bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of the
Pseudomonas aeruginosa
1244 Pilin Glycosylation Site
Authors: Comer Jason E. , Marshall Mark A. , Blanch Vincent J. , Deal Carolyn D. , Castric Peter 
Published: July 2002

ABSTRACT

Previous work (P. Castric, F. J. Cassels, and R. W. Carlson, J. Biol. Chem. 276:26479-26485, 2001) has shown the
Pseudomonas aeruginosa
1244 pilin glycan to be covalently bound to a serine residue. N-terminal sequencing of pilin fragments produced from endopeptidase treatment and identified by reaction with a glycan-specific monoclonal antibody indicated that the glycan was present between residue 75 and the pilin carboxy terminus. Further sequencing of these peptides revealed that serine residues 75, 81, 84, 105, 106, and 108 were not modified. Conversion of serine 148, but not serine 118, to alanine by site-directed mutagenesis, resulted in loss of the ability to carry out pilin glycosylation when tested in an in vivo system. These results showed the pilin glycan to be attached to residue 148, the carboxy-terminal amino acid. The carboxy-proximal portion of the pilin disulfide loop, which is adjacent to the pilin glycan, was found to be a major linear B-cell epitope, as determined by peptide epitope mapping analysis. Immunization of mice with pure pili produced antibodies that recognized the pilin glycan. These sera also reacted with
P. aeruginosa
1244 lipopolysaccharide as measured by Western blotting and enzyme-linked immunosorbent assay.
Purification and Characterization of Sa-Lrp, a DNA-Binding Protein from the Extreme Thermoacidophilic Archaeon
Sulfolobus acidocaldarius
Homologous to the Bacterial Global Transcriptional Regulator Lrp
Authors: Enoru-Eta Julius , Gigot Daniel , Thia-Toong Thia-Lin , Glansdorff Nicolas , Charlier Daniel 
Published: July 2002

ABSTRACT

Archaea
, constituting the third primary domain of life, harbor a basal transcription apparatus of the eukaryotic type, whereas curiously, a large fraction of the potential transcription regulation factors appear to be of the bacterial type. To date, little information is available on these predicted regulators and on the intriguing interplay that necessarily has to occur with the transcription machinery. Here, we focus on
Sa-lrp
of the extremely thermoacidophilic crenarchaeote
Sulfolobus acidocaldarius
, encoding an archaeal homologue of the
Escherichia coli
leucine-responsive regulatory protein Lrp, a global transcriptional regulator and genome organizer.
Sa-lrp
was shown to produce a monocistronic mRNA that was more abundant in the stationary-growth phase and produced in smaller amounts in complex medium, this down regulation being leucine independent. We report on Sa-Lrp protein purification from
S. acidocaldarius
and from recombinant
E. coli
, both identified by N-terminal amino acid sequence determination. Recombinant Sa-Lrp was shown to be homotetrameric and to bind to its own control region; this binding proved to be leucine independent and was stimulated at high temperatures. Interference binding experiments suggested an important role for minor groove recognition in the Sa-Lrp–DNA complex formation, and mutant analysis indicated the importance for DNA binding of the potential helix-turn-helix motif present at the N terminus of Sa-Lrp. The DNA-binding capacity of purified Sa-Lrp was found to be more resistant to irreversible heat inactivation in the presence of
l
-leucine, suggesting a potential physiological role of the amino acid as a cofactor.
Effect of Altered Spacing between
uhpT
Promoter Elements on Transcription Activation
Authors: Chen Qing , Kadner Robert J. 
Published: July 2002

