bluuebunny/crossref_metadata_2025 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

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

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

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

Two 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

The 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

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

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

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

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

An 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

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

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

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

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

The 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.
Characterization of the HspR-Mediated Stress Response inHelicobacter pylori
Authors: Spohn Gunther , Delany Isabel , Rappuoli Rino , Scarlato Vincenzo
Published: July 2002

The major heat shock genes ofHelicobacter pyloriare regulated by the HspR repressor. In the present study we characterize the transcriptional response of the three known HspR-dependent promoters Pcbp, Pgro, and Phrcto different environmental stresses. A temperature shift from 37 to 42°C causes a typical heat shock response at all three promoters characterized by an immediate and strong induction phase of transcription and a subsequent adaptation phase, which is specific for each promoter and whose onset is determined partially by the half-lives of the respective mRNAs. Exposure to high osmolarity induces a similar response on the Pgroand Pcbppromoters while no such response is detectable at the Phrcpromoter. Puromycin treatment induces transcription from all three HspR-dependent promoters, indicating that different environmental stresses are intracellularly sensed by the regulatory machinery through the accumulation of nonnative proteins. The implications of these data for the regulatory network controlling the heat shock response inH. pyloriare discussed.
Up-Regulation of theYersinia enterocolitica yopRegulon by Deletion of the Flagellum Master OperonflhDC
Authors: Bleves Sophie , Marenne Marie-Noëlle , Detry Gautier , Cornelis Guy R.
Published: July 2002

The Yop virulon enables extracellularly locatedYersinia, in close contact with a eukaryotic target cell, to inject bacterial toxic proteins directly into the cytosol of this cell. Several Ysc proteins, forming the Yop secretion apparatus, display homology with proteins of the flagellar basal body. To determine whether this relationship could extend to the regulatory pathways, we analyzed the influence offlhDC, the master regulatory operon of the flagellum, on theyopregulon. In anflhDCmutant, theyopregulon was up-regulated. The transcription ofvirFand the steady-state level of the transcriptional activator VirF were enhanced.yoptranscription was increased at 37°C and could also be detected at a low temperature. Yop secretion was increased at 37°C and occurred even at a low temperature. The Ysc secretion machinery was thus functional at room temperature in the absence of flagella, implying that in wild-type bacteria, FlhD and/or FlhC, or the product of a gene downstream offlhDC, represses theyopregulon. In agreement with this notion, increased expression offlhDCin wild-type bacteria resulted in the oversecretion of flagellins at room temperature and in decreased Yop secretion at 37°C.
Aerobic-Type Ribonucleotide Reductase in the Anaerobe
Bacteroides fragilis
Authors: Smalley Darren , Rocha Edson R. , Smith C. Jeffrey
Published: July 2002

Bacteroides fragilis
, a component of the normal intestinal flora, is an obligate anaerobe capable of long-term survival in the presence of air. Survival is attributed to an elaborate oxidative stress response that controls the induction of more than 28 peptides, but there is limited knowledge concerning the identities of these peptides. In this report, RNA fingerprinting by arbitrarily primed PCR identified five new genes whose expression increased following exposure to O
2
. Nucleotide sequence analysis of the cloned genes indicated that they encoded an outer membrane protein, an aspartate decarboxylase, an efflux pump, heat shock protein HtpG, and an NrdA ortholog constituting the large subunit of a class Ia ribonucleotide reductase (RRase). Attention was focused on the
nrdA
gene since class I RRases are obligate aerobic enzymes catalyzing the reduction of ribonucleoside 5′-diphosphates by a mechanism that requires molecular oxygen for activity. Sequence analysis of the
nrd
locus showed that two genes,
nrdA
and
nrdB
, are located in the same orientation in a 4.5-kb region. Northern hybridization and primer extension experiments confirmed induction of the genes by O
2
and suggested they are an operon. The
B. fragilis nrdA
and
nrdB
genes were overexpressed in
Escherichia coli
, and CDP reductase assays confirmed that they encoded an active enzyme. The enzyme activity was inhibited by hydroxyurea, and ATP was shown to be a positive effector of CDP reductase activity, while dATP was an inhibitor, indicating that the enzyme was a class Ia RRase. A
nrdA
mutant was viable under anaerobic conditions but had decreased survival following exposure to O
2
, and it could not rapidly resume growth after O
2
treatment. The results presented indicate that during aerobic conditions
B. fragilis
NrdAB may have a role in maintaining deoxyribonucleotide pools for DNA repair and growth recovery.
A ToxR Homolog from
Vibrio anguillarum
Serotype O1 Regulates Its Own Production, Bile Resistance, and Biofilm Formation
Authors: Wang Su-Yan , Lauritz Johan , Jass Jana , Milton Debra L.
Published: July 2002

ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium
Vibrio anguillarum
, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment,
toxR
and
toxS
from
V. anguillarum
serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the
Vibrio cholerae
ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the
toxR
mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The
toxR
mutant but not the
toxS
mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from
V. cholerae
, and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a
toxR
mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.
AniA Regulates Reserve Polymer Accumulation and Global Protein Expression in
Rhizobium etli
Authors: Encarnación Sergio , del Carmen Vargas María , Dunn Michael F. , Dávalos Araceli , Mendoza Guillermo , Mora Yolanda , Mora Jaime
Published: July 2002

Previously, it was reported that the oxidative capacity and ability to grow on carbon sources such as pyruvate and glucose were severely diminished in the
Rhizobium etli phaC
::ΩSm
r
/Sp
r
mutant CAR1, which is unable to synthesize poly-β-hydroxybutyric acid (PHB) (M. A. Cevallos, S. Encarnación, A. Leija, Y. Mora, and J. Mora, J. Bacteriol. 178:1646-1654, 1996). By random Tn
5
mutagenesis of the
phaC
strain, we isolated the mutants VEM57 and VEM58, both of which contained single Tn
5
insertions and had recovered the ability to grow on pyruvate or glucose. Nucleotide sequencing of the region surrounding the Tn
5
insertions showed that they had interrupted an open reading frame designated
aniA
based on its high deduced amino acid sequence identity to the
aniA
gene product of
Sinorhizobium meliloti
.
R. etli aniA
was located adjacent to and divergently transcribed from genes encoding the PHB biosynthetic enzymes β-ketothiolase (PhaA) and acetoacetyl coenzyme A reductase (PhaB). An
aniA
::Tn
5
mutant (VEM5854) was constructed and found to synthesize only 40% of the wild type level of PHB. Both VEM58 and VEM5854 produced significantly more extracellular polysaccharide than the wild type. Organic acid excretion and levels of intracellular reduced nucleotides were lowered to wild-type levels in VEM58 and VEM5854, in contrast to those of strain CAR1, which were significantly elevated. Proteome analysis of VEM58 showed a drastic alteration of protein expression, including the absence of a protein identified as PhaB. We propose that the
aniA
gene product plays an important role in directing carbon flow in
R. etli.
Comparative Prevalences of
Brachyspira aalborgi
and
Brachyspira
(
Serpulina
)
pilosicoli
as Etiologic Agents of Histologically Identified Intestinal Spirochetosis in Australia
Authors: Mikosza Andrew S. J. , La Tom , de Boer W. Bastiaan , Hampson David J.
Published: July 2002

DNA from gastrointestinal biopsy specimens from 28 Australian patients with histologic evidence of intestinal spirochetosis (IS) was subjected to PCRs to amplify segments of the 16S rRNA and NADH oxidase genes of
Brachyspira aalborgi
and
Brachyspira (Serpulina) pilosicoli. B. aalborgi
was identified in specimens from 24 (85.7%) patients and
B. pilosicoli
in those from 4 (14.3%) patients (2 of whom were also positive for
B. aalborgi
). For two patients, no product was amplified. This study demonstrates that
B. aalborgi
is much more commonly involved in histologically identified IS in Australian patients than is
B. pilosicoli
. This is the first report of amplification of
B. pilosicoli
DNA from humans with IS.
Validity of Cultures of Fluid Collected through Drainage Catheters versus Those Obtained by Direct Aspiration
Authors: Everts Richard J. , Heneghan Joan P. , Adholla Paul O. , Reller L. Barth
Published: July 2002

