typhi
proteins in the context of a mammalian host cell infection. By using the web-based programs LipoP, SignalP, and Phobius, a total of 191
R. typhi
proteins were predicted to contain signal peptides targeting them to the Sec translocon. Of these putative signal peptides, 102 were tested in an
Escherichia coli
-based alkaline phosphatase (PhoA) gene fusion system. Eighty-four of these candidates exhibited signal peptide activity in
E. coli
, and transcriptional analysis indicated that at least 54 of the
R. typhi
extracytoplasmic proteins undergo active gene expression during infections of HeLa cells. This work highlights a number of interesting proteins possibly involved in rickettsial growth and virulence in mammalian cells.
The Type III Pantothenate Kinase Encoded by
coaX
Is Essential for Growth of
Bacillus anthracis
Authors: Paige Carleitta , Reid Sean D. , Hanna Philip C. , Claiborne Al 
Published: July 2008

ABSTRACT

In
Bacillus anthracis
, the novel type III pantothenate kinase (PanK

Ba

; encoded by
coaX
) catalyzes the first committed step in coenzyme A biosynthesis. We have demonstrated by analyzing the growth characteristics of a conditional
coaX
mutant that PanK

Ba

is an essential enzyme, thus contributing to its validation as a new antimicrobial target.
Doctors with difficulties: why so few women?
Authors: Firth-Cozens J 
Published: July 2008

Abstract
The National Clinical Assessment Service (NCAS), an NHS organisation that assesses doctors and dentists referred to them because of perceived difficulties, has produced a report describing data arising from its first 4 years, showing that male doctors were referred to the service considerably more often than female doctors. Despite women accounting for 42% of the general practitioner medical workforce and 37% of the medical hospital and community (H&C) workforce in 2004, only 13% of GPs and 20% of H&C NCAS referrals were women. When the H&C data were split into specialties, women were under-represented proportionally in all specialties. This paper offers a review of possible reasons for these gender differences and in doing so contributes to the debates concerning problems in performance and also the costs of employing a growing proportion of women doctors. Firstly, it hypothesises that the NCAS data may be non-representative of similar agency data, but finds that in disciplinary organisations of various types around the world, men are consistently over-represented. Secondly, it suggests that perhaps men are referred to such agencies more often than women because their employers are more lenient on women. There is no evidence for this, and it requires primary research to investigate it further. Finally, it considers gender differences in the attributes, beyond technical skills, that underpin a good doctor–patient relationship and finds that, on these attributes, women usually excel over men. In addition, far fewer women are disciplined for addiction. The implications of this for education and rehabilitation are considered. It concludes that any analysis of the economic costs of employing a greater proportion of female doctors must take into account the higher costs of men's litigation, discipline and retraining.
Concentric and radial joint systems within basic sills and their associated porosity enhancement, Neuquén Basin, Argentina
Authors: Bermúdez Adriana , Delpino Daniel H. 
Published: July 2008

Abstract
One of the remarkable characteristics of the Neuquén Basin is that a group of Cenozoic shallow microgabbroic sills exhibit gas and oil shows, and some have produced hydrocarbons for up to 20 years. Their capacity to act as reservoirs is caused by the increase in the effective porosity owing to the simultaneous development of petrological and fracture processes during cooling that generate systematic variations inside the intrusive bodies.
In this work we describe in detail an unusual intracolumnar joint system formed by two joint sets, referred to here as concentric and radial joint sets. This atypical joint system is developed within the small stacked polyhedrons into which certain zones of classic prismatic columns are transformed during the cooling processes. These small polyhedrons are laterally bounded by the vertical planes that limit the columns, and at the top and bottom by transversal joint planes oriented almost perpendicular to the column axis.
As a result of detailed macroscopic analysis, the genesis of the concentric joint sets have been interpreted as resulting from either the development of natural convective flows or from complex processes of cooling influenced by the joint-bounded planes of the columns. When the radial joint set is present, it is clearly related to the development of thermal stresses between the outer and inner parts of the small polyhedrons.
In geology textbooks and previous published papers the genesis of what is informally called ‘onion skin’ joint sets in basic intrusive and extrusive igneous rocks has been attributed to processes related to either weathering or deuteric alteration. However, based on detailed macroscopic observations, our work suggests that, in most cases, any type of alteration process only accentuates the planes previously generated during the cooling process.
Lithological and structural controls on the emplacement and morphology of sills in sedimentary basins
Authors: Thomson Ken , Schofield Nick 
Published: July 2008

Abstract
Three-dimensional seismic data from the NW European Atlantic margin provide detailed data on the relationships between sills, dykes, laccoliths and pre-existing basin structure. The data show that sills are predominantly concave-upwards in shape, being complete or partial versions of radially or bilaterally symmetrical forms that possess flat inner saucers connected to a flat outer rim by a steeply inclined sheet. Sills can be sourced from the steep climbing portions of deeper sills, and can be shown to exploit pre-existing faults to feed shallower sills and laccoliths. Magma flow patterns, as revealed by opacity rendering, suggest that sills propagate upwards and outwards away from the magma feeder. As an individual sill can consist of several leaves emplaced at different stratigraphic levels, and as a sill or dyke can provide magma to other sills and laccoliths, the data suggest that neutral buoyancy concepts may not provide a complete explanation for the mechanism and level of sill emplacement. Instead, the data suggest that the presence of ductile horizons such as overpressured shales may permit sill formation below the neutrally buoyant level. Emplacement below the level of neutral buoyancy would permit sill inflation, and the associated fracturing of the country rock would permit magma to exploit these pathways in order to ascend to shallower levels and feed shallower intrusions.