ABSTRACT

Many bacterial promoters possess multiple sites for binding of transcriptional activator proteins. The
uhpT
promoter, which controls expression of the sugar phosphate transport system in
Escherichia coli
, possesses multiple sites for its specific activator protein, UhpA, and a single site for binding of the global regulator, the catabolite gene activator protein (CAP). The binding of UhpA to the
uhpT
promoter was determined by DNase protection assays; UhpA displayed different affinities for the target sites. The upstream or strong sites, between positions −80 and −50, exhibited a higher affinity for UhpA than did the downstream or weak sites, between positions −50 and −32, adjoining the RNA polymerase-binding site. Phosphorylation of UhpA strongly increased its affinity for both sites. To examine the possible roles of the two sets of UhpA-binding sites, a series of insertion and deletion mutations were introduced at the boundary between them, as suggested from the positions that were protected by UhpA against hydroxyl radical cleavage. Deletions extended in the direction of the weak sites. The insertion or deletion of one helical turn of DNA resulted in the loss of promoter activity and of occupancy by UhpA of the remaining weak-site sequences but was accompanied by normal occupancy of the strong site and no change in the gel retardation behavior of the promoter fragments. However, the deletion of two helical turns of DNA, i.e., 20, 21, or 22 bp, resulted in the novel appearance of UhpA-independent expression and in an additional level of expression that was dependent on UhpA but independent of an inducing signal. The UhpA-independent promoter activity was shown to result from activation by CAP at its more proximal position. UhpA-dependent activity under noninducing conditions appears to result from the binding of unphosphorylated UhpA to the strong sites, which are now in the position normally occupied by the weak sites. Thus, regulated phosphorylation of the response regulator UhpA enhances its occupancy of the weak sites where favorable contacts can allow the binding of RNA polymerase to the promoter.
Identification of Enzymes Homologous to Isocitrate Dehydrogenase That Are Involved in Coenzyme B and Leucine Biosynthesis in Methanoarchaea
Authors: Howell David M. , Graupner Marion , Xu Huimin , White Robert H. 
Published: July 2002

ABSTRACTTwo putativeMethanococcus jannaschiiisocitrate dehydrogenase genes, MJ1596 and MJ0720, were cloned and overexpressed inEscherichia coli, and their gene products were tested for the ability to catalyze the NAD- and NADP-dependent oxidative decarboxylation ofdl-threo-3-isopropylmalic acid,threo-isocitrate,erythro-isocitrate, and homologs ofthreo-isocitrate. Neither enzyme was found to use any of the isomers of isocitrate as a substrate. The protein product of the MJ1596 gene, designated AksF, catalyzed the NAD-dependent decarboxylation of intermediates in the biosynthesis of 7-mercaptoheptanoic acid, a moiety of methanoarchaeal coenzyme B (7-mercaptoheptanylthreonine phosphate). These intermediates included (−)-threo-isohomocitrate [(−)-threo-1-hydroxy-1,2,4-butanetricarboxylic acid], (−)-threo-iso(homo)2citrate [(−)-threo-1-hydroxy-1,2,5-pentanetricarboxylic acid], and (−)-threo-iso(homo)3citrate [(−)-threo-1-hydroxy-1,2,6-hexanetricarboxylic acid]. The protein product of MJ0720 was found to be α-isopropylmalate dehydrogenase (LeuB) and was found to catalyze the NAD-dependent decarboxylation of one isomer ofdl-threo-isopropylmalate to 2-ketoisocaproate; thus, it is involved in the biosynthesis of leucine. The AksF enzyme proved to be thermostable, losing only 10% of its enzymatic activity after heating at 100°C for 10 min, whereas the LeuB enzyme lost 50% of its enzymatic activity after heating at 80°C for 10 min.
Discovery of a Nonclassical Siderophore, Legiobactin, Produced by Strains ofLegionella pneumophila
Authors: Liles Mark R. , Scheel Tracy Aber , Cianciotto Nicholas P. 
Published: July 2002

ABSTRACTThe mechanisms by whichLegionella pneumophila, a facultative intracellular parasite and the agent of Legionnaires' disease, acquires iron are largely unexplained. Several earlier studies indicated thatL. pneumophiladoes not elaborate siderophores. However, we now present evidence that supernatants fromL. pneumophilacultures can contain a nonproteinaceous, high-affinity iron chelator. More specifically, when aerobically grown in a low-iron, chemically defined medium (CDM),L. pneumophilasecretes a substance that is reactive in the chrome azurol S (CAS) assay. Importantly, the siderophore-like activity was only observed when the CDM cultures were inoculated to relatively high density with bacteria that had been grown overnight to log or early stationary phase in CDM or buffered yeast extract. Inocula derived from late-stationary-phase cultures, despite ultimately growing, consistently failed to result in the elaboration of siderophore-like activity. TheLegionellaCAS reactivity was detected in the culture supernatants of the serogroup 1 strains 130b and Philadelphia-1, as well as those from representatives of other serogroups and otherLegionellaspecies. The CAS-reactive substance was resistant to boiling and protease treatment and was associated with the <1-kDa supernatant fraction. As would also be expected for a siderophore, the addition of 0.5 or 2.0 μM iron to the cultures repressed the expression of the CAS-reactive substance. Interestingly, the supernatants were negative in the Arnow, Csáky, and Rioux assays, indicating that theLegionellasiderophore was not a classic catecholate or hydroxamate and, hence, might have a novel structure. We have designated theL. pneumophilasiderophore legiobactin.
Hin Recombinase Mutants Functionally Disrupted in Interactions with Fis
Authors: Nanassy Oliver Z. , Hughes Kelly T. 
Published: July 2002