To examine the validity of cultures of fluid collected through drainage catheters, we reviewed retrospectively fluid specimens that had been collected through catheters in place for at least 2 days. These specimens were taken from patients at a large tertiary-care hospital. A total of 974 specimens representing 620 patient episodes were received. For 554 (89%) episodes there was no reliable imaging evidence for localized infection, rendering the results uninterpretable. The remaining 66 (11%) episodes were followed within 2 days by radiologically guided or open aspiration of one or more fluid collections (predominantly in the abdomen or pelvis) near the drainage catheter, allowing comparison of culture results of 59 direct aspirates with those of prior catheter drainage. In 33 (56%) of these 59 cases, matched culture results were equivalent for therapeutic decision making. However, relying on results of catheter drainage cultures would have led to inadequate antimicrobial therapy in 13 (22%) cases, to excessive therapy in 11 (19%) cases, and to both in 2 cases (3%). We conclude that radiological imaging should be standard practice in the assessment of deep-tissue infections in patients with drainage catheters, and that direct aspiration of potentially infected fluid collections is the most reliable method of obtaining specimens for culture that should be used to guide therapy.
Sequence Polymorphism in the
rrs
Gene of
Mycobacterium tuberculosis
Is Deeply Rooted within an Evolutionary Clade and Is Not Associated with Streptomycin Resistance
Authors: Victor Thomas C. , van Rie Annelies , Jordaan Annemie M. , Richardson Madalene , van der Spuy Gian D. , Beyers Nulda , van Helden Paul D. , Warren Robin
Published: July 2002

A mutation (C-to-T transition) at position 491 of the
rrs
gene was identified in a
Mycobacterium tuberculosis
strain family (
n
= 208 isolates) that was predominant in a suburb of Cape Town, South Africa. This nucleotide change is not involved in streptomycin resistance, and we suggest caution in assuming that all mutations in genes targeted by antituberculosis drugs confer drug resistance.
Prevalence and Antifungal Susceptibility of 442
Candida
Isolates from Blood and Other Normally Sterile Sites: Results of a 2-Year (1996 to 1998) Multicenter Surveillance Study in Quebec, Canada
Authors: St-Germain G. , Laverdière M. , Pelletier R. , Bourgault A.-M. , Libman M. , Lemieux C. , Noël G.
Published: July 2002

During a 2-year surveillance program (1996 to 1998) in Quebec, Canada, 442 strains of
Candida
species were isolated from 415 patients in 51 hospitals. The distribution of species was as follows:
Candida albicans,
54%;
C. glabrata,
15%;
C. parapsilosis,
12%;
C. tropicalis,
9%;
C. lusitaniae,
3%;
C. krusei,
3%; and
Candida
spp., 3%. These data, compared to those of a 1985 survey, indicate variations in species distribution, with the proportions of
C. glabrata
and
C. parapsilosis
increasing by 9 and 4%, respectively, and those of
C. albicans
and
C. tropicalis
decreasing by 10 and 7%, respectively. However, these differences are statistically significant for
C. glabrata
and
C. tropicalis
only. MICs of amphotericin B were ≥4 μg/ml for 3% of isolates, all of which were non-
C. albicans
species. Three percent of
C. albicans
isolates were resistant to flucytosine (≥32 μg/ml). Resistance to itraconazole (≥1 μg/ml) and fluconazole (≥64 μg/ml) was observed, respectively, in 1 and 1% of
C. albicans
, 14 and 9% of
C. glabrata
, 5 and 0% of
C. tropicalis,
and 0% of
C. parapsilosis
and
C. lusitaniae
isolates. Clinical data were obtained for 343 patients. The overall crude mortality rate was 38%, reflecting the multiple serious underlying illnesses found in these patients. Bloodstream infections were documented for 249 patients (73%). Overall, systemic triazoles had been administered to 10% of patients before the onset of candidiasis. The frequency of isolation of non-
C. albicans
species was significantly higher in this group of patients. Overall, only two
C. albicans
isolates were found to be resistant to fluconazole. These were obtained from an AIDS patient and a leukemia patient, both of whom had a history of previous exposure to fluconazole. At present, it appears that resistance to fluconazole in Quebec is rare and is restricted to patients with prior prolonged azole treatment.
Serologic Detection of
Actinobacillus pleuropneumoniae
in Swine by Capsular Polysaccharide-Biotin-Streptavidin Enzyme-Linked Immunosorbent Assay
Authors: Inzana Thomas J. , Fenwick Brad
Published: July 2002

