bluuebunny/crossref_metadata_2025 huggingface dataset:
The specific delivery of proteins to human liver cells by engineered bio‐nanocapsules
Authors: Yu Dongwei , Amano Chie , Fukuda Takayuki , Yamada Tadanori , Kuroda Shun'ichi , Tanizawa Katsuyuki , Kondo Akihiko , Ueda Masakazu , Yamada Hidenori , Tada Hiroko , Seno Masaharu
Published: July 2005

A bio‐nanocapsule (BNC), composed of the surface antigen (sAg) of the hepatitis B virus, is an efficient nanomachine with which to accomplish the liver‐specific delivery of genes and drugs. Approximately 110 molecules of sAg are associated to form a BNC particle with an average diameter of 130 nm. The L protein is an sAg peptide composed mainly of preS and S regions. The preS region, with specific affinity for human hepatocytes, is localized in the N‐terminus. The S region following the preS has two transmembrane regions responsible for the formation of particles. In this study, the fusion of emerald green fluorescent protein (EGFP) at the C‐terminus of the S region was designed to deliver proteins to human hepatocytes. Truncation of the C‐terminus of the S region was required to obtain sufficient expression levels in Cos7 cells. The nanoparticles that were produced delivered EGFP to human hepatoma cells, displaying the EGFP moiety outside, or enclosing it inside. However, only a single orientation characterizes the particle, so that either type of L fusion particle could be effectively and independently separated by an antibody affinity column. The dual C‐terminal topologies of the L fusion particles designed in this study could be applied to various proteins for the C‐terminal moiety of the L fusion proteins, depending on the character of the proteins, such as cytoplasmic proteins, as well as cytokines or ligands to cell surface receptors. We suggest that this fusion design is the most efficient way to prepare a BNC that delivers proteins to specific cells or tissues.
Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D
Authors: Welin Martin , Skovgaard Tine , Knecht Wolfgang , Zhu Chunying , Berenstein Dvora , Munch‐Petersen Birgitte , Piškur Jure , Eklund Hans
Published: July 2005

The Drosophila melanogaster deoxyribonucleoside kinase (Dm‐dNK) double mutant N45D/N64D was identified during a previous directed evolution study. This mutant enzyme had a decreased activity towards the natural substrates and decreased feedback inhibition with dTTP, whereas the activity with 3′‐modified nucleoside analogs like 3′‐azidothymidine (AZT) was nearly unchanged. Here, we identify the mutation N64D as being responsible for these changes. Furthermore, we crystallized the mutant enzyme in the presence of one of its substrates, thymidine, and the feedback inhibitor, dTTP. The introduction of the charged Asp residue appears to destabilize the LID region (residues 167–176) of the enzyme by electrostatic repulsion and no hydrogen bond to the 3′‐OH is made in the substrate complex by Glu172 of the LID region. This provides a binding space for more bulky 3′‐substituents like the azido group in AZT but influences negatively the interactions between Dm‐dNK, substrates and feedback inhibitors based on deoxyribose. The detailed picture of the structure–function relationship provides an improved background for future development of novel mutant suicide genes for Dm‐dNK‐mediated gene therapy.
Sevelamer Hydrochloride and Calcium Bicarbonate Reduce Serum Fibroblast Growth Factor 23 Levels in Dialysis Patients
Authors: Koiwa Fumihiko , Kazama Junichiro J , Tokumoto Akihide , Onoda Noritaka , Kato Hitoshi , Okada Tomoyuki , Nii‐Kono Tomoko , Fukagawa Masafumi , Shigematsu Takashi ,   ROD21 Clinical Research Group
Published: July 2005

Abstract:  Fibroblast growth factor 23 (FGF23) is a member of the fibroblast growth factor superfamily which displays a strong phosphaturic action and an inhibition of vitamin D 1‐α hydroxylase activity. Fourty‐six patients undergoing maintenance hemodialysis therapy participated in the study. They were randomly divided into 2 groups, and treated with either 3 g sevelamer hydrochloride + 3 g of calcium bicarbonate (CaCO3), or 3 g of CaCO3 alone. Serum FGF23 levels were determined by a sandwich enzyme‐linked immunosorbent assay (ELISA) system that detects the intact form of FGF23 molecules. Although the serum inorganic phosphate (Pi) levels were comparable before treatment, the levels were significantly lower in the patients treated with sevelamer hydrochloride + CaCO3 than those with CaCO3 alone after 4 weeks of treatment (P < 0.05). Serum FGF23 levels significantly decreased after 4 weeks of the treatment with sevelamer hydrochloride + CaCO3 from the pretreatment levels (P < 0.05), while no changes were found in the patients treated with CaCO3 alone. Thus, the use of sevelamer hydrochloride and CaCO3 reduced serum FGF23 levels in dialysis patients presumably through inhibiting phosphate load into the intestine.
Higher-Order Pseudoaveraging via Harmonic Balance for Strongly Nonlinear Oscillations
Authors: Nandakumar K. , Chatterjee Anindya
Published: July 2005

