bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thermophilic, Reversible γ-Resorcylate Decarboxylase from
Rhizobium
sp. Strain MTP-10005: Purification, Molecular Characterization, and Expression
Authors: Yoshida Masahiro , Fukuhara Nobuhiro , Oikawa Tadao 
Published: October 2004

ABSTRACT

We found the occurrence of thermophilic reversible γ-resorcylate decarboxylase (γ-RDC) in the cell extract of a bacterium isolated from natural water,
Rhizobium
sp. strain MTP-10005, and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be about 151 kDa by gel filtration, and that of the subunit was 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; in other words, the enzyme was a homotetramer. The enzyme was induced specifically by the addition of γ-resorcylate to the medium. The enzyme required no coenzyme and did not act on 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate, 3,5-dihydroxybenzoate, 2-hydroxybenzoate, or 3-hydroxybenzoate. It was relatively thermostable to heat treatment, and its half-life at 50°C was estimated to be 122 min; furthermore, it catalyzed the reverse carboxylation of resorcinol. The values of
k
cat
/

K
m

(mΜ
−1
· s
−1
) for γ-resorcylate and resorcinol at 30°C and pH 7 were 13.4 and 0.098, respectively. The enzyme contains 327 amino acid residues, and sequence identities were found with those of hypothetical protein AGR C 4595p from
Agrobacterium tumefaciens
strain C58 (96% identity), 5-carboxyvanillate decarboxylase from
Sphingomonas paucimobilis
(32%), and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylases from
Bacillus cereus
ATCC 10987 (26%),
Rattus norvegicus
(26%), and
Homo sapiens
(25%). The genes (
graA
[1,230 bp],
graB
[888 bp], and
graC
[1,056 bp]) that are homologous to those in the resorcinol pathway also exist upstream and downstream of the γ-RDC gene. Judging from these results, the resorcinol pathway also exists in
Rhizobium
sp. strain MTP-10005, and γ-RDC probably catalyzes a reaction just before the hydroxylase in it does.
A pH-Sensitive Function and Phenotype: Evidence that EutH Facilitates Diffusion of Uncharged Ethanolamine in
Salmonella enterica
Authors: Penrod Joseph T. , Mace Christopher C. , Roth John R. 
Published: October 2004

ABSTRACT

The
eutH
gene is part of an operon that allows
Salmonella enterica
to use ethanolamine as a sole source of nitrogen, carbon, and energy. Although the sequence of EutH suggests a role in transport,
eutH
mutants use ethanolamine normally under standard conditions (pH 7.0). These mutants fail to use ethanolamine at a low pH. Evidence is presented that protonated ethanolamine (Eth
0
) does not enter cells, while uncharged ethanolamine (Eth
0
) diffuses freely across the membrane. The external concentration of Eth
0
varies with the pH (pK = 9.5). At pH 7.0, the standard ethanolamine concentration (41 mM) provides enough Eth
0
for an influx rate that can support growth with or without EutH. When a lowered pH and/or ethanolamine concentration reduced the Eth
0
concentration below 25 μM, EutH was needed to facilitate diffusion. EutH
+
cells grew normally at Eth
0
concentrations above 3 μM, close to the Km (9 μM) of the first degradative enzyme, ethanolamine ammonia lyase. It is suggested that EutH facilitates diffusion of Eth
0
. As predicted for a transporter, EutH contributed to the toxicity of ethanolamine seen under some conditions; furthermore, fusion of EutH to fluorescent Yfp protein provided evidence that EutH is a membrane protein.
Chemical Structure, Conjugation, and Cross-Reactivity ofBacillus pumilusSh18 Cell Wall Polysaccharide
Authors: Kubler-Kielb Joanna , Coxon Bruce , Schneerson Rachel 
Published: October 2004

