bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Characterization of the
Sinorhizobium meliloti
Acetate Metabolism Genes
aceA
, SMc00767, and
glcB
Authors: Ramírez-Trujillo J. A. , Encarnación S. , Salazar E. , de los Santos A. García , Dunn M. F. , Emerich D. W. , Calva E. , Hernández-Lucas I. 
Published: May 2007

ABSTRACT

The genes encoding malate synthase (
glcB
) and isocitrate lyase (
aceA
) and a 240-bp open reading frame (SMc00767) located downstream of
aceA
were isolated and functionally characterized in
Sinorhizobium meliloti
. Independent and double interposon mutants of each gene were constructed, and the corresponding phenotypes were analyzed.
aceA
mutants failed to grow on acetate, and mutants deficient in SMc00767 were also affected in acetate utilization. In contrast, mutants deficient in
glcB
grew on acetate similar to wild-type strain Rm5000. Complementation experiments showed that
aceA
and SMc00767 gene constructs were able to restore the growth on acetate in the corresponding single mutants.
aceA
-
glcB
,
aceA
-SMc00767, and
glcB
-SMc00767 double knockouts were also unable to grow on acetate, but this ability was recovered when the wild-type
aceA
-
glcB
or
aceA
-SMc00767 loci were introduced into the double mutants. These data confirm the functional role of
aceA
and SMc00767 and show that
glcB
, in the absence of SMc00767, is required for acetate metabolism. Isocitrate lyase and malate synthase activities were measured in strain Rm5000, the mutant derivatives, and complemented strains.
aceA
and
glcB
were able to complement the enzymatic activity lacking in the corresponding single mutants. The enzymatic activities also showed that SMc00767 represses the activity of isocitrate lyase in cells grown on acetate. Gene fusions confirmed the repressor role of SMc00767, which regulates
aceA
expression at the transcriptional level. Comparison of the transcriptional profiles of the SMc00767 mutant and wild-type strain Rm5000 showed that SMc00767 represses the expression of a moderate number of open reading frames, including
aceA
; thus, we propose that SMc00767 is a novel repressor involved in acetate metabolism in
S. meliloti
. Genetic and functional analyses indicated that
aceA
and SMc00767 constitute a functional two-gene operon, which is conserved in other α-proteobacteria. Alfalfa plants infected with the
aceA
and
glcB
mutants were not impaired in nodulation or nitrogen fixation, and so the glyoxylate cycle is not required in the
Rhizobium
-legume symbiosis.
Differential Interaction ofAggregatibacter(Actinobacillus)actinomycetemcomitansLsrB and RbsB Proteins with Autoinducer 2
Authors: Shao Hanjuan , James Deanna , Lamont Richard J. , Demuth Donald R. 
Published: May 2007

ABSTRACTOur previous studies showed that theAggregatibacter actinomycetemcomitansRbsB protein interacts with cognate and heterologous autoinducer 2 (AI-2) signals and suggested that therbsDABCKoperon encodes a transporter that may internalize AI-2 (D. James et al., Infect. Immun. 74:4021-4029, 2006.). However,A. actinomycetemcomitansalso possesses genes related to thelsroperon ofSalmonella entericaserovar Typhimurium which function to import AI-2. Here, we show thatA. actinomycetemcomitansLsrB protein competitively inhibits the interaction of theVibrio harveyiAI-2 receptor (LuxP) with AI-2 from eitherA. actinomycetemcomitansorV. harveyi. Interestingly, LsrB was a more potent inhibitor of LuxP interaction with AI-2 fromV. harveyiwhereas RbsB competed more effectively with LuxP forA. actinomycetemcomitansAI-2. Inactivation oflsrBin wild-typeA. actinomycetemcomitansor in an isogenic RbsB-deficient strain reduced the rate by which intact bacteria depletedA. actinomycetemcomitansAI-2 from solution. Consistent with the results from the LuxP competition experiments, the LsrB-deficient strain depleted AI-2 to a lesser extent than the RbsB-deficient organism. Inactivation of bothlsrBandrbsBvirtually eliminated the ability of the organism to remove AI-2 from the extracellular environment. These results suggest thatA. actinomycetemcomitanspossesses two proteins that differentially interact with AI-2 and may function to inactivate or facilitate internalization of AI-2.
The Phage φ29 Membrane Protein p16.7, Involved in DNA Replication, Is Required for Efficient Ejection of the Viral Genome
Authors: Alcorlo Martín , González-Huici Víctor , Hermoso José M. , Meijer Wilfried J. J. , Salas Margarita 
Published: May 2007