Some strongly nonlinear conservative oscillators, on slight perturbation, can be studied via averaging of elliptic functions. These and many other oscillations allow harmonic balance-based averaging (HBBA), recently developed as an approximate first-order calculation. Here, we extend HBBA to higher orders. Unlike the usual higher-order averaging for weakly nonlinear oscillations, here both the dynamic variable and time are averaged with respect to an auxiliary variable. Since the harmonic balance approximations introduce technically O(1) errors at each order, the higher-order results are not strictly asymptotic. Nevertheless, as we show with examples, for reasonable values of the small expansion parameter, excellent approximations are obtained.
Extending Den Hartog’s Vibration Absorber Technique to Multi-Degree-of-Freedom Systems
Authors: Ozer Mehmet Bulent , Royston Thomas J.
Published: July 2005

The most common method to design tuned dynamic vibration absorbers is still that of Den Hartog, based on the principle of invariant points. However, this method is optimal only when attaching the absorber to a single-degree-of-freedom undamped main system. In the present paper, an extension of the classical Den Hartog approach to a multi-degree-of-freedom undamped main system is presented. The Sherman-Morrison matrix inversion theorem is used to obtain an expression that leads to invariant points for a multi-degree-of-freedom undamped main system. Using this expression, an analytical solution for the optimal damper value of the absorber is derived. Also, the effect of location of the absorber in the multi-degree-of-freedom system and the effect of the absorber on neighboring modes are discussed.
Hopf Bifurcation of a Magnetic Bearing System with Time Delay
Authors: Ji J. C. , Hansen Colin H.
Published: July 2005

This paper is concerned with a study of the influence of a time delay occurring in a PD feedback control on the dynamic stability of a rotor suspended by magnetic bearings. In the presence of geometric coordinate coupling and time delay, the equations of motion governing the response of the rotor are a set of two-degree-of-freedom nonlinear differential equations with time delay coupling in nonlinear terms. It is found that as the time delay increases beyond a critical value, the equilibrium position of the rotor motion becomes unstable and may bifurcate into two qualitatively different kinds of periodic motion. The resultant Hopf bifurcation is associated with two coincident pairs of complex conjugate eigenvalues crossing the imaginary axis. Based on the reduction of the infinite dimensional problem to the flow on a four-dimensional center manifold, the bifurcating periodic solutions are investigated using a perturbation method.
Convergence Behaviors of Reduced-Order Models For Frictional
Contacts
Authors: Deshmukh D. V. , Berger E. J. , Mackin T. J. , Inglis H.
Published: July 2005

Numerical models to simulate interface behavior of friction connections under cyclic loading are investigated. The question of validity of lower-order models in successfully capturing response of friction joints under cyclic loading is addressed. Single-element macroslip models are not capable of capturing localized interface behavior prior to gross interfacial slip. This paper focuses on the convergence behavior of a multipoint contact microslip model comprised of Iwan-type elements for different physical parameters such as system response amplitude and kinematic state of the friction joint. System dynamics play a significant role in determining the convergence of structural behavior, especially for tuned damper sets in the nonzero damper mass case. This behavior is explored using simple linearized models that explain the response sensitivity in terms of the overall modal density near the forcing frequency. Convergence of the interface response kinematics is also considered, with a focus on the number of damping elements operating in the stick, stick-slip, and slip regimes at steady state. Energy dissipation scaling under light forcing is also examined, with the class of models considered here yielding scaling exponents consistent with experimental observations and analytical predictions from the literature. We show that the interface kinematic behavior converges at a slower rate than the structural response and therefore requires a higher-order interface model. These results suggest that extremely low-order models (i.e., <5 damping elements) provide predictions that are model order dependent, while higher-order models (i.e., >50 damping elements) are not. This result impacts model development and calibration approaches, as well as providing clues for appropriate model reduction strategies.
Wave Reflection and Transmission in Timoshenko Beams and Wave Analysis of Timoshenko Beam Structures
Authors: Mei C. , Mace B. R.
Published: July 2005

This paper concerns wave reflection, transmission, and propagation in Timoshenko beams together with wave analysis of vibrations in Timoshenko beam structures. The transmission and reflection matrices for various discontinuities on a Timoshenko beam are derived. Such discontinuities include general point supports, boundaries, and changes in section. The matrix relations between the injected waves and externally applied forces and moments are also derived. These matrices can be combined to provide a concise and systematic approach to vibration analysis of Timoshenko beams or complex structures consisting of Timoshenko beam components. The approach is illustrated with several numerical examples.
Salicylate Biosynthesis: Overexpression, Purification, and Characterization of Irp9, a Bifunctional Salicylate Synthase fromYersinia enterocolitica
Authors: Kerbarh Olivier , Ciulli Alessio , Howard Nigel I. , Abell Chris
Published: July 2005

In some bacteria, salicylate is synthesized using the enzymes isochorismate synthase and isochorismate pyruvate lyase. In contrast, gene inactivation and complementation experiments withYersinia enterocoliticasuggest the synthesis of salicylate in the biosynthesis of the siderophore yersiniabactin involves a single protein, Irp9, which converts chorismate directly into salicylate. In the present study, Irp9 was for the first time heterologously expressed inEscherichia colias a hexahistidine fusion protein, purified to near homogeneity, and characterized biochemically. The recombinant protein was found to be a dimer, each subunit of which has a molecular mass of 50 kDa. Enzyme assays, reverse-phase high-pressure liquid chromatography and1H nuclear magnetic resonance (NMR) spectroscopic analyses confirmed that Irp9 is a salicylate synthase and converts chorismate to salicylate with aKmfor chorismate of 4.2 μM and akcatof 8 min−1. The reaction was shown to proceed through the intermediate isochorismate, which was detected directly using1H NMR spectroscopy.
Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by
Sphingomonas paucimobilis
SYK-6
Authors: Kasai Daisuke , Masai Eiji , Miyauchi Keisuke , Katayama Yoshihiro , Fukuda Masao
Published: July 2005