ABSTRACT
A previous genetic screen was designed to separate Hin recombinase mutants into distinct classes based on the stage in the recombination reaction at which they are blocked (O. Nanassy, Zoltan, and K. T. Hughes, Genetics 149:1649–1663, 1998). One class of DNA binding-proficient, recombination-deficient mutants was predicted by genetic classification to be defective in the step prior to invertasome formation. Based on the genetic criteria, mutants from this class were also inferred to be defective in interactions with Fis. In order to understand how the genetic classification relates to individual biochemical steps in the recombination reaction these mutants, R123Q, T124I, and A126T, were purified and characterized for DNA cleavage and recombination activities. Both the T124I and A126T mutants were partially active, whereas the R123Q mutant was inactive. The A126T mutant was not as defective for recombination as the T124I allele and could be partially rescued for recombination both in vivo and in vitro by increasing the concentration of Fis protein. Rescue of the A126T allele required the Fis protein to be DNA binding proficient. A model for a postsynaptic role for Fis in the inversion reaction is presented.
Isolation and Characterization of IS
1409
, an Insertion Element of 4-Chlorobenzoate-Degrading
Arthrobacter
sp. Strain TM1, and Development of a System for Transposon Mutagenesis
Authors: Gartemann Karl-Heinz , Eichenlaub Rudolf 
Published: July 2002

ABSTRACT

A new insertion element of 1,449 bp with 25-bp perfect terminal repeats, designated IS
1409
, was identified in the chromosome of 4-chlorobenzoate-degrading
Arthrobacter
sp. strain TM1 NCIB12013. Upon insertion, IS
1409
causes a target duplication of 8 bp. IS
1409
carries only a single open reading frame of 435 codons encoding the transposase TnpA. Both TnpA and the overall organization of IS
1409
are highly similar to those of some related insertion elements of the ISL
3
group (J. Mahillon and M. Chandler, Microbiol. Mol. Biol. Rev. 62:725–774, 1998). IS
1409
was also found in other 4-chlorobenzoate-degrading
Arthrobacter
strains and
Micrococcus luteus
. Based on IS
1409
, a series of transposons carrying resistance genes for chloramphenicol and gentamicin were constructed. These transposons were used to demonstrate transposition events in vivo and to mutagenize
Arthrobacter
sp. strains.
The
coxBAC
Operon Encodes a Cytochrome
c
Oxidase Required for Heterotrophic Growth in the Cyanobacterium
Anabaena variabilis
Strain ATCC 29413
Authors: Schmetterer Georg , Valladares Ana , Pils Dietmar , Steinbach Susanne , Pacher Margit , Muro-Pastor Alicia M. , Flores Enrique , Herrero Antonia 
Published: July 2002

ABSTRACT

Three genes,
coxB, coxA
, and
coxC
, found in a clone from a gene library of the cyanobacterium
Anabaena variabilis
strain ATCC 29413, were identified by hybridization with an oligonucleotide specific for
aa
3
-type cytochrome
c
oxidases. Deletion of these genes from the genome of
A. variabilis
strain ATCC 29413 FD yielded strain CSW1, which displayed no chemoheterotrophic growth and an impaired cytochrome
c
oxidase activity. Photoautotrophic growth of CSW1, however, was unchanged, even with dinitrogen as the nitrogen source. A higher cytochrome
c
oxidase activity was detected in membrane preparations from dinitrogen-grown CSW1 than from nitrate-grown CSW1, but comparable activities of respiratory oxygen uptake were found in the wild type and in CSW1. Our data indicate that the identified
cox
gene cluster is essential for fructose-dependent growth in the dark, but not for growth on dinitrogen, and that other terminal respiratory oxidases are expressed in this cyanobacterium. Transcription analysis showed that
coxBAC
constitutes an operon which is expressed from two transcriptional start points. The use of one of them was stimulated by fructose.
Cloning of a Genetically Unstable Cytochrome P-450 Gene Cluster Involved in Degradation of the Pollutant Ethyl
tert
-Butyl Ether by
Rhodococcus ruber
Authors: Chauvaux Sylvie , Chevalier Fabien , Le Dantec Corinne , Fayolle Françoise , Miras Isabelle , Kunst Frank , Beguin Pierre 
Published: July 2002