ABSTRACT

It is becoming clear that in vivo phage DNA ejection is not a mere passive process. In most cases, both phage and host proteins seem to be involved in pulling at least part of the viral DNA inside the cell. The DNA ejection mechanism of
Bacillus subtilis
bacteriophage φ29 is a two-step process where the linear DNA penetrates the cell with a right-left polarity. In the first step ∼65% of the DNA is pushed into the cell. In the second step, the remaining DNA is actively pulled into the cytoplasm. This step requires protein p17, which is encoded by the right-side early operon that is ejected during the first push step. The membrane protein p16.7, also encoded by the right-side early operon, is known to play an important role in membrane-associated phage DNA replication. In this work we show that, in addition, p16.7 is required for efficient execution of the second pull step of DNA ejection.
Inactivation of the DnaB Helicase Leads to the Collapse and Degradation of the Replication Fork: a Comparison to UV-Induced Arrest
Authors: Belle Jerilyn J. , Casey Andrew , Courcelle Charmain T. , Courcelle Justin 
Published: May 2007

ABSTRACT

Replication forks face a variety of structurally diverse impediments that can prevent them from completing their task. The mechanism by which cells overcome these hurdles is likely to vary depending on the nature of the obstacle and the strand in which the impediment is encountered. Both UV-induced DNA damage and thermosensitive replication proteins have been used in model systems to inhibit DNA replication and characterize the mechanism by which it recovers. In this study, we examined the molecular events that occur at replication forks following inactivation of a thermosensitive DnaB helicase and found that they are distinct from those that occur following arrest at UV-induced DNA damage. Following UV-induced DNA damage, the integrity of replication forks is maintained and protected from extensive degradation by RecA, RecF, RecO, and RecR until replication can resume. By contrast, inactivation of DnaB results in extensive degradation of the nascent and leading-strand template DNA and a loss of replication fork integrity as monitored by two-dimensional agarose gel analysis. The degradation that occurs following DnaB inactivation partially depends on several genes, including
recF
,
recO
,
recR
,
recJ
,
recG
, and
xonA
. Furthermore, the thermosensitive DnaB allele prevents UV-induced DNA degradation from occurring following arrest even at the permissive temperature, suggesting a role for DnaB prior to loading of the RecFOR proteins. We discuss these observations in relation to potential models for both UV-induced and DnaB(Ts)-mediated replication inhibition.
Double-Strand Break Generation under Deoxyribonucleotide Starvation in
Escherichia coli
Authors: Guarino Estrella , Salguero Israel , Jiménez-Sánchez Alfonso , Guzmán Elena C. 
Published: May 2007

ABSTRACT
Stalled replication forks produced by three different ways of depleting deoxynucleoside triphosphate showed different capacities to undergo “replication fork reversal.” This reaction occurred at the stalled forks generated by hydroxyurea treatment, was impaired under thermal inactivation of ribonucleoside reductase, and did not take place under thymine starvation.
Identification ofPseudomonas syringaepv. syringae 61 Type III Secretion System Hrp Proteins That Can Travel the Type III Pathway and Contribute to the Translocation of Effector Proteins into Plant Cells
Authors: Ramos Adela R. , Morello Joanne E. , Ravindran Sandeep , Deng Wen-Ling , Huang Hsiou-Chen , Collmer Alan 
Published: May 2007