Sphingomonas paucimobilis
SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-
O
-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent
O
-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA
O
-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene,
desB
, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in
Escherichia coli
was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The

K
m

for gallate and the
V
max
were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.
The Stringent Response Is Required for Amino Acid and Nitrate Utilization, Nod Factor Regulation, Nodulation, and Nitrogen Fixation in
Rhizobium etli
Authors: Calderón-Flores Arturo , Du Pont Gisela , Huerta-Saquero Alejandro , Merchant-Larios Horacio , Servín-González Luis , Durán Socorro
Published: July 2005

A
Rhizobium etli
Tn
5
insertion mutant, LM01, was selected for its inability to use glutamine as the sole carbon and nitrogen source. The Tn
5
insertion in LM01 was localized to the
rsh
gene, which encodes a member of the RelA/SpoT family of proteins. The LM01 mutant was affected in the ability to use amino acids and nitrate as nitrogen sources and was unable to accumulate (p)ppGpp when grown under carbon and nitrogen starvation, as opposed to the wild-type strain, which accumulated (p)ppGpp under these conditions. The
R. etli rsh
gene was found to restore (p)ppGpp accumulation to a Δ
relA
Δ
spoT
mutant of
Escherichia coli
. The
R. etli
Rsh protein consists of 744 amino acids, and the Tn
5
insertion in LM01 results in the synthesis of a truncated protein of 329 amino acids; complementation experiments indicate that this truncated protein is still capable of (p)ppGpp hydrolysis. A second
rsh
mutant of
R. etli
, strain AC1, was constructed by inserting an Ω element at the beginning of the
rsh
gene, resulting in a null allele. Both AC1 and LM01 were affected in Nod factor production, which was constitutive in both strains, and in nodulation; nodules produced by the
rsh
mutants in
Phaseolus vulgaris
were smaller than those produced by the wild-type strain and did not fix nitrogen. In addition, electron microscopy revealed that the mutant bacteroids lacked poly-β-hydroxybutyrate granules. These results indicate a central role for the stringent response in symbiosis.
Identification, Characterization, and Classification of Genes Encoding Perchlorate Reductase
Authors: Bender Kelly S. , Shang Ching , Chakraborty Romy , Belchik Sara M. , Coates John D. , Achenbach Laurie A.
Published: July 2005

The reduction of perchlorate to chlorite, the first enzymatic step in the bacterial reduction of perchlorate, is catalyzed by perchlorate reductase. The genes encoding perchlorate reductase (
pcrABCD
) in two
Dechloromonas
species were characterized. Sequence analysis of the
pcrAB
gene products revealed similarity to α- and β-subunits of microbial nitrate reductase, selenate reductase, dimethyl sulfide dehydrogenase, ethylbenzene dehydrogenase, and chlorate reductase, all of which are type II members of the microbial dimethyl sulfoxide (DMSO) reductase family. The
pcrC
gene product was similar to a
c
-type cytochrome, while the
pcrD
gene product exhibited similarity to molybdenum chaperone proteins of the DMSO reductase family members mentioned above. Expression analysis of the
pcrA
gene from
Dechloromonas agitata
indicated that transcription occurred only under anaerobic (per)chlorate-reducing conditions. The presence of oxygen completely inhibited
pcrA
expression regardless of the presence of perchlorate, chlorate, or nitrate. Deletion of the
pcrA
gene in
Dechloromonas aromatica
abolished growth in both perchlorate and chlorate but not growth in nitrate, indicating that the
pcrABCD
genes play a functional role in perchlorate reduction separate from nitrate reduction. Phylogenetic analysis of PcrA and other α-subunits of the DMSO reductase family indicated that perchlorate reductase forms a monophyletic group separate from chlorate reductase of
Ideonella dechloratans
. The separation of perchlorate reductase as an activity distinct from chlorate reductase was further supported by DNA hybridization analysis of (per)chlorate- and chlorate-reducing strains using the
pcrA
gene as a probe.
Involvement of AlgQ in Transcriptional Regulation of Pyoverdine Genes in
Pseudomonas aeruginosa
PAO1
Authors: Ambrosi Cecilia , Tiburzi Federica , Imperi Francesco , Putignani Lorenza , Visca Paolo
Published: July 2005