ABSTRACT

Rhodococcus ruber
(formerly
Gordonia terrae
) IFP 2001 is one of a few bacterial strains able to degrade ethyl
tert-
butyl ether (ETBE), which is a major pollutant from gasoline. This strain was found to undergo a spontaneous 14.3-kbp chromosomal deletion, which results in the loss of the ability to degrade ETBE. Sequence analysis of the region corresponding to the deletion revealed the presence of a gene cluster,
ethABCD
, encoding a ferredoxin reductase, a cytochrome P-450, a ferredoxin, and a 10-kDa protein of unknown function, respectively. The EthB and EthD proteins could be easily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were induced by ETBE in the wild-type strain. Upstream of
ethABCD
lies
ethR
, which codes for a putative positive transcriptional regulator of the AraC/XylS family. Transformation of the ETBE-negative mutant by a plasmid carrying the
ethRABCD
genes restored the ability to degrade ETBE. Complementation was abolished if the plasmid carried
ethRABC
only. The
eth
genes are located in a DNA fragment flanked by two identical direct repeats of 5.6 kbp. The ETBE-negative mutants carry a single copy of this 5.6-kbp repeat, suggesting that the 14.3-kbp chromosomal deletion resulted from a recombination between the two identical sequences. The 5.6-kbp repeat is a class II transposon carrying a TnpA transposase, a truncated form of the recombinase TnpR, and a terminal inverted repeat of 38 bp. The truncated TnpR is encoded by an IS
3
-interrupted
tnpR
gene.
Hha Is a Negative Modulator of Transcription ofhilA, theSalmonella entericaSerovar Typhimurium Invasion Gene Transcriptional Activator
Authors: Fahlen Thomas F. , Wilson Rebecca L. , Boddicker Jennifer D. , Jones Bradley D. 
Published: July 2002

ABSTRACTAn early step in the establishment ofSalmonella entericaserovar Typhimurium murine infection is the penetration of the intestinal mucosa of the small intestine. The majority of the genes responsible for theSalmonellainvasive phenotype are encoded onSalmonellapathogenicity island 1, and their transcription is controlled by thehilAtranscriptional activator. The expression ofhilAis regulated by environmental signals including oxygen, osmolarity, pH, and growth phase such that the presence of any one suboptimal condition results in repression ofhilAexpression and the invasive phenotype. We have conducted a search for negative regulators ofhilAby introduction of aSalmonella entericaserovar Typhimurium chromosomal DNA gene bank into aSalmonella entericaserovar TyphimuriumhilA::Tn5lacZYreporter strain. This screen has identified thehhagene as a regulator that exerts a negative influence onhilAexpression. Plasmid-encodedhhasignificantly reduceshilA::Tn5lacZYchromosomal expression, as well as expression of the invasion genesinvF,prgH, andsipC. Anhhanull mutation results in substantial derepression of both chromosomally encoded and plasmid-encodedhilA::Tn5lacZYexpression. Introduction of plasmid-encodedhhainto strain SL1344 results in attenuation of invasion using in vitro and in vivo assays. Importantly, purified Hha protein was found to bind to ahilADNA promoter fragment, suggesting that the regulatory activity of the Hha protein occurs at thehilApromoter. These data add detail to the developing model of the regulation ofSalmonellainvasion genes.
Membrane-Associated Quinoprotein Formaldehyde Dehydrogenase from
Methylococcus capsulatus
Bath
Authors: Zahn James A. , Bergmann David J. , Boyd Jeffery M. , Kunz Ryan C. , DiSpirito Alan A. 
Published: July 2002

ABSTRACT

A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph
Methylococcus capsulatus
Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 μmol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)
+
-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159–167, 1999; Vorholt et al., J. Bacteriol. 180:5351–5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome
b
559/569
complex as the physiological electron acceptor.
Evidence that a Linear Megaplasmid Encodes Enzymes of Aliphatic Alkene and Epoxide Metabolism and Coenzyme M (2-Mercaptoethanesulfonate) Biosynthesis in
Xanthobacter
Strain Py2
Authors: Krum Jonathan G. , Ensign Scott A. 
Published: July 2002