ABSTRACTPseudomonas syringaetranslocates effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). T3SS components HrpB, HrpD, HrpF, and HrpP were shown to be pathway substrates and to contribute to elicitation of the plant hypersensitive response and to translocation and secretion of the model effector AvrPto1.
FepA- and TonB-Dependent Bacteriophage H8: Receptor Binding and Genomic Sequence
Authors: Rabsch Wolfgang , Ma Li , Wiley Graham , Najar Fares Z. , Kaserer Wallace , Schuerch Daniel W. , Klebba Joseph E. , Roe Bruce A. , Gomez Jenny A. Laverde , Schallmey Marcus , Newton Salete M. C. , Klebba Phillip E. 
Published: May 2007

ABSTRACT

H8 is derived from a collection of
Salmonella enterica
serotype Enteritidis bacteriophage. Its morphology and genomic structure closely resemble those of bacteriophage T5 in the family
Siphoviridae
. H8 infected
S. enterica
serotypes Enteritidis and Typhimurium and
Escherichia coli
by initial adsorption to the outer membrane protein FepA. Ferric enterobactin inhibited H8 binding to
E. coli
FepA (50% inhibition concentration, 98 nM), and other ferric catecholate receptors (Fiu, Cir, and IroN) did not participate in phage adsorption. H8 infection was TonB dependent, but
exbB
mutations in
Salmonella
or
E. coli
did not prevent infection; only
exbB tolQ
or
exbB tolR
double mutants were resistant to H8. Experiments with deletion and substitution mutants showed that the receptor-phage interaction first involves residues distributed over the protein's outer surface and then narrows to the same charged (R316) or aromatic (Y260) residues that participate in the binding and transport of ferric enterobactin and colicins B and D. These data rationalize the multifunctionality of FepA: toxic ligands like bacteriocins and phage penetrate the outer membrane by parasitizing residues in FepA that are adapted to the transport of the natural ligand, ferric enterobactin. DNA sequence determinations revealed the complete H8 genome of 104.4 kb. A total of 120 of its 143 predicted open reading frames (ORFS) were homologous to ORFS in T5, at a level of 84% identity and 89% similarity. As in T5, the H8 structural genes clustered on the chromosome according to their function in the phage life cycle. The T5 genome contains a large section of DNA that can be deleted and that is absent in H8: compared to T5, H8 contains a 9,000-bp deletion in the early region of its chromosome, and nine potentially unique gene products. Sequence analyses of the tail proteins of phages in the same family showed that relative to pb5 (Oad) of T5 and Hrs of BF23, the FepA-binding protein (Rbp) of H8 contains unique acidic and aromatic residues. These side chains may promote binding to basic and aromatic residues in FepA that normally function in the adsorption of ferric enterobactin. Furthermore, a predicted H8 tail protein showed extensive identity and similarity to pb2 of T5, suggesting that it also functions in pore formation through the cell envelope. The variable region of this protein contains a potential TonB box, intimating that it participates in the TonB-dependent stage of the phage infection process.
An Anhydro-
N
-Acetylmuramyl-
l
-Alanine Amidase with Broad Specificity Tethered to the Outer Membrane of
Escherichia coli
Authors: Uehara Tsuyoshi , Park James T. 
Published: May 2007

ABSTRACT

From its amino acid sequence homology with AmpD, we recognized YbjR, now renamed AmiD, as a possible second 1,6-anhydro-
N
-acetylmuramic acid (anhMurNAc)-
l
-alanine amidase in
Escherichia coli
. We have now confirmed that AmiD is an anhMurNAc-
l
-Ala amidase and demonstrated that AmpD and AmiD are the only enzymes present in
E. coli
that are able to cleave the anhMurNAc-
l
-Ala bond. The activity was present only in the outer membrane fraction obtained from an
ampD
mutant. In contrast to AmpD, which is specific for the anhMurNAc-
l
-alanine bond, AmiD also cleaved the bond between MurNAc and
l
-alanine in both muropeptides and murein sacculi. Unlike the periplasmic murein amidases, AmiD did not participate in cell separation.
ampG
mutants, which are unable to import GlcNAc-anhMurNAc-peptides into the cytoplasm, released mainly peptides into the medium due to AmiD activity, whereas an
ampG amiD
double mutant released a large amount of intact GlcNAc-anhMurNAc-peptides into the medium.
Bifunctional CTP:Inositol-1-Phosphate Cytidylyltransferase/CDP-Inositol:Inositol-1-Phosphate Transferase, the Key Enzyme for Di-myo-Inositol-Phosphate Synthesis in Several (Hyper)thermophiles
Authors: Rodrigues Marta V. , Borges Nuno , Henriques Mafalda , Lamosa Pedro , Ventura Rita , Fernandes Chantal , Empadinhas Nuno , Maycock Christopher , da Costa Milton S. , Santos Helena 
Published: May 2007