In response to iron limitation,
Pseudomonas aeruginosa
produces the fluorescent siderophore pyoverdine. Transcription of pyoverdine biosynthetic (
pvd
) genes is driven by the iron starvation sigma factor PvdS, which is negatively regulated by the Fur-Fe(II) holorepressor. We studied the effect of AlgQ, the
Escherichia coli
Rsd orthologue, on pyoverdine production by
P. aeruginosa
PAO1. AlgQ is a global regulatory protein which activates alginate, ppGpp, and inorganic polyphosphate synthesis through a cascade involving nucleoside diphosphate kinase (Ndk). AlgQ is also capable of interacting with region 4 of RpoD. In a reconstituted
E. coli
system, PvdS-dependent transcription from the
pvdA
promoter was doubled by the multicopy
algQ
gene. The
P. aeruginosa
Δ
algQ
mutant exhibited a moderate but reproducible reduction in pyoverdine production compared with wild-type PAO1, as a result of a decline in transcription of
pvd
genes. PvdS expression was not affected by the
algQ
mutation. Single-copy
algQ
fully restored pyoverdine production and expression of
pvd
genes in the Δ
algQ
mutant, while
ndk
did not. An increased intracellular concentration of RpoD mimicked the Δ
algQ
phenotype, whereas PvdS overexpression suppressed the
algQ
mutation.
E. coli rsd
could partially substitute for
algQ
in transcriptional modulation of
pvd
genes. We propose that AlgQ acts as an anti-sigma factor for RpoD, eliciting core RNA polymerase recruitment by PvdS and transcription initiation at
pvd
promoters. AlgQ provides a link between the pyoverdine and alginate regulatory networks. These systems have similarities in responsiveness and physiological function: both depend on alternative sigma factors, respond to nutrient starvation, and act as virulence determinants for
P. aeruginosa
.
Transcriptional Analysis of the
Bacillus anthracis
Capsule Regulators
Authors: Drysdale Melissa , Bourgogne Agathe , Koehler Theresa M.
Published: July 2005

The poly-
d
-glutamic acid capsule of
Bacillus anthracis
is essential for virulence. Control of capsule synthesis occurs at the level of transcription and involves positive regulation of the capsule biosynthetic operon
capBCAD
by a CO
2
/bicarbonate signal and three plasmid-borne regulators:
atxA
,
acpA
, and
acpB
. Although the molecular mechanism for control of
cap
transcription is unknown,
atxA
affects
cap
expression via positive control of
acpA
and
acpB
, two genes with partial functional similarity. Transcriptional analyses of a genetically complete strain indicate that
capB
expression is several hundred-fold higher during growth in 5% CO
2
compared to growth in air.
atxA
was expressed appreciably during growth in air and induced only 2.5-fold by CO
2
. In contrast, expression of
acpA
and
acpB
was induced up to 23-fold and 59-fold, respectively, by CO
2
. The 5′-end mapping of gene transcripts revealed
atxA
-regulated and
atxA
-independent apparent transcription start sites for
capB
,
acpA
, and
acpB
. Transcripts mapping to all
atxA
-regulated start sites were increased during growth in elevated CO
2
. The
acpA
gene has one
atxA
-regulated and one
atxA
-independent start site.
acpB
lies downstream of
capBCAD
. A single
atxA
-independent start site maps immediately upstream of
acpB. atxA
-mediated control of
acpB
appears to occur via transcriptional read-through from
atxA
-dependent start sites 5′ of
capB
. One
atxA
-independent and two
atxA
-regulated start sites map upstream of
capB
. Transcription from the
atxA-
regulated start sites of
capBCAD
was reduced significantly in an
acpA acpB
double mutant but unaffected in mutants with deletion of only
acpA
or
acpB
, in agreement with the current model for epistatic relationships between the regulators.
Distribution, Expression, and Motif Variability of Ankyrin Domain Genes inWolbachia pipientis
Authors: Iturbe-Ormaetxe Iñaki , Burke Gaelen R. , Riegler Markus , O'Neill Scott L.
Published: July 2005

The endosymbiotic bacteriumWolbachia pipientisinfects a wide range of arthropods, in which it induces a variety of reproductive phenotypes, including cytoplasmic incompatibility (CI), parthenogenesis, male killing, and reversal of genetic sex determination. The recent sequencing and annotation of the firstWolbachiagenome revealed an unusually high number of genes encoding ankyrin domain (ANK) repeats. These ANK genes are likely to be important in mediating theWolbachia-host interaction. In this work we determined the distribution and expression of the different ANK genes found in the sequencedWolbachia wMel genome in nineWolbachiastrains that induce different phenotypic effects in their hosts. A comparison of the ANK genes ofwMel and the non-CI-inducingwAuWolbachiastrain revealed significant differences between the strains. This was reflected in sequence variability in shared genes that could result in alterations in the encoded proteins, such as motif deletions, amino acid insertions, and in some cases disruptions due to insertion of transposable elements and premature stops. In addition, onewMel ANK gene, which is part of an operon, was absent in thewAu genome. These variations are likely to affect the affinity, function, and cellular location of the predicted proteins encoded by these genes.
Bacillus subtilisPhosphorylated PhoP: Direct Activation of the EσA- and Repression of the EσE-ResponsivephoB-PS+VPromoters during Pho Response
Authors: Abdel-Fattah Wael R. , Chen Yinghua , Eldakak Amr , Hulett F. Marion
Published: July 2005