ABSTRACT

The bacterial metabolism of propylene proceeds by epoxidation to epoxypropane followed by a sequence of three reactions resulting in epoxide ring opening and carboxylation to form acetoacetate. Coenzyme M (2-mercaptoethanesulfonic acid) (CoM) plays a central role in epoxide carboxylation by serving as the nucleophile for epoxide ring opening and the carrier of the C
3
unit that is ultimately carboxylated to acetoacetate, releasing CoM. In the present work, a 320-kb linear megaplasmid has been identified in the gram-negative bacterium
Xanthobacter
strain Py2, which contains the genes encoding the key enzymes of propylene oxidation and epoxide carboxylation. Repeated subculturing of
Xanthobacter
strain Py2 under nonselective conditions, i.e., with glucose or acetate as the carbon source in the absence of propylene, resulted in the loss of the propylene-positive phenotype. The propylene-negative phenotype correlated with the loss of the 320-kb linear megaplasmid, loss of induction and expression of alkene monooxgenase and epoxide carboxylation enzyme activities, and the loss of CoM biosynthetic capability. Sequence analysis of a hypothetical protein (XecG), encoded by a gene located downstream of the genes for the four enzymes of epoxide carboxylation, revealed a high degree of sequence identity with proteins of as-yet unassigned functions in the methanogenic archaea
Methanobacterium thermoautotrophicum
and
Methanococcus jannaschii
and in
Bacillus subtilis
. The
M. jannaschii
homolog of XecG, MJ0255, is located next to a gene, MJ0256, that has been shown to encode a key enzyme of CoM biosynthesis (M. Graupner, H. Xu, and R. H. White, J. Bacteriol. 182: 4862–4867, 2000). We propose that the propylene-positive phenotype of
Xanthobacter
strain Py2 is dependent on the selective maintenance of a linear megaplasmid containing the genes for the key enzymes of alkene oxidation, epoxide carboxylation, and CoM biosynthesis.
Independence of Circadian Timing from Cell Division in Cyanobacteria
Authors: Mori Tetsuya , Johnson Carl Hirschie 
Published: July 2002

ABSTRACT

In the cyanobacterium
Synechococcus elongatus
, cell division is regulated by a circadian clock. Deletion of the circadian clock gene,
kai
C, abolishes rhythms of gene expression and cell division timing. Overexpression of the
ftsZ
gene halted cell division but not growth, causing cells to grow as filaments without dividing. The nondividing filamentous cells still exhibited robust circadian rhythms of gene expression. This result indicates that the circadian timing system is independent of rhythmic cell division and, together with other results, suggests that the cyanobacterial circadian system is stable and well sustained under a wide range of intracellular conditions.
Efficiency of Recombination Reactions Catalyzed by Class 1 Integron Integrase IntI1
Authors: Collis Christina M. , Recchia Gavin D. , Kim Mi-Jurng , Stokes H. W. , Hall Ruth M. 
Published: July 2002

ABSTRACT

The class 1 integron integrase, IntI1, recognizes two distinct types of recombination sites,
attI
sites, found in integrons, and members of the 59-be family, found in gene cassettes. The efficiencies of the integrative version of the three possible reactions, i.e., between two 59-be, between
attI1
and a 59-be, or between two
attI1
sites, were compared. Recombination events involving two
attI1
sites were significantly less efficient than the reactions in which a 59-be participated, and the
attI1
× 59-be reaction was generally preferred over the 59-be × 59-be reaction. Recombination of
attI1
with secondary sites was less efficient than the 59-be × secondary site reaction.
Functional Analysis of the Signal Recognition Particle inEscherichia coliby Characterization of a Temperature-SensitiveffhMutant
Authors: Park Sei-Kyoung , Jiang Fenglei , Dalbey Ross E. , Phillips Gregory J. 
Published: July 2002

ABSTRACTThe Ffh protein ofEscherichia coliis a 48-kDa polypeptide that is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP). Efforts to understand the function of Ffh in bacteria have depended largely on the use ofE. colistrains that allow depletion of the wild-type gene product. As an alternative approach to studying Ffh, a temperature-sensitiveffhmutant was isolated. Theffh-10(Ts) mutation results in two amino acid changes in conserved regions of the Ffh protein, and characterization of the mutant revealed that the cells rapidly lose viability at the nonpermissive temperature of 42°C as well as show reduced growth at the permissive temperature of 30°C. While theffhmutant is defective in insertion of inner membrane proteins, the export of proteins with cleavable signal sequences is not impaired. The mutant also shows elevated expression of heat shock proteins and accumulates insoluble proteins, especially at 42°C. It was further observed that the temperature sensitivity of theffhmutant was suppressed by overproduction of 4.5S RNA, the RNA component of the bacterial SRP, by stabilizing the thermolabile protein. Collectively, these results are consistent with a model in which Ffh is required only for localization of proteins integral to the cytoplasmic membrane and suggest new genetic approaches to the study of how the structure of the SRP contributes to its function.