bluuebunny/crossref_metadata_2025 huggingface dataset:
Cocaine Differentially Modulates Chemokine Production by Mononuclear Cells from Normal Donors and Human Immunodeficiency Virus Type 1-Infected Patients
Authors: Nair Madhavan P. N. , Chadha Kailash C. , Hewitt Ross G. , Mahajan Supriya , Sweet Ann , Schwartz Stanley A.
Published: April 2009

Earlier studies have supported a significant role for cocaine in the susceptibility to and the progression of human immunodeficiency virus type 1 (HIV-1) infection. Recently, several unique HIV-1 entry coreceptors (e.g., CCR5 and CCR3) and a trio of HIV-1-specific suppressor chemokines, namely, RANTES (regulated-upon-activation T expressed and secreted), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β, were identified. Although cocaine has been linked to the immunopathogenesis of HIV-1 infection, the corresponding cellular and molecular mechanism(s) have not been well defined. We hypothesize that cocaine mediates these pathologic effects through the downregulation of HIV-1-suppressing chemokines and/or upregulating HIV-1 entry coreceptors in HIV-1-infected subjects, resulting in disease progression to AIDS. Our results show that cocaine selectively downregulates endogenous MIP-1β secretion by normal peripheral blood mononuclear cells (PBMC), while cocaine did not affect the MIP-1β production by PBMC from AIDS patients. Cocaine also selectively suppresses lipopolysaccharide-induced MIP-1β production by PBMC from HIV-infected patients. Further, cocaine significantly downregulates endogenous MIP-1β gene expression, while it upregulates HIV-1 entry coreceptor CCR5 by normal PBMC. These studies suggests a role for cocaine as a cofactor in the pathogenesis of HIV infection and support the premise that cocaine increases susceptibility to and progression of HIV-1 infection by inhibiting the synthesis of HIV-1 protective chemokines and/or upregulating the HIV-1 entry coreceptor, CCR5.
Using Morpholinos for Gene Knockdown in
Giardia intestinalis
Authors: Carpenter Meredith L. , Cande W. Zacheus
Published: April 2009

We used translation-blocking morpholinos to reduce protein levels in
Giardia intestinalis
. Twenty-four hours after electroporation with morpholinos targeting either green fluorescent protein or kinesin-2b, levels of these proteins were reduced by 60%. An epitope-tagged transgene can also be used as a reporter for morpholino efficacy with targets lacking specific antibodies.
Functional Domains of ExsA, the Transcriptional Activator of the
Pseudomonas aeruginosa
Type III Secretion System
Authors: Brutinel Evan D. , Vakulskas Christopher A. , Yahr Timothy L.
Published: April 2009

The opportunistic pathogen
Pseudomonas aeruginosa
utilizes a type III secretion system (T3SS) to evade phagocytosis and damage eukaryotic cells. Transcription of the T3SS regulon is controlled by ExsA, a member of the AraC/XylS family of transcriptional regulators. These family members generally consist of an ∼100-amino acid carboxy-terminal domain (CTD) with two helix-turn-helix DNA binding motifs and an ∼200-amino acid amino-terminal domain (NTD) with known functions including oligomerization and ligand binding. In the present study, we show that the CTD of ExsA binds to ExsA-dependent promoters in vitro and activates transcription from ExsA-dependent promoters both in vitro and in vivo. Despite possessing these activities, the CTD lacks the cooperative binding properties observed for full-length ExsA at the P

exsC

promoter. In addition, the CTD is unaffected by the negative regulatory activity of ExsD, an inhibitor of ExsA activity. Binding studies confirm that ExsD interacts directly with the NTD of ExsA. Our data are consistent with a model in which a single ExsA molecule first binds to a high-affinity site on the P

exsC

promoter. Protein-protein interactions mediated by the NTD then recruit an additional ExsA molecule to a second site on the promoter to form a complex capable of stimulating wild-type levels of transcription. These findings provide important insight into the mechanisms of transcriptional activation by ExsA and inhibition of ExsA activity by ExsD.
Functional Genomics Reveals Extended Roles of the
Mycobacterium tuberculosis
Stress Response Factor σ
H
Authors: Mehra Smriti , Kaushal Deepak
Published: April 2009

Mycobacterium tuberculosis
is one of the most successful pathogens of humankind. During infection,
M. tuberculosis
must cope with and survive against a variety of different environmental conditions. Sigma factors likely facilitate the modulation of the pathogen's gene expression in response to changes in its extracellular milieu during infection. σ
H
, an alternate sigma factor encoded by the
M. tuberculosis
genome, is induced by thiol-oxidative stress, heat shock, and phagocytosis. In response to these conditions, σ
H
induces the expression of σ
B
, σ
E
, and the thioredoxin regulon. In order to more effectively characterize the transcriptome controlled by σ
H
, we studied the long-term effects of the induction of σ
H
on global transcription in
M. tuberculosis
. The
M. tuberculosis
isogenic mutant of σ
H
(Δ-σ
H
) is more susceptible to diamide stress than wild-type
M. tuberculosis
. To study the long-term effects of σ
H
induction, we exposed both strains to diamide, rapidly washed it away, and resumed culturing in diamide-free medium (post-diamide stress culturing). Analysis of the effects of σ
H
induction in this experiment revealed a massive temporal programming of the
M. tuberculosis
transcriptome. Immediately after the induction of σ
H
, genes belonging to the functional categories “virulence/detoxification” and “regulatory proteins” were induced in large numbers. Fewer genes belonging to the “lipid metabolism” category were induced, while a larger number of genes belonging to this category were downregulated. σ
H
caused the induction of the ATP-dependent
clp
proteolysis regulon, likely mediated by a transcription factor encoded by Rv2745c, several members of the
mce1
virulence regulon, and the sulfate acquisition/transport network.
Phosphate-Enhanced Stationary-Phase Fitness of
Escherichia coli
Is Related to Inorganic Polyphosphate Level
Authors: Schurig-Briccio Lici A. , Farías Ricardo N. , Rintoul María R. , Rapisarda Viviana A.
Published: April 2009