ABSTRACTBacillus pumilusstrain Sh18 cell wall polysaccharide (CWP), cross-reactive with the capsular polysaccharide ofHaemophilus influenzaetype b, was purified and its chemical structure was elucidated using fast atom bombardment mass spectrometry, nuclear magnetic resonance techniques, and sugar-specific degradation procedures. Two major structures, 1,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate), with the latter partially substituted by 2-acetamido-2-deoxy-α-galactopyranose (13%) and 2-acetamido-2-deoxy-α-glucopyranose (6%) on positionO-2, were found. A minor component was established to be a polymer of →3-O-(2-acetamido-2-deoxy-β-glucopyranosyl)-1→4-ribitol-1-OPO3→. The ratios of the three components were 56, 34, and 10 mol%, respectively. The Sh18 CWP was covalently bound to carrier proteins, and the immunogenicity of the resulting conjugates was evaluated in mice. Two methods of conjugation were compared: (i) binding of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-activated hydroxyl groups of the CWP to adipic acid dihydrazide (ADH)-derivatized protein, and (ii) binding of the carbodiimide-activated terminal phosphate group of the CWP to ADH-derivatized protein. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with the homologous polysaccharide and with a number of other bacterial polysaccharides containing ribitol and glycerol phosphates, includingH. influenzaetypes a and b and strains ofStaphylococcus aureusandStaphylococcus epidermidis.
Three Parallel Quorum-Sensing Systems Regulate Gene Expression in
Vibrio harveyi
Authors: Henke Jennifer M. , Bassler Bonnie L. 
Published: October 2004

ABSTRACT

In a process called quorum sensing, bacteria communicate using extracellular signal molecules termed autoinducers. Two parallel quorum-sensing systems have been identified in the marine bacterium
Vibrio harveyi
. System 1 consists of the LuxM-dependent autoinducer HAI-1 and the HAI-1 sensor, LuxN. System 2 consists of the LuxS-dependent autoinducer AI-2 and the AI-2 detector, LuxPQ. The related bacterium,
Vibrio cholerae
, a human pathogen, possesses System 2 (LuxS, AI-2, and LuxPQ) but does not have obvious homologues of
V. harveyi
System 1. Rather, System 1 of
V. cholerae
is made up of the CqsA-dependent autoinducer CAI-1 and a sensor called CqsS. Using a
V. cholerae
CAI-1 reporter strain we show that many other marine bacteria, including
V. harveyi
, produce CAI-1 activity. Genetic analysis of
V. harveyi
reveals
cqsA
and
cqsS
, and phenotypic analysis of
V. harveyi cqsA
and
cqsS
mutants shows that these functions comprise a third
V. harveyi
quorum-sensing system that acts in parallel to Systems 1 and 2. Together these communication systems act as a three-way coincidence detector in the regulation of a variety of genes, including those responsible for bioluminescence, type III secretion, and metalloprotease production.
Crystal Structures ofEscherichia coliATP-Dependent Glucokinase and Its Complex with Glucose
Authors: Lunin Vladimir V. , Li Yunge , Schrag Joseph D. , Iannuzzi Pietro , Cygler Miroslaw , Matte Allan 
Published: October 2004

ABSTRACTIntracellular glucose inEscherichia colicells imported by phosphoenolpyruvate-dependent phosphotransferase system-independent uptake is phosphorylated by glucokinase by using ATP to yield glucose-6-phosphate. Glucokinases (EC 2.7.1.2) are functionally distinct from hexokinases (EC 2.7.1.1) with respect to their narrow specificity for glucose as a substrate. While structural information is available for ADP-dependent glucokinases fromArchaea, no structural information exists for the large sequence family of eubacterial ATP-dependent glucokinases. Here we report the first structure determination of a microbial ATP-dependent glucokinase, that fromE. coliO157:H7. The crystal structure ofE. coliglucokinase has been determined to a 2.3-Å resolution (apo form) and refined to finalRwork/Rfreefactors of 0.200/0.271 and to 2.2-Å resolution (glucose complex) with finalRwork/Rfreefactors of 0.193/0.265.E. coliGlK is a homodimer of 321 amino acid residues. Each monomer folds into two domains, a small α/β domain (residues 2 to 110 and 301 to 321) and a larger α+β domain (residues 111 to 300). The active site is situated in a deep cleft between the two domains.E. coliGlK is structurally similar toSaccharomyces cerevisiaehexokinase and human brain hexokinase I but is distinct from the ADP-dependent GlKs. Bound glucose forms hydrogen bonds with the residues Asn99, Asp100, Glu157, His160, and Glu187, all of which, except His160, are structurally conserved in human hexokinase 1. Glucose binding results in a closure of the small domains, with a maximal Cα shift of ∼10 Å. A catalytic mechanism is proposed that is consistent with Asp100 functioning as the general base, abstracting a proton from the O6 hydroxyl of glucose, followed by nucleophilic attack at the γ-phosphoryl group of ATP, yielding glucose-6-phosphate as the product.
Effect of Dimer Dissociation on Activity and Thermostability of the α-Glucuronidase fromGeobacillus stearothermophilus: Dissecting the Different Oligomeric Forms of Family 67 Glycoside Hydrolases
Authors: Shallom Dalia , Golan Gali , Shoham Gil , Shoham Yuval 
Published: October 2004