ThephoBgene ofBacillus subtilisencodes an alkaline phosphatase (PhoB, formerly alkaline phosphatase III) that is expressed from separate promoters during phosphate deprivation in a PhoP-PhoR-dependent manner and at stage two of sporulation under phosphate-sufficient conditions independent of PhoP-PhoR. Isogenic strains containing either the completephoBpromoter or individualphoBpromoter fusions were used to assess expression from each promoter under both induction conditions. ThephoBpromoter responsible for expression during sporulation,phoB-PS, was expressed in a wild-type strain during phosphate deprivation, but induction occurred >3 h later than induction of Pho regulon genes and the levels were approximately 50-fold lower than that observed for the PhoPR-dependent promoter,phoB-PV. EσEwas necessary and sufficient for PSexpression in vitro. PSexpression in aphoPRmutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of σEis delayed in aphoPRmutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoP∼P) repressed PSin vitro via direct binding to the promoter, the first example of an EσE-responsive promoter that is repressed by PhoP∼P. Whereas either PhoP or PhoP∼P in the presence of EσAwas sufficient to stimulate transcription from thephoB-PVpromoter in vitro, roughly 10- and 17-fold-higher concentrations of PhoP than of PhoP∼P were required for PVpromoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon,ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during Pi-replete growth and PhoP concentrations increased only threefold in vivo duringphoPRautoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely.
Transcriptional Analysis of a Gene Cluster Involved in Glucose Tolerance in
Zymomonas mobilis
: Evidence for an Osmoregulated Promoter
Authors: Christogianni Anastasia , Douka Eugenia , Koukkou Anna I. , Hatziloukas Efstathios , Drainas Constantin
Published: July 2005

Exponentially growing cells of
Zymomonas mobilis
normally exhibit a lag period of up to 3 h when they are transferred from a liquid medium containing 2% glucose to a liquid medium containing 10% glucose. A mutant of
Z. mobilis
(CU1) exhibited a lag period of more than 20 h when it was grown under the same conditions, whereas it failed to grow on a solid medium containing 10% glucose. The glucose-defective phenotype of mutant CU1 was due to a spontaneous insertion in a putative gene (ORF4) identified as part of an operon (
glc
) which includes three additional putative genes (ORF1, ORF2, and ORF3) with no obvious involvement in the glucose tolerance mechanism. The common promoter controlling
glc
operon transcription, designated P

glc

, was found to be osmoregulated and stimulated by the putative product of ORF4 in an autoregulated fashion, as indicated by expression of the
gfp
reporter gene. Additionally, reverse transcriptase PCR analysis showed that the gene cluster produces a single mRNA, which verified the operon organization of this transcription unit. Further transcriptional analysis demonstrated that
glc
operon expression is regulated by the concentration of glucose, which supported the hypothesis that this operon is directly involved in the uncharacterized glucose tolerance mechanism of
Z. mobilis
.
Functional Characterization of a Na
+
-Coupled Dicarboxylate Carrier Protein from
Staphylococcus aureus
Authors: Hall Jason A. , Pajor Ana M.
Published: July 2005

We have cloned and functionally characterized a Na
+
-coupled dicarboxylate transporter, SdcS, from
Staphylococcus aureus
. This carrier protein is a member of the divalent anion/Na
+
symporter (DASS) family and shares significant sequence homology with the mammalian Na
+
/dicarboxylate cotransporters NaDC-1 and NaDC-3. Analysis of SdcS function indicates transport properties consistent with those of its eukaryotic counterparts. Thus, SdcS facilitates the transport of the dicarboxylates fumarate, malate, and succinate across the cytoplasmic membrane in a Na
+
-dependent manner. Furthermore, kinetic work predicts an ordered reaction sequence with Na
+
(
K
0.5
of 2.7 mM) binding before dicarboxylate (

K
m

of 4.5 μM). Because this transporter and its mammalian homologs are functionally similar, we suggest that SdcS may serve as a useful model for DASS family structural analysis.
Resistance ofNeisseria meningitidisto the Toxic Effects of Heme Iron and Other Hydrophobic Agents Requires Expression ofght
Authors: Rasmussen Andrew W. , Alexander Heather L. , Perkins-Balding Donna , Shafer William M. , Stojiljkovic Igor
Published: July 2005

Several genetic systems that allow the use of iron-protoporphyrin IX (heme) have been described for the pathogenic bacteriumNeisseria meningitidis. However, many questions about the process of heme acquisition and utilization remain to be answered. To isolate and analyze unidentified genes that play a role in heme iron uptake and utilization, aHimar1transposon mutant library was screened inN. meningitidisserogroup A strain IR4162. One locus identified by transposon mutagenesis conferred protection against heme toxicity. A mutant with a deletion in a gene termedght(gene of hydrophobic agent tolerance) within this locus was susceptible to heme and other hydrophobic agents compared to the parental strain. Transcriptional analysis indicated thatghtis cotranscribed with an upstream open reading frame NMA2149. Uncharacterized orthologues ofghtwere identified in many other gram-negative bacteria. We present genetic evidence for the importance ofghtin resistance to hydrophobic agents and its potential role in interaction with other hydrophobic agent resistance mechanisms withinN. meningitidis.
Characterization of a Multigene-Encoded Sodium/Hydrogen Antiporter (Sha) from
Pseudomonas aeruginosa
: Its Involvement in Pathogenesis
Authors: Kosono Saori , Haga Koki , Tomizawa Rui , Kajiyama Yusuke , Hatano Kazuo , Takeda Shinobu , Wakai Yoshimi , Hino Motohiro , Kudo Toshiaki
Published: July 2005