We found that
Escherichia coli
grown in media with >37 mM phosphate maintained a high polyphosphate level in late stationary phase, which could account for changes in gene expression and enzyme activities that enhance stationary-phase fitness.
Transcriptome and Functional Analysis of the Eukaryotic-Type Serine/Threonine Kinase PknB inStaphylococcus aureus
Authors: Donat Stefanie , Streker Karin , Schirmeister Tanja , Rakette Sonja , Stehle Thilo , Liebeke Manuel , Lalk Michael , Ohlsen Knut
Published: April 2009

The function of theStaphylococcus aureuseukaryotic-like serine/threonine protein kinase PknB was investigated by performing transcriptome analysis using DNA microarray technology and biochemical assays. The transcriptional profile revealed a strong regulatory impact of PknB on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis. Functional activity of overexpressed and purified PknB kinase was demonstrated using the myelin basic protein as a surrogate substrate. Phosphorylation occurred in a time-dependent manner with Mn2+as a preferred cofactor. Furthermore, biochemical characterization revealed regulation of adenylosuccinate synthase (PurA) activity by phosphorylation. Phosphorylated PurA showed a 1.8-fold decrease in enzymatic activity compared to unphosphorylated PurA. Loss of PknB led to formation of larger cell clusters, and apknBdeletion strain showed 32-fold-higher sensitivity to the cell wall-active antibiotic tunicamycin. The results of this study strongly indicate that PknB has a role in regulation of purine biosynthesis, autolysis, and central metabolic processes inS. aureus.
Comparison of the Complete Genome Sequences of
Bifidobacterium animalis
subsp.
lactis
DSM 10140 and Bl-04
Authors: Barrangou Rodolphe , Briczinski Elizabeth P. , Traeger Lindsay L. , Loquasto Joseph R. , Richards Melissa , Horvath Philippe , Coûté-Monvoisin Anne-Claire , Leyer Gregory , Rendulic Snjezana , Steele James L. , Broadbent Jeffery R. , Oberg Taylor , Dudley Edward G. , Schuster Stephan , Romero Dennis A. , Roberts Robert F.
Published: April 2009

Bifidobacteria are important members of the human gut flora, especially in infants. Comparative genomic analysis of two
Bifidobacterium animalis
subsp.
lactis
strains revealed evolution by internal deletion of consecutive spacer-repeat units within a novel clustered regularly interspaced short palindromic repeat locus, which represented the largest differential content between the two genomes. Additionally, 47 single nucleotide polymorphisms were identified, consisting primarily of nonsynonymous mutations, indicating positive selection and/or recent divergence. A particular nonsynonymous mutation in a putative glucose transporter was linked to a negative phenotypic effect on the ability of the variant to catabolize glucose, consistent with a modification in the predicted protein transmembrane topology. Comparative genome sequence analysis of three
Bifidobacterium
species provided a core genome set of 1,117 orthologs complemented by a pan-genome of 2,445 genes. The genome sequences of the intestinal bacterium
B. animalis
subsp.
lactis
provide insights into rapid genome evolution and the genetic basis for adaptation to the human gut environment, notably with regard to catabolism of dietary carbohydrates, resistance to bile and acid, and interaction with the intestinal epithelium. The high degree of genome conservation observed between the two strains in terms of size, organization, and sequence is indicative of a genomically monomorphic subspecies and explains the inability to differentiate the strains by standard techniques such as pulsed-field gel electrophoresis.
Transcriptomic Analysis of
Rhizobium leguminosarum
Biovar viciae in Symbiosis with Host Plants
Pisum sativum
and
Vicia cracca
Authors: Karunakaran R. , Ramachandran V. K. , Seaman J. C. , East A. K. , Mouhsine B. , Mauchline T. H. , Prell J. , Skeffington A. , Poole P. S.
Published: April 2009

Rhizobium leguminosarum
bv. viciae forms nitrogen-fixing nodules on several legumes, including pea (
Pisum sativum
) and vetch (
Vicia cracca
), and has been widely used as a model to study nodule biochemistry. To understand the complex biochemical and developmental changes undergone by
R. leguminosarum
bv. viciae during bacteroid development, microarray experiments were first performed with cultured bacteria grown on a variety of carbon substrates (glucose, pyruvate, succinate, inositol, acetate, and acetoacetate) and then compared to bacteroids. Bacteroid metabolism is essentially that of dicarboxylate-grown cells (i.e., induction of dicarboxylate transport, gluconeogenesis and alanine synthesis, and repression of sugar utilization). The decarboxylating arm of the tricarboxylic acid cycle is highly induced, as is γ-aminobutyrate metabolism, particularly in bacteroids from early (7-day) nodules. To investigate bacteroid development, gene expression in bacteroids was analyzed at 7, 15, and 21 days postinoculation of peas. This revealed that bacterial rRNA isolated from pea, but not vetch, is extensively processed in mature bacteroids. In early development (7 days), there were large changes in the expression of regulators, exported and cell surface molecules, multidrug exporters, and heat and cold shock proteins.
fix
genes were induced early but continued to increase in mature bacteroids, while
nif
genes were induced strongly in older bacteroids. Mutation of 37 genes that were strongly upregulated in mature bacteroids revealed that none were essential for nitrogen fixation. However, screening of 3,072 mini-Tn
5
mutants on peas revealed previously uncharacterized genes essential for nitrogen fixation. These encoded a potential magnesium transporter, an AAA domain protein, and proteins involved in cytochrome synthesis.
ATP-Binding Site Lesions in FtsE Impair Cell Division
Authors: Arends S. J. Ryan , Kustusch Ryan J. , Weiss David S.
Published: April 2009

