bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metal Ion Dependence of RecombinantEscherichia coliAllantoinase
Authors: Mulrooney Scott B. , Hausinger Robert P. 
Published: December 2002

ABSTRACTAllantoinase is a suspected dinuclear metalloenzyme that catalyzes the hydrolytic cleavage of the five-member ring of allantoin (5-ureidohydantoin) to form allantoic acid. RecombinantEscherichia coliallantoinase purified from overproducing cultures amended with 2.5 mM zinc, 1 mM cobalt, or 1 mM nickel ions was found to possess ∼1.4 Zn, 0.0 Co, 0.0 Ni, and 0.4 Fe; 0.1 Zn, 1.0 Co, 0.0 Ni, and 0.2 Fe; and 0.0 Zn, 0.0 Co, 0.6 Ni, and 0.1 Fe per subunit, respectively, whereas protein obtained from nonamended cultures contains near stoichiometric levels of iron. We conclude that allantoinase is incompletely activated in the recombinant cells, perhaps due to an insufficiency of a needed accessory protein. Enzyme isolated from nonsupplemented cultures possesses very low activity (kcat= 34.7 min−1) compared to the zinc-, cobalt-, and nickel-containing forms of allantoinase (kcatvalues of 5,000 and 28,200 min−1and 200 min−1, respectively). These rates and correspondingKmvalues (17.0, 19.5, and 80 mM, respectively) are significantly greater than those that have been reported previously. Absorbance spectroscopy of the cobalt species reveals a band centered at 570 nm consistent with five-coordinate geometry. Dithiothreitol is a competitive inhibitor of the enzyme, with significantKidifferences for the zinc and cobalt species (237 and 795 μM, respectively). Circular dichroism spectroscopy revealed that the zinc enzyme utilizes only theSisomer of allantoin, whereas the cobalt allantoinase prefers theSisomer, but also hydrolyzes theRisomer at about 1/10 the rate. This is the first report for metal content of allantoinase from any source.
Cadaverine Inhibition of Porin Plays a Role in Cell Survival at Acidic pH
Authors: Samartzidou Hrissi , Mehrazin Mahsa , Xu Zhaohui , Benedik Michael J. , Delcour Anne H. 
Published: December 2002

ABSTRACT

When grown at acidic pH,
Escherichia coli
cells secrete cadaverine, a polyamine known to inhibit porin-mediated outer membrane permeability. In order to understand the physiological significance of cadaverine excretion and the inhibition of porins, we isolated an OmpC mutant that showed resistance to spermine during growth and polyamine-resistant porin-mediated fluxes. Here, we show that the addition of exogenous cadaverine allows wild-type cells to survive a 30-min exposure to pH 3.6 better than cells expressing the cadaverine-insensitive OmpC porin. Competition experiments between strains expressing either wild-type or mutant OmpC showed that the lack of sensitivity of the porin to cadaverine confers a survival disadvantage to the mutant cells at reduced pH. On the basis of these results, we propose that the inhibition of porins by excreted cadaverine represents a novel mechanism that provides bacterial cells with the ability to survive acid stress.
Arsenite Oxidase
aox
Genes from a Metal-Resistant β-Proteobacterium
Authors: Muller Daniel , Lièvremont Didier , Simeonova Diliana Dancheva , Hubert Jean-Claude , Lett Marie-Claire 
Published: December 2002

ABSTRACT

The β-proteobacterial strain ULPAs1, isolated from an arsenic-contaminated environment, is able to efficiently oxidize arsenite [As(III)] to arsenate [As(V)]. Mutagenesis with a
lacZ
-based reporter transposon yielded two knockout derivatives deficient in arsenite oxidation. Sequence analysis of the DNA flanking the transposon insertions in the two mutants identified two adjacent open reading frames, named
aoxA
and
aoxB
, as well as a putative promoter upstream of the
aoxA
gene. Reverse transcription-PCR data indicated that these genes are organized in an operonic structure. The proteins encoded by
aoxA
and
aoxB
share 64 and 72% identity with the small Rieske subunit and the large subunit of the purified and crystallized arsenite oxidase of
Alcaligenes faecalis
, respectively (P. J. Ellis, T. Conrads, R. Hille, and P. Kuhn, Structure [Cambridge] 9:125-132, 2001). Importantly, almost all amino acids involved in cofactor interactions in both subunits of the
A. faecalis
enzyme were conserved in the corresponding sequences of strain ULPAs1. An additional Tat (twin-arginine translocation) signal peptide sequence was detected at the N terminus of the protein encoded by
aoxA
, strongly suggesting that the Tat pathway is involved in the translocation of the arsenite oxidase to its known periplasmic location.
Growth of Mycobacteria on Carbon Monoxide and Methanol
Authors: Park Sae W. , Hwang Eun H. , Park Hyuck , Kim Jeong A. , Heo Jinho , Lee Key H. , Song Taeksun , Kim Eungbin , Ro Young T. , Kim Si W. , Kim Young M. 
Published: December 2002