Sha (also known as Mrp/Mnh/Pha) is a Na
+
/H
+
antiporter encoded by a cluster of six or seven genes that probably form a multisubunit transport complex. The Sha system is important for the homeostasis of H
+
, Na
+
, and other monovalent cations and plays a critical role in various functions, including alkaliphily, sporulation, and symbiosis. Here, we characterized the
sha
homologue genes from the opportunistic pathogen
Pseudomonas aeruginosa
, which exist as a cluster of six genes (PA1054 to PA1059). The gene cluster PA1054 to PA1059, but not the cluster with a deletion of PA1054, complemented a growth defect in the presence of 0.2 M NaCl and a defect in Na
+
/H
+
antiport activity of the
Escherichia coli
TO114 mutant lacking the three major Na
+
/H
+
antiporters, indicating that genes PA1054 to PA1059 are responsible for Na
+
/H
+
antiport activity. We disrupted PA1054 (a
shaA
homologue gene) and determined its effect on Na
+
tolerance during growth, Na
+
efflux, and pathogenicity in mice. Disruption of PA1054 resulted in severe Na
+
sensitivity during growth and decreased Na
+
efflux activity. In mice, the deletion mutant of PA1054 also exhibited an attenuated virulence in systemic, pulmonary, and urinary tract infections and also a decrease in colonization of the infected organs. From these results, we conclude that the genes PA1054 to PA1059 encode a Na
+
/H
+
antiporter that is largely responsible for Na
+
extrusion in
P. aeruginosa
and has a role in the infection of the pathogen. We propose to designate PA1054 to PA1059 as the
sha
(sodium hydrogen antiporter) genes,
shaABCDEFG
.
Identification of Genes Required for Recycling Reducing Power during Photosynthetic Growth
Authors: Tavano Christine L. , Podevels Angela M. , Donohue Timothy J.
Published: July 2005

Photosynthetic organisms have the unique ability to transform light energy into reducing power. We study the requirements for photosynthesis in the α-proteobacterium
Rhodobacter sphaeroides
. Global gene expression analysis found that ∼50 uncharacterized genes were regulated by changes in light intensity and O
2
tension, similar to the expression of genes known to be required for photosynthetic growth of this bacterium. These uncharacterized genes included RSP4157 to -4159, which appeared to be cotranscribed and map to plasmid P004. A mutant containing a polar insertion in RSP4157, CT01, was able to grow via photosynthesis under autotrophic conditions using H
2
as an electron donor and CO
2
as a carbon source. However, CT01 was unable to grow photoheterotrophically in a succinate-based medium unless compounds that could be used to recycle reducing power (the external electron acceptor dimethyl sulfoxide (DMSO) or CO
2
) were provided. This suggests that the insertion in RSP4157 caused a defect in recycling reducing power during photosynthetic growth when a fixed carbon source was present. CT01 had decreased levels of RNA for genes encoding putative glycolate degradation functions. We found that exogenous glycolate also rescued photoheterotrophic growth of CT01, leading us to propose that CO
2
produced from glycolate metabolism can be used by the Calvin cycle to recycle reducing power generated in the photosynthetic apparatus. The ability of glycolate, CO
2
, or DMSO to support photoheterotrophic growth of CT01 suggests that one or more products of RSP4157 to -4159 serve a previously unknown role in recycling reducing power under photosynthetic conditions.
Functional Analysis of the
glycero-manno
-Heptose 7-Phosphate Kinase Domain from the Bifunctional HldE Protein, Which Is Involved in ADP-
l
-
glycero
-
d
-
manno
-Heptose Biosynthesis
Authors: McArthur Fiona , Andersson C. Evalena , Loutet Slade , Mowbray Sherry L. , Valvano Miguel A.
Published: July 2005

The core oligosaccharide component of the lipopolysaccharide can be subdivided into inner and outer core regions. In
Escherichia coli
, the inner core consists of two 3-
deoxy
-
d
-
manno-
octulosonic acid and three
glycero
-
manno
-heptose residues. The HldE protein participates in the biosynthesis of ADP-
glycero
-
manno
-heptose precursors used in the assembly of the inner core. HldE comprises two functional domains: an N-terminal region with homology to the ribokinase superfamily (HldE1 domain) and a C-terminal region with homology to the cytidylyltransferase superfamily (HldE2 domain). We have employed the structure of the
E. coli
ribokinase as a template to model the HldE1 domain and predict critical amino acids required for enzyme activity. Mutation of these residues renders the protein inactive as determined in vivo by functional complementation analysis. However, these mutations did not affect the secondary or tertiary structure of purified HldE1, as judged by fluorescence spectroscopy and circular dichroism. Furthermore, in vivo coexpression of wild-type, chromosomally encoded HldE and mutant HldE1 proteins with amino acid substitutions in the predicted ATP binding site caused a dominant negative phenotype as revealed by increased bacterial sensitivity to novobiocin. Copurification experiments demonstrated that HldE and HldE1 form a complex in vivo. Gel filtration chromatography resulted in the detection of a dimer as the predominant form of the native HldE1 protein. Altogether, our data support the notions that the HldE functional unit is a dimer and that structural components present in each HldE1 monomer are required for enzymatic activity.
Rot Repression of Enterotoxin B Expression in
Staphylococcus aureus
Authors: Tseng Ching Wen , Stewart George C.
Published: July 2005