FtsE and FtsX ofEscherichia coliconstitute an apparent ABC transporter that localizes to the septal ring. In the absence of FtsEX, cells divide poorly and several membrane proteins essential for cell division are largely absent from the septal ring, including FtsK, FtsQ, FtsI, and FtsN. These observations, together with the fact thatftsEandftsXare cotranscribed withftsY, which helps to target some proteins for insertion into the cytoplasmic membrane, suggested that FtsEX might contribute to insertion of division proteins into the membrane. Here we show that this hypothesis is probably wrong, because cells depleted of FtsEX had normal amounts of FtsK, FtsQ, FtsI, and FtsN in the membrane fraction. We also show that FtsX localizes to septal rings in cells that lack FtsE, arguing that FtsX targets the FtsEX complex to the ring. Nevertheless, both proteins had to be present to recruit further Fts proteins to the ring. Mutant FtsE proteins with lesions in the ATP-binding site supported septal ring assembly (when produced together with FtsX), but these rings constricted poorly. This finding implies that FtsEX uses ATP to facilitate constriction rather than assembly of the septal ring. Finally, topology analysis revealed that FtsX has only four transmembrane segments, none of which contains a charged amino acid. This structure is not what one would expect of a substrate-specific transmembrane channel, leading us to suggest that FtsEX is not really a transporter even though it probably has to hydrolyze ATP to support cell division.
Microarray Identification of
Clostridium difficile
Core Components and Divergent Regions Associated with Host Origin
Authors: Janvilisri Tavan , Scaria Joy , Thompson Angela D. , Nicholson Ainsley , Limbago Brandi M. , Arroyo Luis G. , Songer J. Glenn , Gröhn Yrjö T. , Chang Yung-Fu
Published: April 2009

Clostridium difficile
is a gram-positive, spore-forming enteric anaerobe which can infect humans and a wide variety of animal species. Recently, the incidence and severity of human
C. difficile
infection has markedly increased. In this study, we evaluated the genomic content of 73
C. difficile
strains isolated from humans, horses, cattle, and pigs by comparative genomic hybridization with microarrays containing coding sequences from
C. difficile
strains 630 and QCD-32g58. The sequenced genome of
C. difficile
strain 630 was used as a reference to define a candidate core genome of
C. difficile
and to explore correlations between host origins and genetic diversity. Approximately 16% of the genes in strain 630 were highly conserved among all strains, representing the core complement of functional genes defining
C. difficile
. Absent or divergent genes in the tested strains were distributed across the entire
C. difficile
630 genome and across all the predicted functional categories. Interestingly, certain genes were conserved among strains from a specific host species, but divergent in isolates with other host origins. This information provides insight into the genomic changes which might contribute to host adaptation. Due to a high degree of divergence among
C. difficile
strains, a core gene list from this study offers the first step toward the construction of diagnostic arrays for
C. difficile
.
In Vivo Analysis of Cobinamide Salvaging inRhodobacter sphaeroidesStrain 2.4.1
Authors: Gray Michael J. , Escalante-Semerena Jorge C.
Published: April 2009

The genome ofRhodobacter sphaeroidesencodes the components of two distinct pathways for salvaging cobinamide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme B12). One pathway, conserved among bacteria, depends on a bifunctional kinase/guanylyltransferase (CobP) enzyme to convert adenosylcobinamide (AdoCbi) to AdoCbi-phosphate (AdoCbi-P), an intermediate in de novo AdoCbl biosynthesis. The other pathway, of archaeal origin, depends on an AdoCbi amidohydrolase (CbiZ) enzyme to generate adenosylcobyric acid (AdoCby), which is converted to AdoCbi-P by the AdoCbi-P synthetase (CobD) enzyme. Here we report thatR. sphaeroidesstrain 2.4.1 synthesizes AdoCbl de novo and that it salvages Cbi using both of the predicted Cbi salvaging pathways. AdoCbl produced byR. sphaeroideswas identified and quantified by high-performance liquid chromatography and bioassay. The deletion ofcobB(encoding an essential enzyme of the de novo corrin ring biosynthetic pathway) resulted in a strain ofR. sphaeroidesthat would not grow on acetate in the absence of exogenous corrinoids. The results from a nutritional analysis showed that the presence of either CbiZ or CobP was necessary and sufficient for Cbi salvaging, that CbiZ-dependent Cbi salvaging depended on the presence of CobD, and that CobP-dependent Cbi salvaging occurred in acbiZ+strain. Possible reasons whyR. sphaeroidesmaintains two distinct pathways for Cbi salvaging are discussed.
Occurrence, Plasticity, and Evolution of the
vpma
Gene Family, a Genetic System Devoted to High-Frequency Surface Variation in
Mycoplasma agalactiae
Authors: Nouvel Laurent-Xavier , Marenda Marc , Sirand-Pugnet Pascal , Sagné Eveline , Glew Michelle , Mangenot Sophie , Barbe Valérie , Barré Aurélien , Claverol Stéphane , Citti Christine
Published: April 2009