Serologic detection of
Actinobacillus pleuropneumoniae
infections in swine have been problematic due to antigenic cross-reactivity of Apx toxins, lipopolysaccharide, and outer membrane proteins between
A. pleuropneumoniae
serotypes and other bacterial species. To maximize serologic specificity and sensitivity, we developed an assay that uses highly purified
A. pleuropneumoniae
capsular polysaccharide (CP) conjugated to biotin, which is then bound to streptavidin-coated enzyme-linked immunosorbent assay (CP-BS-ELISA) plates. This assay was used to test a panel of 240 serum samples from pigs prior to challenge, after challenge with bacterial species other than
A. pleuropneumoniae
, or after challenge with
A. pleuropneumoniae
serotype 1, 5, or 7. Overall assay results for the individual sera tested were reproducible on the same day and on separate days. The sensitivity of the assay was 100% by ELISAs with biotin-CPs of serotypes 1 and 7 and 87.5% by ELISAs with biotin-CP of serotype 5. Specificity was 100% by ELISAs with biotin-CPs of serotypes 1 and 5 and 94.5% by ELISAs with biotin-CP of serotype 7. The biotin-CPs of at least three
A. pleuropneumoniae
serotypes could be combined for use in a screening assay to detect antibodies to CPs from strains of different serotypes. In conclusion, the CP-BS-ELISA proved to be a serotype-specific and species-specific assay with high sensitivity for the identification of pigs exposed to
A. pleuropneumoniae
.
Weissella confusa
(Basonym:
Lactobacillus confusus
) Bacteremia: a Case Report
Authors: Olano Arrel , Chua Jimmy , Schroeder Suzanne , Minari Afaf , La Salvia Margaret , Hall Gerri
Published: July 2002

Infection with
Lactobacillus
is rare, and only a handful of species have been identified as being clinically significant:
Lactobacillus casei, Lactobacillus rhamnosus
, and
Lactobacillus leichmannii
. The literature contains one case report of bacteremia caused by
Weissella confusa
(basonym:
Lactobacillus confusus
), but the clinical significance of the infection was unclear. We describe a case of
W. confusa
bacteremia in a 46-year-old man with a history of abdominal aortic dissection and repair. This procedure was complicated by gut ischemia, which necessitated massive small bowel resection. He subsequently developed short-bowel syndrome, which required him to have total parenteral nutrition. He later developed an
Enterococcus faecalis
aortic valve endocarditis that required a coronary artery bypass graft and aortic root replacement with homograft and 6 weeks of intravenous ampicillin and gentamicin. Three months prior to his most recent admission, he was diagnosed with
Klebsiella pneumoniae
bacteremia and candidemia. At the present admission, he had fever (
T
max
, 39.5°C) and chills of 2 days' duration and was admitted to the intensive care unit because of hemodynamic instability. Blood cultures grew
K. pneumoniae
and
W. confusa
in four of four blood culture bottles (both aerobe and anaerobe bottles). Imaging studies failed to find any foci of infection. A transesophageal echocardiogram revealed no vegetations. A culture of the patient's Hickman catheter tip was negative. The patient was treated with piperacillin-tazobactam and gentamicin. His condition improved, and he was discharged home, where he completed 4 weeks of piperacillin-tazobactam therapy. Lactobacillemia seldom results in mortality; however, it may be a marker of a serious underlying disease. It is usually seen in patients who have a complex medical history or in patients who receive multiple antibiotics.
Lactobacillus
spp. are generally associated with polymicrobial infections, and when isolated from the blood, they need to be considered possible pathogens. The presence of a vancomycin-resistant, gram-positive coccobacilli on a blood culture should alert clinicians to the possibility of bacteremia caused by
W. confusa
or other small gram-positive rods.
Evaluation of Biosite Triage
Clostridium difficile
Panel for Rapid Detection of
Clostridium difficile
in Stool Samples
Authors: Landry Marie L. , Topal Jeffrey , Ferguson David , Giudetti Darlene , Tang Yajarayma
Published: July 2002