ABSTRACTSeveral mycobacterial strains, such asMycobacterium flavescens,Mycobacterium gastri,Mycobacterium neoaurum,Mycobacterium parafortuitum,Mycobacterium peregrinum,Mycobacterium phlei,Mycobacterium smegmatis,Mycobacterium tuberculosis, andMycobacterium vaccae, were found to grow on carbon monoxide (CO) as the sole source of carbon and energy. These bacteria, except forM. tuberculosis, also utilized methanol as the sole carbon and energy source. A CO dehydrogenase (CO-DH) assay, staining by activity of CO-DH, and Western blot analysis using an antibody raised against CO-DH ofMycobacteriumsp. strain JC1 (formerlyAcinetobactersp. strain JC1 [J. W. Cho, H. S. Yim, and Y. M. Kim, Kor. J. Microbiol.23:1-8, 1985]) revealed that CO-DH is present in extracts of the bacteria prepared from cells grown on CO. Ribulose bisphosphate carboxylase/oxygenase (RubisCO) activity was also detected in extracts prepared from all cells, exceptM. tuberculosis, grown on CO. The mycobacteria grown on methanol, except forM. gastri, which showed hexulose phosphate synthase activity, did not exhibit activities of classic methanol dehydrogenase, hydroxypyruvate reductase, or hexulose phosphate synthase but exhibitedN,N-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase and RuBisCO activities. Cells grown on methanol were also found to have dihydroxyacetone synthase. Double immunodiffusion revealed that the antigenic sites of CO-DHs, RuBisCOs, and dihydroxyacetone synthases in all mycobacteria tested are identical with those of theMycobacteriumsp. strain JC1 enzymes.
Protease-Deficient DegP Suppresses Lethal Effects of a Mutant OmpC Protein by Its Capture
Authors: CastilloKeller Maria , Misra Rajeev 
Published: December 2002

ABSTRACT

The expression of assembly-defective outer membrane proteins can confer lethality if they are not degraded by envelope proteases. We report here that the expression of a mutant OmpC protein, OmpC
2Cys
, which forms disulfide bonds in the periplasm due to the presence of two non-native cysteine residues, is lethal in cells lacking the major periplasmic protease, DegP. This lethality is not observed in
dsbA
strains that have diminished ability to form periplasmic disulfide bonds. Our data show that this OmpC
2Cys
-mediated lethality in a
degP
::Km
r
dsbA
+
background can be reversed by a DegP variant, DegP
S210A
, that is devoid of its proteolytic activity but retains its reported chaperone activity. However, DegP
S210A
does not reverse the lethal effect of OmpC
2Cys
by correcting its assembly but rather by capturing misfolded mutant OmpC polypeptides and thus removing them from the assembly pathway. Displacement of OmpC
2Cys
by DegP
S210A
also alleviates the negative effect that the mutant OmpC protein has on wild-type OmpF.
Transcriptome Analysis of
Neisseria meningitidis
during Infection
Authors: Dietrich Guido , Kurz Sebastian , Hübner Claudia , Aepinus Christian , Theiss Stephanie , Guckenberger Matthias , Panzner Ursula , Weber Jacqueline , Frosch Matthias 
Published: December 2002

ABSTRACT

Neisseria meningitidis
is the cause of septicemia and meningococcal meningitis. During the course of infection,
N
.
meningitidis
encounters multiple environments within its host, which makes rapid adaptation to environmental changes a crucial factor for neisserial pathogenicity. Employing oligonucleotide-based DNA microarrays, we analyzed the transcriptome of
N
.
meningitidis
during two key steps of meningococcal infection, i.e., the interaction with epithelial cells (HeLa cells) and endothelial cells (human brain microvascular endothelial cells). Seventy-two genes were differentially regulated after contact with epithelial cells, and 48 genes were differentially regulated after contact with endothelial cells, including a considerable proportion of well-known virulence genes. While a considerable number of genes were in concordance between bacteria adherent to both cell types, we identified several open reading frames that were differentially regulated in only one system. The data obtained with this novel approach may provide insight into the pathogenicity mechanisms of
N
.
meningitidis
and could demonstrate the importance of gene regulation on the transcriptional level during different stages of meningococcal infection.
Subunit Topology of Two 20S Proteasomes from
Haloferax volcanii
Authors: Kaczowka Steven J. , Maupin-Furlow Julie A. 
Published: December 2002