Mycoplasma agalactiae
, an important pathogen of small ruminants, exhibits a very versatile surface architecture by switching multiple, related lipoproteins (Vpmas) on and off. In the type strain, PG2, Vpma phase variation is generated by a cluster of six
vpma
genes that undergo frequent DNA rearrangements via site-specific recombination. To further comprehend the degree of diversity that can be generated at the
M. agalactiae
surface, the
vpma
gene repertoire of a field strain, 5632, was analyzed and shown to contain an extended repertoire of 23
vpma
genes distributed between two loci located 250 kbp apart. Loci I and II include 16 and 7
vpma
genes, respectively, with all
vpma
genes of locus II being duplicated at locus I. Several Vpmas displayed a chimeric structure suggestive of homologous recombination, and a global proteomic analysis further indicated that at least 13 of the 16 Vpmas can be expressed by the 5632 strain. Because a single promoter is present in each
vpma
locus, concomitant Vpma expression can occur in a strain with duplicated loci. Consequently, the number of possible surface combinations is much higher for strain 5632 than for the type strain. Finally, our data suggested that insertion sequences are likely to be involved in 5632
vpma
locus duplication at a remote chromosomal position. The role of such mobile genetic elements in chromosomal shuffling of genes encoding major surface components may have important evolutionary and epidemiological consequences for pathogens, such as mycoplasmas, that have a reduced genome and no cell wall.
A Role for the EAL-Like Protein STM1344 in Regulation of CsgD Expression and Motility inSalmonella entericaSerovar Typhimurium
Authors: Simm Roger , Remminghorst Uwe , Ahmad Irfan , Zakikhany Katherina , Römling Ute
Published: April 2009

The bacterial second messenger cyclic di-GMP (c-di-GMP) regulates the transition between sessility and motility. InSalmonella entericaserovar Typhimurium, the expression of CsgD, the regulator of multicellular rdar morphotype behavior, is a major target of c-di-GMP signaling. CsgD expression is positively regulated by at least two diguanylate cyclases, GGDEF domain proteins, and negatively regulated by at least four phosphodiesterases, EAL domain proteins. Here, we show that in contrast to EAL domain proteins acting as phosphodiesterases, the EAL-like protein STM1344 regulated CsgD expression positively and motility negatively. STM1344, however, did not have a role in c-di-GMP turnover and also did not bind the nucleotide. STM1344 acted upstream of the phosphodiesterases STM1703 and STM3611, previously identified to participate in CsgD downregulation, where it repressed their expression. Consequently, although STM1344 has not retained a direct role in c-di-GMP metabolism, it still participates in the regulation of c-di-GMP turnover and has a role in the transition between sessility and motility.
Endotoxin, Capsule, and Bacterial Attachment Contribute to
Neisseria meningitidis
Resistance to the Human Antimicrobial Peptide LL-37
Authors: Jones Allison , Geörg Miriam , Maudsdotter Lisa , Jonsson Ann-Beth
Published: April 2009

Pathogenic bacteria have evolved numerous mechanisms to evade the human immune system and have developed widespread resistance to traditional antibiotics. We studied the human pathogen
Neisseria meningitidis
and present evidence of novel mechanisms of resistance to the human antimicrobial peptide LL-37. We found that bacteria attached to host epithelial cells are resistant to 10 μM LL-37 whereas bacteria in solution or attached to plastic are killed, indicating that the cell microenvironment protects bacteria. The bacterial endotoxin lipooligosaccharide and the polysaccharide capsule contribute to LL-37 resistance, probably by preventing LL-37 from reaching the bacterial membrane, as more LL-37 reaches the bacterial membrane on both lipooligosaccharide-deficient and capsule-deficient mutants whereas both mutants are also more susceptible to LL-37 killing than the wild-type strain.
N. meningitidis
bacteria respond to sublethal doses of LL-37 and upregulate two of their capsule genes,
siaC
and
siaD
, which further results in upregulation of capsule biosynthesis.
SoxRS-Mediated Lipopolysaccharide Modification Enhances Resistance against Multiple Drugs in
Escherichia coli
Authors: Lee Joon-Hee , Lee Kang-Lok , Yeo Won-Sik , Park Su-Jin , Roe Jung-Hye
Published: April 2009

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of gram-negative bacteria that serves as a barrier against harmful molecules, including antibiotics. The
waaYZ
locus that encodes the LPS core biosynthetic function in
Escherichia coli
was found to be induced strongly by superoxide generators but not by H
2
O
2
, ethanol, or heat shock. This induction was dependent on SoxRS, a superoxide and nitric oxide sensing system, through a soxbox in the
waaY
promoter that binds SoxS. A Δ
waaYZ
mutant became more sensitive to some superoxide generators, and the activation of SoxR by these drugs became more sensitized in the mutant. Through phenotypic microarray analysis, we found that the mutant became sensitive to a wide variety of chemicals not restricted to oxidizing agents. We found that the mutant is under envelope stress and is altered in LPS composition, as monitored by the level of σ
E
activation and changes in the electrophoretic mobility of LPS, respectively.
waaY
expression was also regulated by MarA (multiple-antibiotic resistance regulator), which shares a binding site (soxbox) with SoxS, and was induced by salicylate, a nonoxidative compound. These results demonstrate a novel way of protecting gram-negative bacteria against various compounds by modifying LPS, possibly through phosphorylation. Since either oxidant or nonoxidant compounds elicit resistance toward themselves and other toxic drugs, this mechanism could serve as an efficient way for pathogenic bacteria to enhance survival during antibiotic treatment within an oxidant-rich host immune environment.
Role of the Extracytoplasmic Function Sigma Factor RpoE4 in Oxidative and Osmotic Stress Responses in
Rhizobium etli
Authors: Martínez-Salazar Jaime M. , Salazar Emmanuel , Encarnación Sergio , Ramírez-Romero Miguel A. , Rivera Javier
Published: April 2009