ABSTRACTThe oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. α-Glucuronidases are family 67 glycosidases that cleave the α-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of α-glucuronidases are available, those fromGeobacillus stearothermophilus(AguA) and fromCellvibrio japonicus(GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the α-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial α-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in α-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35°C, compared to 65°C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9°C, was almost identical to that of the wild-type, 73.4°C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region and reduce the activity. Structural and mechanistic explanations for these effects are discussed.
Genomic Studies with
Escherichia coli
MelR Protein: Applications of Chromatin Immunoprecipitation and Microarrays
Authors: Grainger David C. , Overton Timothy W. , Reppas Nikos , Wade Joseph T. , Tamai Eiji , Hobman Jon L. , Constantinidou Chrystala , Struhl Kevin , Church George , Busby Stephen J. W. 
Published: October 2004

ABSTRACT

Escherichia coli
MelR protein is a transcription activator that is essential for melibiose-dependent expression of the
melAB
genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the
melAB
promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the
melAB
promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the
melAB
promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the
melAB
promoter is the major, if not the sole, target in
E. coli
for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without
melR
.
Use of Fluorescent-Protein Tagging To Determine the Subcellular Localization of
Mycoplasma pneumoniae
Proteins Encoded by the Cytadherence Regulatory Locus
Authors: Kenri Tsuyoshi , Seto Shintaro , Horino Atsuko , Sasaki Yuko , Sasaki Tsuguo , Miyata Makoto 
Published: October 2004

ABSTRACT

Mycoplasma pneumoniae
lacks a cell wall but has internal cytoskeleton-like structures that are assumed to support the attachment organelle and asymmetric cell shape of this bacterium. To explore the fine details of the attachment organelle and the cytoskeleton-like structures, a fluorescent-protein tagging technique was applied to visualize the protein components of these structures. The focus was on the four proteins—P65, HMW2, P41, and P24—that are encoded in the
crl
operon (for “cytadherence regulatory locus”), which is known to be essential for the adherence of
M. pneumoniae
to host cells. When the P65 and HMW2 proteins were fused to enhanced yellow fluorescent protein (EYFP), a variant of green fluorescent protein, the fused proteins became localized at the attachment organelle, enabling visualization of the organelles of living cells by fluorescence microscopy. The leading end of gliding
M. pneumoniae
cells, expressing the EYFP-P65 fusion, was observed as a focus of fluorescence. On the other hand, when the P41 and P24 proteins were labeled with EYFP, the fluorescence signals of these proteins were observed at the proximal end of the attachment organelle. Coexpression of the P65 protein labeled with enhanced cyan fluorescent protein clearly showed that the sites of localization of P41 and P24 did not overlap that of P65. The localization of P41 and P24 suggested that they are also cytoskeletal proteins that function in the formation of unknown structures at the proximal end of the attachment organelle. The fluorescent-protein fusion technique may serve as a powerful tool for identifying components of cytoskeleton-like structures and the attachment organelle. It can also be used to analyze their assembly.
Complete Genome Sequence of the Genetically Tractable Hydrogenotrophic MethanogenMethanococcus maripaludis
Authors: Hendrickson E. L. , Kaul R. , Zhou Y. , Bovee D. , Chapman P. , Chung J. , Conway de Macario E. , Dodsworth J. A. , Gillett W. , Graham D. E. , Hackett M. , Haydock A. K. , Kang A. , Land M. L. , Levy R. , Lie T. J. , Major T. A. , Moore B. C. , Porat I. , Palmeiri A. , Rouse G. , Saenphimmachak C. , Söll D. , Van Dien S. , Wang T. , Whitman W. B. , Xia Q. , Zhang Y. , Larimer F. W. , Olson M. V. , Leigh J. A. 
Published: October 2004