The accessory gene regulator (Agr) system is a quorum-sensing system of
Staphylococcus aureus
responsible for upregulation of certain exoprotein genes and downregulation of certain cell-wall associated proteins during the post-exponential phase of growth. The enterotoxin B (
seb
) determinant is upregulated by the Agr system. Agr-regulated
ci
s elements within the
seb
promoter region were examined by deletion analyses of the
seb
promoter by a hybrid promoter approach utilizing the staphylococcal
lac
operon promoter. To identify the regulatory pathway for enterotoxin B expression, the
seb
promoter fused to the chloramphenicol acetyltransferase reporter gene was introduced into mutants of
S. aureus
lacking
agr
or different members of the Sar family of transcriptional regulators. Agr control of
seb
promoter activity was found to be dependent upon the presence of a functional Rot protein, and Rot was shown to be able to bind to the
seb
promoter. Therefore, the Agr-mediated post-exponential-phase increase in
seb
transcription results from the Agr system's inactivation of Rot repressor activity.
Swarming Differentiation and Swimming Motility inBacillus subtilisAre Controlled byswrA, a Newly Identified Dicistronic Operon
Authors: Calvio Cinzia , Celandroni Francesco , Ghelardi Emilia , Amati Giuseppe , Salvetti Sara , Ceciliani Fabrizio , Galizzi Alessandro , Senesi Sonia
Published: July 2005

The number and disposition of flagella harbored by eubacteria are regulated by a specific trait successfully maintained over generations. The genes governing the number of flagella inBacillus subtilishave never been identified, although theifmlocus has long been recognized to influence the motility phenotype of this microorganism. The characterization of a spontaneousifmmutant ofB. subtilis, displaying diverse degrees of cell flagellation in both liquid and solid media, raised the question of how theifmlocus governs the number and assembly of functional flagella. The major finding of this investigation is the characterization of a newly identified dicistronic operon, namedswrA, that controls both swimming motility and swarming differentiation inB. subtilis. Functional analysis of theswrAoperon allowedswrAA(previously namedswrA[D. B. Kearns, F. Chu, R. Rudner, and R. Losick, Mol. Microbiol.52:357-369, 2004]) to be the first gene identified inB. subtilisthat controls the number of flagella in liquid environments and the assembly of flagella in response to cell contact with solid surfaces. Evidence is given that the second gene of the operon,swrAB, is essential for enabling the surface-adhering cells to undergo swarming differentiation. Preliminary data point to a molecular interaction between the two gene products.
Expression of Canonical SOS Genes Is Not under LexA Repression inBdellovibrio bacteriovorus
Authors: Campoy Susana , Salvador Noelia , Cortés Pilar , Erill Ivan , Barbé Jordi
Published: July 2005

The here-reported identification of the LexA-binding sequence ofBdellovibrio bacteriovorus, a bacterial predator belonging to the δ-Proteobacteria, has made possible a detailed study of its LexA regulatory network. Surprisingly, only thelexAgene and a multiple gene cassette includingdinPanddnaEhomologues are regulated by the LexA protein in this bacterium. In vivo expression analyses have confirmed that this gene cassette indeed forms a polycistronic unit that, like thelexAgene, is DNA damage inducible inB. bacteriovorus. Conversely, genes such asrecA,uvrA,ruvCAB, andssb, which constitute the canonical core of theProteobacteriaSOS system, are not repressed by the LexA protein in this organism, hinting at a persistent selective pressure to maintain both thelexAgene and its regulation on the reported multiple gene cassette. In turn, in vitro experiments show that theB. bacteriovorusLexA-binding sequence is not recognized by other δ-ProteobacteriaLexA proteins but binds to the cyanobacterial LexA repressor. This placesB. bacteriovorusLexA at the base of the δ-ProteobacteriaLexA family, revealing a high degree of conservation in the LexA regulatory sequence prior to the diversification and specialization seen in deeper groups of theProteobacteriaphylum.
NanA, a Neuraminidase fromStreptococcus pneumoniae, Shows High Levels of Sequence Diversity, at Least in Part through Recombination withStreptococcus oralis
Authors: King Samantha J. , Whatmore Adrian M. , Dowson Christopher G.
Published: July 2005

Streptococcus pneumoniae, an important human pathogen, contains at least two genes,nanAandnanB, that express sialidase activity. NanA is a virulence determinant of pneumococci which is important in animal models of colonization and middle ear infections. The gene encoding NanA was detected in all 106 pneumococcal strains screened that represented 59 restriction profiles. Sequencing confirmed a high level of diversity, up to 17.2% at the nucleotide level and 14.8% at the amino acid level. NanA diversity is due to a number of mechanisms including insertions, point mutations, and recombination generating mosaic genes. The level of nucleotide divergence for each recombinant block is greater than 30% and much higher than the 20% identified within mosaicpbpgenes, suggesting that a high selective pressure exists for these alterations. These data indicate that at least one of the four recombinant blocks identified originated from aStreptococcus oralisisolate, demonstrating for the first time that protein virulence determinants of pneumococci have, as identified previously for genes encoding penicillin binding proteins, evolved by recombination with oral streptococci. No amino acid alterations were identified within the aspartic boxes or predicted active site, suggesting that sequence variation may be important in evading the adaptive immune response. Furthermore, this suggests thatnanAis an important target of the immune system in the interaction between the pneumococcus and host.
The Holin Protein of Bacteriophage PRD1 Forms a Pore for Small-Molecule and Endolysin Translocation
Authors: Žiedaitė Gabija , Daugelavičius Rimantas , Bamford Jaana K. H. , Bamford Dennis H.
Published: July 2005