The aims of this study were to functionally characterize and analyze the transcriptional regulation and transcriptome of the
Rhizobium etli rpoE4
gene. An
R. etli rpoE4
mutant was sensitive to oxidative, saline, and osmotic stresses. Using transcriptional fusions, we determined that RpoE4 controls its own transcription and that it is negatively regulated by
rseF
(
r
egulator of
s
igma
rpo
E
4
; CH03274), which is cotranscribed with
rpoE4
.
rpoE4
expression was induced not only after oxidative, saline, and osmotic shocks, but also under microaerobic and stationary-phase growth conditions. The transcriptome analyses of an
rpoE4
mutant and an
rpoE4
-overexpressing strain revealed that the RpoE4 extracytoplasmic function sigma factor regulates about 98 genes; 50 of them have the
rpoE4
promoter motifs in the upstream regulatory regions. Interestingly, 16 of 38 genes upregulated in the
rpoE4
-overexpressing strain encode unknown putative cell envelope proteins. Other genes controlled by RpoE4 include
rpoH2
, CH00462, CH02434, CH03474, and
xthA1
, which encode proteins involved in the stress response (a heat shock sigma factor, a putative Mn-catalase, an alkylation DNA repair protein, pyridoxine phosphate oxidase, and exonuclease III, respectively), as well as several genes, such as CH01253, CH03555, and PF00247, encoding putative proteins involved in cell envelope biogenesis (a putative peptidoglycan binding protein, a cell wall degradation protein, and phospholipase D, respectively). These results suggest that
rpoE4
has a relevant function in cell envelope biogenesis and that it plays a role as a general regulator in the responses to several kinds of stress.
Differential Accumulation of Form I RubisCO in
Rhodopseudomonas palustris
CGA010 under Photoheterotrophic Growth Conditions with Reduced Carbon Sources
Authors: Joshi Gauri S. , Romagnoli Simona , VerBerkmoes Nathan C. , Hettich Robert L. , Pelletier Dale , Tabita F. Robert
Published: April 2009

Rhodopseudomonas palustris
is unique among characterized nonsulfur purple bacteria because of its capacity for anaerobic photoheterotrophic growth using aromatic acids. Like growth with other reduced electron donors, this growth typically requires the presence of bicarbonate/CO
2
or some other added electron acceptor in the growth medium. Proteomic studies indicated that there was specific accumulation of form I ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) subunit proteins (CbbL and CbbS), as well as the CbbX protein, in cells grown on benzoate without added bicarbonate; such cells used the small amounts of dissolved CO
2
in the medium to support growth. These proteins were not observed in extracts from cells grown in the presence of high levels (10 mM) of added bicarbonate. To confirm the results of the proteomics studies, it was shown that the total RubisCO activity levels were significantly higher (five- to sevenfold higher) in wild-type (CGA010) cells grown on benzoate with a low level (0.5 mM) of added bicarbonate. Immunoblots indicated that the increase in RubisCO activity levels was due to a specific increase in the amount of form I RubisCO (CbbLS) and not in the amount of form II RubisCO (CbbM), which was constitutively expressed. Deletion of the main transcriptional regulator gene,
cbbR
, resulted in impaired growth on benzoate-containing low-bicarbonate media, and it was established that form I RubisCO synthesis was absolutely and specifically dependent on CbbR. To understand the regulatory role of the CbbRRS two-component system, strains with nonpolar deletions of the
cbbRRS
genes were grown on benzoate. Distinct from the results obtained with photoautotrophic growth conditions, the results of studies with various CbbRRS mutant strains indicated that this two-component system did not affect the observed enhanced synthesis of form I RubisCO under benzoate growth conditions. These studies indicate that diverse growth conditions differentially affect the ability of the CbbRRS two-component system to influence
cbb
transcription.
Efficient Strand Transfer by the RadA Recombinase from the Hyperthermophilic Archaeon
Desulfurococcus amylolyticus
Authors: Kil Yuri V. , Baitin Dmitry M. , Masui Ryoji , Bonch-Osmolovskaya Elizaveta A. , Kuramitsu Seiki , Lanzov Vladislav A.
Published: April 2009

The
radA
gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon,
Desulfurococcus amylolyticus
, was cloned, sequenced, and overexpressed in
Escherichia coli
cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the
E. coli
RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65°C (catalytic rate constant of 1.2 to 2.5 min
−1
at 80 to 95°C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90°C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92°C).
Opa Expression Correlates with Elevated Transformation Rates in
Neisseria gonorrhoeae
Authors: Hill Stuart A.
Published: April 2009