ABSTRACTThe genome sequence of the genetically tractable, mesophilic, hydrogenotrophic methanogenMethanococcus maripaludiscontains 1,722 protein-coding genes in a single circular chromosome of 1,661,137 bp. Of the protein-coding genes (open reading frames [ORFs]), 44% were assigned a function, 48% were conserved but had unknown or uncertain functions, and 7.5% (129 ORFs) were unique toM. maripaludis. Of the unique ORFs, 27 were confirmed to encode proteins by the mass spectrometric identification of unique peptides. Genes for most known functions and pathways were identified. For example, a full complement of hydrogenases and methanogenesis enzymes was identified, including eight selenocysteine-containing proteins, with each being paralogous to a cysteine-containing counterpart. At least 59 proteins were predicted to contain iron-sulfur centers, including ferredoxins, polyferredoxins, and subunits of enzymes with various redox functions. Unusual features included the absence of a Cdc6 homolog, implying a variation in replication initiation, and the presence of a bacterial-like RNase HI as well as an RNase HII typical of theArchaea. The presence of alanine dehydrogenase and alanine racemase, which are uniquely present among theArchaea, explained the ability of the organism to usel- andd-alanine as nitrogen sources. Features that contrasted with the related organismMethanocaldococcus jannaschiiincluded the absence of inteins, even though close homologs of most intein-containing proteins were encoded. Although two-thirds of the ORFs had their highest Blastp hits inMethanocaldococcus jannaschii, lateral gene transfer or gene loss has apparently resulted in genes, which are often clustered, with top Blastp hits in more distantly related groups.
lpt6
, a Gene Required for Addition of Phosphoethanolamine to Inner-Core Lipopolysaccharide of
Neisseria meningitidis
and
Haemophilus influenzae
Authors: Wright J. Claire , Hood Derek W. , Randle Gaynor A. , Makepeace Katherine , Cox Andrew D. , Li Jianjun , Chalmers Ronald , Richards James C. , Moxon E. Richard 
Published: October 2004

ABSTRACT

We previously described a gene,
lpt3
, required for the addition of phosphoethanolamine (PEtn) at the 3 position on the β-chain heptose (HepII) of the inner-core
Neisseria meningitidis
lipopolysaccharide (LPS), but it has long been recognized that the inner-core LPS of some strains possesses PEtn at the 6 position (PEtn-6) on HepII. We have now identified a gene,
lpt6
(NMA0408), that is required for the addition of PEtn-6 on HepII. The
lpt6
gene is located in a region previously identified as Lgt-3 and is associated with other LPS biosynthetic genes. We screened 113 strains, representing all serogroups and including disease and carriage strains, for the
lpt3
and
lpt6
genes and showed that 36% contained both genes, while 50% possessed
lpt3
only and 12% possessed
lpt6
only. The translated amino acid sequence of
lpt6
has a homologue (72.5% similarity) in a product of the
Haemophilus influenzae
Rd genome sequence. Previous structural studies have shown that all
H. influenzae
strains investigated have PEtn-6 on HepII. Consistent with this, we found that, among 70 strains representing all capsular serotypes and nonencapsulated
H. influenzae
strains, the
lpt6
homologue was invariably present. Structural analysis of LPS from
H. influenzae
and
N. meningitidis
strains where
lpt6
had been insertionally inactivated revealed that PEtn-6 on HepII could not be detected. The translated amino acid sequences from the
N. meningitidis
and
H. influenzae lpt6
genes have conserved residues across their lengths and are part of a family of proven or putative PEtn transferases present in a wide range of gram-negative bacteria.
ParB ofPseudomonas aeruginosa: Interactions with Its Partner ParA and Its TargetparSand Specific Effects on Bacterial Growth
Authors: Bartosik Aneta A. , Lasocki Krzysztof , Mierzejewska Jolanta , Thomas Christopher M. , Jagura-Burdzy Grazyna 
Published: October 2004