ABSTRACTThe pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts ofArchaeoglobus fulgidusand structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol.188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression inEscherichia coliof the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3′-phosphate-1′-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus,Pyrococcus furiosus,Thermococcus kodakaraensis,Aquifex aeolicus, andRubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP andl-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol andl-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP andl-myo-inositol-1-P; the DIPP synthase domain used onlyl-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted inAeropyrum pernix,Thermotoga maritima, andHyperthermus butylicus. Additionally, usingl-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3′-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.
The LysR-Type Transcriptional Regulator YofA Controls Cell Division through the Regulation of Expression of
ftsW
in
Bacillus subtilis
Authors: Lu Zuolei , Takeuchi Michio , Sato Tsutomu 
Published: May 2007

ABSTRACT

We have carried out a functional analysis of LysR family transcriptional regulators in
Bacillus subtilis
. The cell density of cultures of a
yofA
insertion mutant declined sharply after the end of exponential growth, as measured by optical density at 600 nm. Complementation in
trans
and analysis of isopropyl-β-
d
-thiogalactopyranoside (IPTG)-dependent growth of an inducible
yofA
strain confirmed that YofA contributes to the cell density of a culture after the end of exponential growth. Microscopic observation suggested that cell division is inhibited or delayed in the
yofA
mutant during entry into stationary phase. Analysis of the transcription of cell division genes revealed that the expression of
ftsW
is inhibited in
yofA
mutants, and overexpression of
yofA
, driven by a multiple-copy plasmid, enhances the induction of
ftsW
expression. These results suggest that YofA is required for the final round of cell division before entry into stationary phase and that YofA positively regulates
ftsW
expression. The defects caused by mutation of
yofA
were suppressed in strains carrying P

spac

-
ftsW
in the presence of IPTG. Furthermore, maximal expression of
yofA
was observed at the onset of stationary phase, which coincided with the maximal
ftsW
expression. Our data indicate that YofA is involved in cell division through positive regulation of the expression of
ftsW
in
B. subtilis
.
Drug-Induced Conformational Changes in Multidrug Efflux Transporter AcrB from
Haemophilus influenzae
Authors: Dastidar Vishakha , Mao Weimin , Lomovskaya Olga , Zgurskaya Helen I. 
Published: May 2007

ABSTRACT

In gram-negative bacteria, transporters belonging to the resistance-nodulation-cell division (RND) superfamily of proteins are responsible for intrinsic multidrug resistance.
Haemophilus influenzae
, a gram-negative pathogen causing respiratory diseases in humans and animals, constitutively produces the multidrug efflux transporter AcrB (AcrB
HI
). Similar to other RND transporters AcrB
HI
associates with AcrA
HI
, the periplasmic membrane fusion protein, and the outer membrane channel TolC
HI
. Here, we report that AcrAB
HI
confers multidrug resistance when expressed in
Escherichia coli
and requires for its activity the
E. coli
TolC (TolC
EC
) protein. To investigate the intracellular dynamics of AcrAB
HI
, single cysteine mutations were constructed in AcrB
HI
in positions previously identified as important for substrate recognition. The accessibility of these strategically positioned cysteines to the hydrophilic thiol-reactive fluorophore fluorescein-5-maleimide (FM) was studied in vivo in the presence of various substrates of AcrAB
HI
and in the presence or absence of AcrA
HI
and TolC
EC
. We report that the reactivity of specific cysteines with FM is affected by the presence of some but not all substrates. Our results suggest that substrates induce conformational changes in AcrB
HI
.
IcsA Surface Presentation in
Shigella flexneri
Requires the Periplasmic Chaperones DegP, Skp, and SurA
Authors: Purdy Georgiana E. , Fisher Carolyn R. , Payne Shelley M. 
Published: May 2007