One hundred two stool samples were tested by both the rapid Triage
Clostridium difficile
Panel (Triage Panel) and the cytotoxin cell culture assay. Five samples positive by both the
C. difficile
toxin A (Tox A) and common antigen components of the Triage Panel had cytotoxin titers of ≥10,000. Twenty-three samples were Triage Panel Tox A negative but common antigen positive. Ten of these had cytotoxin titers of 10 to 1,000, but 13 were cytotoxin negative. Bacterial isolates obtained from 8 of these 13 specimens were analyzed for Tox A and B genes by PCR, and only two contained toxigenic bacteria. Thus, the majority of samples positive only for
C. difficile
common antigen contained nontoxigenic bacteria. A Triage Panel Tox A-positive result indicated a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 33.3, 100, 100, and 88.2%, respectively. A Triage Panel common antigen-positive result indicated a sensitivity, specificity, PPV, and NPV of 100, 82.7, 53.6, and 100%, respectively. The high NPV of the Triage Panel common antigen, together with rapid reporting of results, should prove useful in avoiding unnecessary use of contact precautions and antibiotic treatment for
C. difficile
-negative patients. However, with Triage Panel common antigen-positive patients, a sensitive cytotoxin assay should be used to distinguish true cytotoxin-positive patients from
C. difficile
carriers.
Mycobacterium triplex
Infection in a Liver Transplant Patient
Authors: Hoff E. , Sholtis M. , Procop G. , Sabella C. , Goldfarb J. , Wyllie R. , Cunningham R. , Stockman L. , Hall G.
Published: July 2002

Mycobacterium triplex
was first named in 1996 as an acid-fast bacillus with features that most resemble
Mycobacterium simiae
and
Mycobacterium avium-intracellulare
complex but which possesses a distinct mycolic acid pattern as well as a distinctive 16S rRNA hypervariable region. It has been isolated from lymph node, sputum, and cerebrospinal fluid specimens, but to date only rare clinical cases of this organism have been reported in the literature. The following is a case report of
M. triplex
that was isolated from the pericardial and peritoneal fluid of a 13-year-old female liver transplant patient.
Molecular Cloning and Sequencing of the Circumsporozoite Protein Gene fromPlasmodium falciparumStrain FCC-1/HN and Expression of the Gene in Mycobacteria
Authors: Zheng Chunfu , Xie Peimei , Chen Yatang
Published: July 2002

Mycobacterium bovisbacillus Calmette-Guérin (BCG) has been used as a live bacterial vaccine to immunize more than 2 billion people against tuberculosis. In an attempt to use this vaccine strain as a vehicle for protective antigens, thePlasmodium falciparumgene from strain FCC-1/HN encoding circumsporozoite protein (CSP) was amplified from theP. falciparumgenome, sequenced, and expressed inM. bovisBCG under the control of an expression cassette carrying the promoter of heat shock protein 70 (HSP70) fromMycobacterium tuberculosis. The recombinant shuttle plasmid pBCG/CSP was introduced into mycobacteria by electroporation, and the recombinant mycobacteria harboring pBCG/CSP could be induced by heating to express CSP; the molecular mass of recombinant CSP was about 42 kDa. This report of expression of the almost-full-lengthP. falciparumCSP gene in BCG provides scientific evidence for the application of the HSP70 promoter in expressing a foreign gene in BCG and in development of BCG as a multivalent vectoral vaccine for malaria.
Outbreak of Infection with Extended-Spectrum β-Lactamase-Producing
Klebsiella pneumoniae
in a Mexican Hospital
Authors: Silva Jesus , Gatica Rodolfo , Aguilar Cecilia , Becerra Zita , Garza-Ramos Ulises , Velázquez Manuel , Miranda Guadalupe , Leaños Blanca , Solórzano Fortino , Echániz Gabriela
Published: July 2002

Thirty-one strains of
Klebsiella pneumoniae
(including 10 duplicates) from 21 septicemic pediatric patients (age, <2 months) were studied during a 4-month period (June to October 1996) in which the fatality rate was 62% (13 of 21). These isolates identified by the API 20E system yielded the same biotype. Pulsed-field gel electrophoresis experiments revealed the same clone in 31 strains. The isolates were multidrug-resistant but were still susceptible to ciprofloxacin, imipenem, and cefoxitin. A 135-kb plasmid was harbored in all of the isolates. No transconjugants were obtained that were resistant to ampicillin, cefotaxime, tetracycline, or gentamicin. Isoelectric focusing for β-lactamases was performed on all strains, and three bands with pIs of 5.4, 7.6, and 8.2 were obtained. Of these, the pI 8.2 β-lactamase had an extended-spectrum β-lactamase phenotype. PCR amplification of both TEM- and SHV-type genes was obtained. The sequence analysis of the SHV PCR product indicated a mutation corresponding to the SHV-5 β-lactamase.