PRD1 is a bacteriophage with an icosahedral outer protein layer surrounding the viral membrane, which encloses the linear double-stranded DNA genome. PRD1 infects gram-negative cells harboring a conjugative IncP plasmid. Here we studied the lytic functions of PRD1. Using infected cells and plasmid-borne lysis genes, we demonstrated that a two-component lysis system (holin-endolysin) operates to release progeny phage particles from the host cell. Monitoring of ion fluxes and the ATP content of the infected cells allowed us to build a model of the sequence of lysis-related physiological changes. A decrease in the intracellular level of ATP is the earliest indicator of cell lysis, followed by the leakage of K
+
from the cytosol approximately 20 min prior to the decrease in culture turbidity. However, the K
+
efflux does not immediately lead to the depolarization of the cytoplasmic membrane or leakage of the intracellular ATP. These effects are observed only ∼5 to 10 min prior to cell lysis. Similar results were obtained using cells expressing the holin and endolysin genes from plasmids.
YycH Regulates the Activity of the Essential YycFG Two-Component System in
Bacillus subtilis
Authors: Szurmant Hendrik , Nelson Kristine , Kim Eun-Ja , Perego Marta , Hoch James A.
Published: July 2005

Of the numerous two-component signal transduction systems found in bacteria, only a very few have proven to be essential for cell viability. Among these is the YycF (response regulator)-YycG (histidine kinase) system, which is highly conserved in and specific to the low-G+C content gram-positive bacteria. Given the pathogenic nature of several members of this class of bacteria, the YycF-YycG system has been suggested as a prime antimicrobial target. In an attempt to identify genes involved in regulation of this two-component system, a transposon mutagenesis study was designed to identify suppressors of a temperature-sensitive YycF mutant in
Bacillus subtilis
. Suppressors could be identified, and the prime target was the
yycH
gene located adjacent to
yycG
and within the same operon. A
lacZ
reporter assay revealed that YycF-regulated gene expression was elevated in a
yycH
strain, whereas disruption of any of the three downstream genes within the operon,
yycI
,
yycJ
, and
yycK
, showed no such effect. The concentrations of both YycG and YycF, assayed immunologically, remained unchanged between the wild-type and the
yycH
strain as determined by immunoassay. Alkaline phosphatase fusion studies showed that YycH is located external to the cell membrane, suggesting that it acts in the regulation of the sensor domain of the YycG sensor histidine kinase. The
yycH
strain showed a characteristic cell wall defect consistent with the previously suggested notion that the YycF-YycG system is involved in regulating cell wall homeostasis and indicating that either up- or down-regulation of YycF activity affects this homeostatic mechanism.
Amino Acids in Positions 48, 52, and 73 Differentiate the Substrate Specificities of the Highly Homologous Chlorocatechol 1,2-Dioxygenases CbnA and TcbC
Authors: Liu Shenghao , Ogawa Naoto , Senda Toshiya , Hasebe Akira , Miyashita Kiyotaka
Published: July 2005

Chlorocatechol 1,2-dioxygenase (CCD) is the first-step enzyme of the chlorocatechol
ortho
-cleavage pathway, which plays a central role in the degradation of various chloroaromatic compounds. Two CCDs, CbnA from the 3-chlorobenzoate-degrader
Ralstonia eutropha
NH9 and TcbC from the 1,2,4-trichlorobenzene-degrader
Pseudomonas
sp. strain P51, are highly homologous, having only 12 different amino acid residues out of identical lengths of 251 amino acids. But CbnA and TcbC are different in substrate specificities against dichlorocatechols, favoring 3,5-dichlorocatechol (3,5-DC) and 3,4-dichlorocatechol (3,4-DC), respectively. A study of chimeric mutants constructed from the two CCDs indicated that the N-terminal parts of the enzymes were responsible for the difference in the substrate specificities. Site-directed mutagenesis studies further identified the amino acid in position 48 (Leu in CbnA and Val in TcbC) as critical in differentiating the substrate specificities of the enzymes, which agreed well with molecular modeling of the two enzymes. Mutagenesis studies also demonstrated that Ile-73 of CbnA and Ala-52 of TcbC were important for their high levels of activity towards 3,5-DC and 3,4-DC, respectively. The importance of Ile-73 for 3,5-DC specificity determination was also shown with other CCDs such as TfdC from
Burkholderia
sp. NK8 and TfdC from
Alcaligenes
sp. CSV90 (identical to TfdC from
R. eutropha
JMP134), which convert 3,5-DC preferentially. Together with amino acid sequence comparisons indicating high conservation of Leu-48 and Ile-73 among CCDs, these results suggested that TcbC of strain P51 had diverged from other CCDs to be adapted to conversion of 3,4-DC.
Functional Replacement of the Oligomerization Domain of H-NS by the Hha Protein of
Escherichia coli
Authors: Rodríguez Sonia , Nieto José María , Madrid Cristina , Juárez Antonio
Published: July 2005

Members of the H-NS family of proteins play a relevant role as modulators of gene expression in gram-negative bacteria. Interaction of these proteins with members of the Hha/YmoA family of proteins has been previously reported. It has been hypothesized that the latter proteins are functionally equivalent to the N-terminal domain of H-NS-like proteins. In this report we test this assumption by replacing the N-terminal domain of
Escherichia coli
H-NS by Hha. It has been possible to obtain a functional protein that can compensate for some of the
hns-
induced phenotypes. These results highlight the relevance of H-NS-Hha interactions to generate heterooligomeric complexes that modulate gene expression in gram-negative bacteria.