ABSTRACT

Haloferax volcanii
, a halophilic archaeon, synthesizes three different proteins (α1, α2, and β) which are classified in the 20S proteasome superfamily. The α1 and β proteins alone form active 20S proteasomes; the role of α2, however, is not clear. To address this, α2 was synthesized with an epitope tag and purified by affinity chromatography from recombinant
H. volcanii
. The α2 protein copurified with α1 and β in a complex with an overall structure and peptide-hydrolyzing activity comparable to those of the previously described α1-β proteasome. Supplementing buffers with 10 mM CaCl
2
stabilized the halophilic proteasomes in the absence of salt and enabled them to be separated by native gel electrophoresis. This facilitated the discovery that wild-type
H. volcanii
synthesizes more than one type of 20S proteasome. Two 20S proteasomes, the α1-β and α1-α2-β proteasomes, were identified during stationary phase. Cross-linking of these enzymes, coupled with available structural information, suggested that the α1-β proteasome was a symmetrical cylinder with α1 rings on each end. In contrast, the α1-α2-β proteasome appeared to be asymmetrical with homo-oligomeric α1 and α2 rings positioned on separate ends. Inter-α-subunit contacts were only detected when the ratio of α1 to α2 was perturbed in the cell using recombinant technology. These results support a model that the ratio of α proteins may modulate the composition and subunit topology of 20S proteasomes in the cell.
Biochemical and Mutational Characterization of the Heme Chaperone CcmE Reveals a Heme Binding Site
Authors: Enggist Elisabeth , Schneider Michael J. , Schulz Henk , Thöny-Meyer Linda 
Published: December 2002

ABSTRACT

CcmE is a heme chaperone that binds heme transiently in the periplasm of
Escherichia coli
and delivers it to newly synthesized and exported
c
-type cytochromes. The chemical nature of the covalent bond between heme and H130 is not known. We have purified soluble histidine-tagged CcmE and present its spectroscopic characteristics in the visible range. Alanine scanning mutagenesis of conserved amino acids revealed that H130 is the only residue found to be strictly required for heme binding and delivery. Mutation of the hydrophobic amino acids F37, F103, L127, and Y134 to alanine affected CcmE more than mutation of charged and polar residues. Our data are in agreement with the recently solved nuclear magnetic resonance structure of apo-CcmE (PDB code 1LIZ) and suggest that heme is bound to a hydrophobic platform at the surface of the protein and then attached to H130 by a covalent bond. Replacement of H130 with cysteine led to the formation of a covalent bond between heme and C130 at a low level. However, the H130C mutant CcmE was not active in cytochrome
c
maturation. Isolation and characterization of the heme-binding peptides obtained after a tryptic digest of wild-type and H130C CcmE support the hypothesis that heme is bound covalently at a vinyl group.
Transcriptional Organization of thePseudomonas putida tol-oprLGenes
Authors: Llamas María A. , Ramos Juan L. , Rodríguez-Herva José J. 
Published: December 2002

ABSTRACTProteins of the Tol system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. InPseudomonas putida, the seven genes,orf1,tolQ,tolR,tolA,tolB,oprL, andorf2, which encode the proteins of this complex, are clustered in a 5.8-kb region of chromosomal DNA. Analysis of polar mutations, reverse transcriptase PCR assays, and transcriptional fusion constructs with a promoterlesslacZgene revealed that the genes are arranged in two operons:orf1 tolQ tolR tolA tolBandoprL orf2. We were also able to find a transcript that was initiated at theorf1promoter and covered the two operons in a single mRNA. On the basis of the OprL protein level, we surmised that this transcript contributed only about 10 to 15% of the total OprL protein. Primer extension analysis identified theoprL orf2operon promoter within thetolBgene, and the −10 and −35 regions exhibited some similarity to those of σ70-recognized promoters. The transcription start point oforf1was located 91 bp upstream of theorf1start codon, and the −10/−35 region also exhibited σ70−10/−35 recognition sequences. The expression from both promoters in rich and minimal media was constitutive and was very little influenced by the growth phase or iron-deficient conditions. In addition, analyses of the β-galactosidase activities of different translational fusion constructs revealed that translation oftolAandorf2genes was dependent on the translation of their corresponding upstream genes (tolRandoprL, respectively).
Recruitment of MinC, an Inhibitor of Z-Ring Formation, to the Membrane in
Escherichia coli
: Role of MinD and MinE
Authors: Hu Zonglin , Saez Cristian , Lutkenhaus Joe 
Published: December 2002