ABSTRACT

A
Shigella flexneri degP
mutant, which was defective for plaque formation in Henle cell monolayers, had a reduced amount of IcsA detectable on the bacterial surface with antibody. However, the mutant secreted IcsA to the outer membrane at wild-type levels. This suggests that IcsA adopts an altered conformation in the outer membrane of the
degP
mutant with reduced exposure on the cell surface. IcsA is, therefore, unlikely to be accessible to actin-nucleating proteins within the eukaryotic cell cytoplasm, which is required for bacterial movement within the host cell and cell-to-cell spread. The
degP
mutant was somewhat more sensitive to detergents, antibiotics, and the antimicrobial peptide magainin, indicating that the
degP
phenotype was not limited to IcsA surface presentation. The plaque defect of the
degP
mutant, which is independent of DegP protease activity, was suppressed by overexpression of the periplasmic chaperone Skp but not by SurA.
S. flexneri skp
and
surA
mutants failed to form plaques in Henle cell monolayers and were defective in cell surface presentation and polar localization of IcsA. Therefore, the three periplasmic folding factors DegP, Skp, and SurA were all required for IcsA localization and plaque formation by
S. flexneri
.
Gene Cloning and Characterization of the Very Large NAD-Dependentl-Glutamate Dehydrogenase from the PsychrophileJanthinobacterium lividum, Isolated from Cold Soil
Authors: Kawakami Ryushi , Sakuraba Haruhiko , Ohshima Toshihisa 
Published: May 2007

ABSTRACTNAD-dependentl-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophileJanthinobacterium lividumUTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed byStreptomyces clavuligerusandPseudomonas aeruginosa(about 180 kDa). The entire amino acid sequence of theJ. lividumenzyme showed about 40% identity with the sequences fromS. clavuligerusandP. aeruginosaenzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination ofl-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such asl-aspartate (1,735%) andl-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids.
Pyocyanin Alters Redox Homeostasis and Carbon Flux through Central Metabolic Pathways in
Pseudomonas aeruginosa
PA14
Authors: Price-Whelan Alexa , Dietrich Lars E. P. , Newman Dianne K. 
Published: May 2007

ABSTRACT

The opportunistic pathogen
Pseudomonas aeruginosa
produces colorful, redox-active antibiotics called phenazines. Excretion of pyocyanin, the best-studied natural phenazine, is responsible for the bluish tint of sputum and pus associated with
P. aeruginosa
infections in humans. Although the toxicity of pyocyanin for other bacteria, as well as its role in eukaryotic infection, has been studied extensively, the physiological relevance of pyocyanin metabolism for the producing organism is not well understood. Pyocyanin reduction by
P. aeruginosa
PA14 is readily observed in standing liquid cultures that have consumed all of the oxygen in the medium. We investigated the physiological consequences of pyocyanin reduction by assaying intracellular concentrations of NADH and NAD
+
in the wild-type strain and a mutant defective in phenazine production. We found that the mutant accumulated more NADH in stationary phase than the wild type. This increased accumulation correlated with a decrease in oxygen availability and was relieved by the addition of nitrate. Pyocyanin addition to a phenazine-null mutant also decreased intracellular NADH levels, suggesting that pyocyanin reduction facilitates redox balancing in the absence of other electron acceptors. Analysis of extracellular organic acids revealed that pyocyanin stimulated stationary-phase pyruvate excretion in
P. aeruginosa
PA14, indicating that pyocyanin may also influence the intracellular redox state by decreasing carbon flux through central metabolic pathways.