Neisseria gonorrhoeae
is naturally competent for DNA transformation. Under most conditions encountered in vivo, gonococci express one or more opacity (Opa) proteins on their surfaces. Recently, it was shown that DNA preferentially binds to the surfaces of Opa-expressing organisms compared to those of isogenic Opa-negative strains, presumably due to the numerous cationic residues in the predicted surface-exposed loops of the Opa protein. This study examined whether Opa-DNA interactions actually influence DNA transformation of the gonococcus. The data show that Opa-expressing gonococci are more efficient recipients of DNA for transformation and are more susceptible to exogenous DNase I treatment at early stages during the DNA transformation process than non-Opa expressors. Furthermore, inhibition of the transformation process was demonstrable for Opa
+
populations when either nonspecific DNA or the polyanion heparin was used. Overall, the data suggest that Opa expression, with its presumptive positive surface charge contribution, promotes DNA transformation by causing a more prolonged sequestration of donor DNA at the cell surface, which translates into more efficient transformation over time.
Monoubiquitination Is Sufficient To Signal Internalization of the Maltose Transporter in
Saccharomyces cerevisiae
Authors: Lucero Pilar , Peñalver Élida , Vela Laura , Lagunas Rosario
Published: April 2009

Monoubiquitination of the 12-transmembrane segment (12-TMS)
Saccharomyces cerevisiae
maltose transporter promoted the maximal internalization rate of this protein. This modification is similar to that of the 7-TMS α-factor receptor but different from that of the 12-TMS uracil and general amino acid permeases. This result shows that binding of ubiquitin-Lys63 chains is not required for maximal internalization of all 12-TMS-containing proteins.
Elongation Factor Tu and DnaK Are Transferred from the Cytoplasm to the Periplasm of
Escherichia coli
during Osmotic Downshock Presumably via the Mechanosensitive Channel MscL
Authors: Berrier Catherine , Garrigues Alexia , Richarme Gilbert , Ghazi Alexandre
Published: April 2009

Upon osmotic downshock, a few cytoplasmic proteins, including thioredoxin, elongation factor Tu (EF-Tu), and DnaK, are released from Tris-EDTA-treated
Escherichia coli
cells by an unknown mechanism. We have shown previously that deletion of
mscL
, the gene coding for the mechanosensitive channel of the plasma membrane with the highest conductance, prevents the release of thioredoxin. We confirm and extend the implication of MscL in this process by showing that the release of EF-Tu and DnaK is severely impaired in MscL-deficient strains. Release of these proteins is not observed in the absence of a Tris-EDTA treatment which disrupts the outer membrane, indicating that, in intact cells, they are transferred to the periplasm upon shock, presumably through the MscL channel.
DNA-Binding Properties of the
Fremyella diplosiphon
RpbA Repressor
Authors: Manna Pradip , Nieder Roxanne P. , Schaefer Michael R.
Published: April 2009

Mutant strain FdBM1 of the cyanobacterium
Fremyella diplosiphon
is characterized by elevated transcription of the
cpcB1A1
gene set due to inactivation of
rpbA
by Tn
5469
. The predicted RpbA protein contains two regions resembling the characterized helix-turn-helix (HTH) motif involved in DNA recognition by many phage and bacterial transcription regulator proteins. It was therefore hypothesized that RpbA functions as a DNA-binding repressor involved in the control of transcription from
cpcB1A1
. A histidine-tagged form of RpbA, designated RpbA-His
6
, was examined for its ability to bind to the defined promoter region for
cpcB1A1
. Gel mobility shift assays showed that RpbA-His
6
specifically binds to a DNA fragment containing the
cpcB1A1
promoter and that significant binding can be achieved with equimolar amounts of RpbA-His
6
and the
cpcB1A1
promoter probe. DNase I footprint analysis localized the RpbA-His
6
binding site to an asymmetric 21-bp region that overlaps the putative −10 promoter sequence. A mutational analysis suggested that binding by RpbA-His
6
to its cognate DNA may involve both putative HTH motif-like regions. We conclude that RpbA functions as a transcriptional repressor for
cpcB1A1
and suggest that binding by RpbA to its cognate DNA may represent an atypical protein-DNA interaction.
Pal Lipoprotein ofEscherichia coliPlays a Major Role in Outer Membrane Integrity
Authors: Cascales Eric , Bernadac Alain , Gavioli Marthe , Lazzaroni Jean-Claude , Lloubes Roland
Published: April 2009

The Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared inEscherichia coli. lppandtol-palmutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of anlppstrain but that overproduced Lpp has no effect in apalstrain. Together with the previously reported observation that overproduced TolA complements anlppbut not apalstrain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.
Role of GlnK in NifL-Mediated Regulation of NifA Activity inAzotobacter vinelandii
Authors: Rudnick Paul , Kunz Christopher , Gunatilaka Malkanthi K. , Hines Eric R. , Kennedy Christina
Published: April 2009

In several diazotrophic species ofProteobacteria, PIIsignal transduction proteins have been implicated in the regulation of nitrogen fixation in response to NH4+by several mechanisms. InAzotobacter vinelandii, expression ofnifA, encoding thenif-specific activator, is constitutive, and thus, regulation of NifA activity by the flavoprotein NifL appears to be the primary level of nitrogen control. In vitro and genetic evidence suggests that the nitrogen response involves the PII-like GlnK protein and GlnD (uridylyltransferase/uridylyl-removing enzyme), which reversibly uridylylates GlnK in response to nitrogen limitation. Here, the roles of GlnK and GlnK-UMP inA. vinelandiiwere studied to determine whether the Nif−phenotype ofglnDstrains was due to an inability to modify GlnK, an effort previously hampered becauseglnKis an essential gene in this organism. AglnKY51Fmutation, encoding an unuridylylatable form of the protein, was stable only in a strain in which glutamine synthetase activity is not inhibited by NH4+, suggesting that GlnK-UMP is required to signal adenylyltransferase/adenylyl-removing enzyme-mediated deadenylylation.glnKY51Fstrains were significantly impaired for diazotrophic growth and expression of anifH-lacZfusion. NifL interacted with GlnK and GlnKY51F in a yeast two-hybrid system. Together, these data are consistent with those obtained from in vitro experiments (Little et al., EMBO J., 19:6041–6050, 2000) and support a model for regulation of NifA activity in which unmodified GlnK stimulates NifL inhibition and uridylylation of GlnK in response to nitrogen limitation prevents this function. This model is distinct from one proposed for the related bacteriumKlebsiella pneumoniae, in which unmodified GlnK relieves NifL inhibition instead of stimulating it.
Shigella
Spa32 Is an Essential Secretory Protein for Functional Type III Secretion Machinery and Uniformity of Its Needle Length
Authors: Tamano Koichi , Katayama Eisaku , Toyotome Takahito , Sasakawa Chihiro
Published: April 2009