ABSTRACT

In
Escherichia coli
, the
min
system prevents division away from midcell through topological regulation of MinC, an inhibitor of Z-ring formation. The topological regulation involves oscillation of MinC between the poles of the cell under the direction of the MinDE oscillator. Since the mechanism of MinC involvement in the oscillation is unknown, we investigated the interaction of MinC with the other Min proteins. We observed that MinD dimerized in the presence of ATP and interacted with MinC. In the presence of a phospholipid bilayer, MinD bound to the bilayer and recruited MinC in an ATP-dependent manner. Addition of MinE to the MinCD-bilayer complex resulted in release of both MinC and MinD. The release of MinC did not require ATP hydrolysis, indicating that MinE could displace MinC from the MinD-bilayer complex. In contrast, MinC was unable to displace MinE bound to the MinD-bilayer complex. These results suggest that MinE induces a conformational change in MinD bound to the bilayer that results in the release of MinC. Also, it is argued that binding of MinD to the membrane activates MinC.
Cinnamate:Coenzyme A Ligase from the Filamentous Bacterium
Streptomyces coelicolor
A3(2)
Authors: Kaneko Masafumi , Ohnishi Yasuo , Horinouchi Sueharu 
Published: December 2002

ABSTRACT

4-Coumarate:coenzyme A ligase (4CL) plays a key role in phenylpropanoid metabolism, providing precursors for a large variety of important plant secondary metabolites, such as lignin, flavonoids, and phytoalexins. Although 4CLs have been believed to be specific to plants, a gene encoding a 4CL-like enzyme which shows more than 40% identity in amino acid sequence to plant 4CLs was found in the genome of the gram-positive, filamentous bacterium
Streptomyces coelicolor
A3(2). The recombinant enzyme, produced in
Escherichia coli
with a histidine tag at its N-terminal end, showed distinct 4CL activity. The optimum pH and temperature of the reaction were pH 8.0 and 30°C, respectively. The

K
m

value for 4-coumarate and
k
cat
were determined as 131 ± 4 μM and 0.202 ± 0.007 s
−1
, respectively. The

K
m

value was comparable to those of plant 4CLs. The substrate specificity of this enzyme was, however, distinctly different from those of plant 4CLs. The enzyme efficiently converted cinnamate (

K
m

, 190 ± 2 μM;
k
cat
, 0.475 ± 0.012 s
−1
), which is a very poor substrate for plant 4CLs. Furthermore, the enzyme showed only low activity toward caffeate and no activity toward ferulate, both of which are generally good substrates for plant 4CLs. The enzyme was therefore named ScCCL for
S. coelicolor
A3(2) cinnamate CoA ligase. To determine the amino acid residues providing the unique substrate specificity of ScCCL, eight ScCCL mutant enzymes having a mutation(s) at amino acid residues that probably line up along the substrate-binding pocket were generated. Mutant A294G used caffeate as a substrate more efficiently than ScCCL, and mutant A294G/A318G used ferulate, which ScCCL could not use as a substrate, suggesting that Ala
294
and Ala
318
are involved in substrate recognition. Furthermore, the catalytic activities of A294G and A294G/A318G toward cinnamate and 4-coumarate were greatly enhanced compared with those of the wild-type enzyme.
Requirement of ArcA for Redox Regulation in
Escherichia coli
under Microaerobic but Not Anaerobic or Aerobic Conditions
Authors: Alexeeva Svetlana , Hellingwerf Klaas J. , Teixeira de Mattos M. Joost 
Published: December 2002