ABSTRACTThepargenes ofPseudomonas aeruginosahave been studied to increase the understanding of their mechanism of action and role in the bacterial cell. Key properties of the ParB protein have been identified and are associated with different parts of the protein. The ParB- ParB interaction domain was mapped in vivo and in vitro to the C-terminal 56 amino acids (aa); 7 aa at the C terminus play an important role. The dimerization domain ofP. aeruginosaParB is interchangeable with the dimerization domain of KorB from plasmid RK2 (IncP1 group). The C-terminal part of ParB is also involved in ParB-ParA interactions. Purified ParB binds specifically to DNA containing a putativeparSsequence based on the consensus sequence found in the chromosomes ofBacillus subtilis, Pseudomonas putida, andStreptomyces coelicolor. The overproduction of ParB was shown to inhibit the function of genes placed nearparS. This “silencing” was dependent on theparSsequence and its orientation. The overproduction ofP. aeruginosaParB or its N-terminal part also causes inhibition of the growth ofP. aeruginosaandP. putidabut notEscherichia colicells. Since this inhibitory determinant is located well away from ParB segments required for dimerization or interaction with the ParA counterpart, this result may suggest a role for the N terminus ofP. aeruginosaParB in interactions with host cell components.
Thirty-Eight C-Terminal Amino Acids of the Coupling Protein TraD of the F-Like Conjugative Resistance Plasmid R1 Are Required and Sufficient To Confer Binding to the Substrate Selector Protein TraM
Authors: Beranek Andreas , Zettl Markus , Lorenzoni Klaus , Schauer Alexandra , Manhart Michael , Koraimann Günther 
Published: October 2004

ABSTRACT

Coupling proteins (CPs) are present in type IV secretion systems of plant, animal, and human pathogens and are essential for DNA transfer in bacterial conjugation systems. CPs connect the DNA-processing machinery to the mating pair-forming transfer apparatus. In this report we present in vitro and in vivo data that demonstrate specific binding of CP TraD of the IncFII R1 plasmid transfer system to relaxosomal protein TraM. With overlay assays and enzyme-linked immunosorbent assays we showed that a truncated version of TraD, termed TraD11 (ΔN155), interacted strongly with TraM. The apparent TraD11-TraM association constant was determined to be 2.6 × 10
7
liters/mol. Electrophoretic mobility shift assays showed that this variant of TraD also strongly bound to TraM when it was in complex with its target DNA. When 38 amino acids were additionally removed from the C terminus of TraD, no binding to TraM was observed. TraD15, comprising the 38 amino-acid-long C terminus of TraD, bound to TraM, indicating that the main TraM interaction domain resides in these 38 amino acids of TraD. TraD15 exerted a dominant negative effect on DNA transfer but not on phage infection by pilus-specific phage R17, indicating that TraM-TraD interaction is important for conjugative DNA transfer but not for phage infection. We also observed that TraD encoded by the closely related F factor bound to TraM encoded by the R1 plasmid. Our results thus provide evidence that substrate selection within the IncF plasmid group is based on TraM's capability to select the correct DNA molecule for transport and not on substrate selection by the CP.
Genome-Wide Transcriptional Analysis of the Cold Shock Response in Wild-Type and Cold-Sensitive, Quadruple-
csp
-Deletion Strains of
Escherichia coli
Authors: Phadtare Sangita , Inouye Masayori 
Published: October 2004

ABSTRACT

A DNA microarray-based global transcript profiling of
Escherichia coli
in response to cold shock showed that in addition to the known cold shock-inducible genes, new genes such as the flagellar operon, those encoding proteins involved in sugar transport and metabolism, and remarkably, genes encoding certain heat shock proteins are induced by cold shock. In the light of strong reduction in metabolic activity of the cell after temperature downshift, the induction of sugar metabolism machinery is unexpected. The deletion of four
csp
s (
cspA
,
cspB
,
cspG
, and
cspE
) affected cold shock induction of mostly those genes that are transiently induced in the acclimation phase, emphasizing that CspA homologues are essential in the acclimation phase. Relevance of these findings with respect to the known RNA chaperone function of CspA homologues is discussed.
Evidence for Polyadenylated mRNA in
Pseudomonas aeruginosa
Authors: Saravanamuthu Senthil Selvan , von Götz Franz , Salunkhe Prabhakar , Chozhavendan Rathinam , Geffers Robert , Buer Jan , Tümmler Burkhard , Steinmetz Ivo 
Published: October 2004

ABSTRACT

In this paper, we report the synthesis of
Pseudomonas aeruginosa
cDNA in the presence of oligo(dT) primers. Hybridization of oligonucleotide DNA microarrays indicates that under the experimental conditions used, at least 43.7% of the expressed genes from
P. aeruginosa
PAO1, representing many different functional classes, can be detected by using oligo(dT)-primed cDNAs.