The
Shigella
type III secretion machinery is responsible for delivering to host cells the set of effectors required for invasion. The type III secretion complex comprises a needle composed of MxiH and MxiI and a basal body made up of MxiD, MxiG, and MxiJ. In
S. flexneri
, the needle length has a narrow range, with a mean of approximately 45 nm, suggesting that it is strictly regulated. Here we show that Spa32, encoded by one of the
spa
genes, is an essential protein translocated via the type III secretion system and is involved in the control of needle length as well as type III secretion activity. When the
spa32
gene was mutated, the type III secretion complexes possessed needles of various lengths, ranging from 40 to 1,150 nm. Upon introduction of a cloned
spa32
into the
spa32
mutant, the bacteria produced needles of wild-type length. The
spa32
mutant overexpressing MxiH produced extremely long (>5 μm) needles. Spa32 was secreted into the medium via the type III secretion system, but secretion did not depend on activation of the system. The
spa32
mutant and the mutant overexpressing MxiH did not secrete effectors such as Ipa proteins into the medium or invade HeLa cells. Upon introduction of
Salmonella invJ
, encoding InvJ, which has 15.4% amino acid identity with Spa32, into the
spa32
mutant, the bacteria produced type III needles of wild-type length and efficiently entered HeLa cells. These findings suggest that Spa32 is an essential secreted protein for a functional type III secretion system in
Shigella
spp. and is involved in the control of needle length. Furthermore, its function is interchangeable with that of
Salmonella
InvJ.
λ Integrase Complementation at the Level of DNA Binding and Complex Formation
Authors: Nunes-Düby Simone E. , Radman-Livaja Marta , Kuimelis Robert G. , Pearline Rachel V. , McLaughlin Larry W. , Landy Arthur
Published: April 2009

Site-specific recombinases of the λ Int family carry out two single-strand exchanges by binding as head-to-head dimers on inverted core-type DNA sites. Each protomer may cleave its own site as a monomer incis(as for Cre recombinase), or it may recruit the tyrosine from its partner intransto form a composite active site (as for Flp recombinase). The crystal structure of the λ Int catalytic domain is compatible with both cleavage mechanisms, but two previous biochemical studies on λ integrase (Int) generated data that were not in agreement. Support forcisandtranscleavage came from assays with bispecific DNA substrates for λ and HK022 Ints and from functional complementation between recombination-deficient mutants, respectively. The data presented here do not provide new evidence forciscleavage, but they strongly suggest that the previously described complementation results cannot be used in support of atrans-cleavage mechanism. We show here that IntR212Q retains some residual catalytic function but is impaired in binding to core-type DNA on linear substrates and in forming higher-orderattL intasome structures. The binding-proficient mutant IntY342F can stabilize IntR212Q binding to core-type DNA through protein-protein interactions. Similarly, the formation of higher-order Int complexes with arm- and core-type DNA is boosted with both mutants present. This complementation precedes cleavage and thus precludes any conclusions about the mechanism of catalysis. Cross-core stimulation of wild-type HK022-Int cleavage on its cognate site (incis) by mutant λ Ints on bispecific core DNA suicide substrates is shown to be independent of the catalytic tyrosine but appears to be proportional to the respective core-binding affinities of the λ Int mutants.
Disruption oflolCDE, Encoding an ATP-Binding Cassette Transporter, Is Lethal forEscherichia coliand Prevents Release of Lipoproteins from the Inner Membrane
Authors: Narita Shin-ichiro , Tanaka Kimie , Matsuyama Shin-ichi , Tokuda Hajime
Published: April 2009

ATP-binding cassette transporter LolCDE was previously identified, by using reconstituted proteoliposomes, as an apparatus catalyzing the release of outer membrane-specific lipoproteins from the inner membrane ofEscherichia coli. Mutations resulting in defective LolD were previously shown to be lethal forE. coli. The amino acid sequences of LolC and LolE are similar to each other, but the necessity of both proteins for lipoprotein release has not been proved. Moreover, previous reconstitution experiments did not clarify whether or not LolCDE is the sole apparatus for lipoprotein release. To address these issues, a chromosomallolC-lolD-lolEnull mutant harboring a helper plasmid that carries thelolCDEgenes and a temperature-sensitive replicon was constructed. The mutant failed to grow at a nonpermissive temperature because of the depletion of LolCDE. In addition to functional LolD, both LolC and LolE were required for growth. At a nonpermissive temperature, the outer membrane lipoproteins were mislocalized in the inner membrane since LolCDE depletion inhibited the release of lipoproteins from the inner membrane. Furthermore, both LolC and LolE were essential for the release of lipoproteins. On the other hand, LolCDE depletion did not affect the translocation of a lipoprotein precursor across the inner membrane and subsequent processing to the mature lipoprotein. From these results, we conclude that the LolCDE complex is an essential ABC transporter forE. coliand the sole apparatus mediating the release of outer membrane lipoproteins from the inner membrane.
Mutations in the Thiol-Disulfide Oxidoreductases BdbC and BdbD Can Suppress CytochromecDeficiency of CcdA-DefectiveBacillus subtilisCells
Authors: Erlendsson Lýđur S. , Hederstedt Lars
Published: April 2009