ABSTRACT

In
Escherichia coli,
the two-component regulatory ArcAB system functions as a major control system for the regulation of expression of genes encoding enzymes involved in both aerobic and anaerobic catabolic pathways. Previously, we have described the physiological response of wild-type
E. coli
to changes in oxygen availability through the complete range from anaerobiosis to full aerobiosis (S. Alexeeva, B. de Kort, G. Sawers, K. J. Hellingwerf, and M. J. Teixeira de Mattos, J. Bacteriol. 182:4934-4940, 2000, and S. Alexeeva, K. J. Hellingwerf, and M. J. Teixeira de Mattos, J. Bacteriol. 184:1402-1406, 2002). Here, we address the question of the contribution of the ArcAB-dependent transcriptional regulation to this response. Wild-type
E. coli
and a mutant lacking the ArcA regulator were grown in glucose-limited chemostat cultures at controlled levels of oxygen availability ranging from full aerobiosis to complete anaerobiosis. A flux analysis of the distribution of catabolic fluxes over parallel pathways was carried out, and the intracellular redox state (as reflected by the NADH/NAD ratio) was monitored for all steady states. Deletion of ArcA neither significantly altered the in vivo activity of the pyruvate dehydrogenase complex and pyruvate formate lyase nor significantly affected catabolism under fully aerobic and fully anaerobic conditions. In contrast, profound effects of the absence of ArcA were seen under conditions of oxygen-restricted growth: increased respiration, an altered electron flux distribution over the cytochrome
o
- and
d
-terminal oxidases, and a significant change in the intracellular redox state were observed. Thus, the ArcA regulator was found to exert major control on flux distribution, and it is concluded that the ArcAB system should be considered a microaerobic redox regulator.
Targeted Gene Disruption by Homologous Recombination in the Hyperthermophilic ArchaeonThermococcus kodakaraensisKOD1
Authors: Sato Takaaki , Fukui Toshiaki , Atomi Haruyuki , Imanaka Tadayuki 
Published: December 2002

ABSTRACTIn contrast to the high accumulation in sequence data for hyperthermophilic archaea, methodology for genetically manipulating these strains is still at an early stage. This study aimed to develop a gene disruption system for the hyperthermophilic euryarchaeonThermococcus kodakaraensisKOD1. Uracil-auxotrophic mutants with mutations in the orotidine-5′-monophosphate decarboxylase gene (pyrF) were isolated by positive selection using 5-fluoroorotic acid (5-FOA) and used as hosts for further transformation experiments. We then attempted targeted disruption of thetrpElocus in the host strain by homologous recombination, as disruption oftrpEwas expected to result in tryptophan auxotrophy, an easily detectable phenotype. A disruption vector harboring thepyrFmarker withintrpEwas constructed for double-crossover recombination. The host cells were transformed with the exogenous DNA using the CaCl2method, and several transformants could be selected based on genetic complementation. Genotypic and phenotypic analyses of a transformant revealed the unique occurrence of targeted disruption, as well as a phenotypic change of auxotrophy from uracil to tryptophan caused by integration of the wild-typepyrFinto the host chromosome attrpE. As with the circular plasmid, gene disruption with linear DNA was also possible when the homologous regions were relatively long. Shortening these regions led to predominant recombination between thepyrFmarker in the exogenous DNA and the mutated allele on the host chromosome. In contrast, we could not obtaintrpEdisruptants by insertional inactivation using a vector designed for single-crossover recombination. The gene targeting system developed in this study provides a long-needed tool in the research on hyperthermophilic archaea and will open the way to a systematic, genetic approach for the elucidation of unknown gene function in these organisms.
Contrasting Sensitivities of
Escherichia coli
Aconitases A and B to Oxidation and Iron Depletion
Authors: Varghese Shery , Tang Yue , Imlay James A. 
Published: December 2002

ABSTRACT

Superoxide damages dehydratases that contain catalytic [4Fe-4S]
2+
clusters. Aconitases are members of that enzyme family, and previous work showed that most aconitase activity is lost when
Escherichia coli
is exposed to superoxide stress. More recently it was determined that
E. coli
synthesizes at least two isozymes of aconitase, AcnA and AcnB. Synthesis of AcnA, the less-abundant enzyme, is positively controlled by SoxS, a protein that is activated in the presence of superoxide-generating chemicals. We have determined that this arrangement exists because AcnA is resistant to superoxide in vivo. Surprisingly, purified AcnA is extremely sensitive to superoxide and other chemical oxidants unless it is combined with an uncharacterized factor that is present in cell extracts. In contrast, AcnB is highly sensitive to a variety of chemical oxidants in vivo, in extracts, and in its purified form. Thus, the induction of AcnA during oxidative stress provides a mechanism to circumvent a block in the tricarboxylic acid cycle. AcnA appears to be as catalytically competent as AcnB, so the retention of the latter as the primary housekeeping enzyme must provide some other advantage. We observed that the [4Fe-4S] cluster of AcnB is in dynamic equilibrium with the surrounding iron pool, so that AcnB is rapidly demetallated when intracellular iron pools drop. AcnA and other dehydratases do not show this trait. Demetallated AcnB is known to bind its cognate mRNA. The absence of AcnB activity also causes the accumulation and excretion of citrate, an iron chelator for which
E. coli
synthesizes a transport system. Thus, AcnB may be retained as the primary aconitase because the lability of its exposed cluster allows
E. coli
to sense and respond to iron depletion.