Cytochromes of thectype in the gram-positive bacteriumBacillus subtilisare all membrane anchored, with their heme domains exposed on the outer side of the cytoplasmic membrane. They are distinguished from other cytochromes by having heme covalently attached by two thioether bonds. The cysteinyls in the heme-binding site (CXXCH) in apocytochromecmust be reduced in order for the covalent attachment of the heme to occur. It has been proposed that CcdA, a membrane protein, transfers reducing equivalents from thioredoxin in the cytoplasm to proteins on the outer side of the cytoplasmic membrane. Strains deficient in the CcdA protein are defective in cytochromecand spore synthesis. We have discovered that mutations in thebdbCandbdbDgenes can suppress the defects caused by lack of CcdA. BdbC and BdbD are thiol-disulfide oxidoreductases. Our experimental findings indicate that theseB. subtilisproteins functionally correspond to the well-characterizedEscherichia coliDsbB and DsbA proteins, which catalyze the formation of disulfide bonds in proteins in the periplasmic space.
The Flavoenzyme Ferredoxin (Flavodoxin)-NADP(H) Reductase Modulates NADP(H) Homeostasis during the
soxRS
Response of
Escherichia coli
Authors: Krapp Adriana R. , Rodriguez Ramiro E. , Poli Hugo O. , Paladini Darío H. , Palatnik Javier F. , Carrillo Néstor
Published: April 2009

Escherichia coli
cells from strain
fpr
, deficient in the
soxRS
-induced ferredoxin (flavodoxin)-NADP(H) reductase (FPR), display abnormal sensitivity to the bactericidal effects of the superoxide-generating reagent methyl viologen (MV). Neither bacteriostatic effects nor inactivation of oxidant-sensitive hydrolyases could be detected in
fpr
cells exposed to MV. FPR inactivation did not affect the MV-driven
soxRS
response, whereas FPR overexpression led to enhanced stimulation of the regulon, with concomitant oxidation of the NADPH pool. Accumulation of a site-directed FPR mutant that uses NAD(H) instead of NADP(H) had no effect on
soxRS
induction and failed to protect
fpr
cells from MV toxicity, suggesting that FPR contributes to NADP(H) homeostasis in stressed bacteria.
Sensitive Screening Tests for Suspected Class A Carbapenemase Production in Species of
Enterobacteriaceae
Authors: Pasteran Fernando , Mendez Tania , Guerriero Leonor , Rapoport Melina , Corso Alejandra
Published: April 2009

The detection of class A serine-carbapenemases among species of
Enterobacteriaceae
remains a challenging issue. Methods of identification for routine use in clinical microbiology laboratories have not been standardized to date. We developed a novel screening methodology suitable for countries with high basal levels of carbapenem resistance due to non-carbapenemase-mediated mechanisms and standardized several simple confirmatory methods that allow the recognition of bacteria producing class A carbapenemases, including KPC, Sme, IMI, NMC-A, and GES, by using boronic acid (BA) derivatives. A total of 28 genetically unrelated
Enterobacteriaceae
strains producing several class A carbapenemases were tested. Thirty-eight genetically unrelated negative controls were included. The isolates were tested against imipenem (IPM), meropenem (MEM), and ertapenem (ETP) by MIC and disk diffusion assays in order to select appropriate tools to screen for suspected carbapenemase production. It was possible to differentiate class A carbapenemase-producing bacteria from non-carbapenemase-producing bacteria by using solely the routine IPM susceptibility tests. The modified Hodge test was evaluated and found to be highly sensitive, although false-positive results were documented. Novel BA-based methods (a double-disk synergy test and combined-disk and MIC tests) using IPM, MEM, and ETP, in combination with 3-aminophenylboronic acid as an inhibitor, were designed as confirmatory tools. On the basis of the performance of these methods, a sensitive flow chart for suspicion and confirmation of class A carbapenemase production in species of
Enterobacteriaceae
was designed. By using this methodology, isolates producing KPC, GES, Sme, IMI, and NMC-A carbapenemases were successfully distinguished from those producing other classes of β-lactamases (extended-spectrum β-lactamases, AmpCs, and metallo-β-lactamases, etc). These methods will rapidly provide useful information needed for targeting antimicrobial therapy and appropriate infection control.
Enrichment Broth Improved Detection of Extended-Spectrum-Beta-Lactamase-Producing Bacteria in Throat and Rectal Surveillance Cultures of Samples from Patients in Intensive Care Units
Authors: Murk Jean-Luc A. N. , Heddema Edou R. , Hess Dorine L. J. , Bogaards Johannes A. , Vandenbroucke-Grauls Christina M. J. E. , Debets-Ossenkopp Yvette J.
Published: April 2009

We evaluated the use of a trypticase soy broth (TSB) for improving detection of extended-spectrum-beta-lactamase-producing (ESBL
+
) bacteria. Preenrichment of throat and rectal swabs in TSB prior to inoculation on solid medium doubled the number of ESBL
+
bacteria detected in samples obtained from patients in our intensive care unit.