bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Macrolide-Specific ABC-Type Efflux Transporter in
Escherichia coli
Authors: Kobayashi Nobuyoshi , Nishino Kunihiko , Yamaguchi Akihito 
Published: July 2002

ABSTRACT

In the
Escherichia coli
genome, five putative open reading frame (ORF) clusters,
mdlAB
,
ybjYZ
,
yddA
,
yojHI
, and
yhiH
, have been assumed to be possible genes for ABC drug efflux transporters (I. T. Paulsen, M. K. Sliwinski, and M. H. Saier, Jr., J. Mol. Biol. 277:573–592, 1998). We cloned all of these ORFs in multicopy plasmids and investigated the drug resistance of drug-supersensitive host cells lacking constitutive multidrug efflux transporter genes
acrAB
. Among them, only
ybjYZ
gave significant erythromycin resistance and significantly decreased the accumulation of [
14
C]erythromycin. Therefore,
ybjYZ
was renamed
macAB
(macrolide-specific ABC-type efflux carrier). Plasmids carrying both the
macA
and -
B
genes conferred resistance against macrolides composed of 14- and 15-membered lactones but no or weak resistance against 16-membered ones. Neither of the two genes produced resistance alone. The DNA sequence suggests that MacB is an integral membrane protein with four transmembrane segments and one nucleotide-binding domain, while MacA belongs to a membrane fusion protein (MFP) family with a signal-like sequence at its N terminus. The expression of the histidine-tagged proteins confirmed that MacB is an integral membrane protein and MacA is a peripheral membrane protein. In addition, MacAB required TolC for its function in a way similar to that of most of the MFP-dependent transporters in
E. coli
. MacB is thus a novel ABC-type macrolide efflux transporter which functions by cooperating with the MFP MacA and the multifunctional outer membrane channel TolC. This is the first case of an experimentally identified ABC antibiotic efflux transporter in gram-negative organisms.
Identification of the Structural Gene for the TDP-Fuc4NAc:Lipid II Fuc4NAc Transferase Involved in Synthesis of Enterobacterial Common Antigen in
Escherichia coli
K-12
Authors: Rahman Arifur , Barr Kathleen , Rick Paul D. 
Published: July 2002

ABSTRACT

The polysaccharide chains of enterobacterial common antigen (ECA) are comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy
-d-
galactose, ManNAcA is
N
-acetyl
-d-
mannosaminuronic acid, and GlcNAc is
N
-acetyl
-d-
glucosamine. Individual trisaccharide repeat units are assembled as undecaprenyl-linked intermediates in a sequence of reactions that culminate in the transfer of Fuc4NAc from TDP-Fuc4NAc to ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) to yield Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III), the donor of trisaccharide repeat units for ECA polysaccharide chain elongation. Most of the genes known to be involved in ECA assembly are located in the
wec
gene cluster located at ca. 85.4 min on the
Escherichia coli
chromosome. The available data suggest that the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase also resides in the
wec
gene cluster; however, the location of this gene has not been unequivocally defined. Previous characterization of the nucleotide sequence of the
wec
gene cluster in the region between
o416
and
wecG
revealed that it contained three open reading frames:
o74
,
o204
, and
o450
. In contrast, the results of experiments described in the current investigation revealed that it contains only two open reading frames,
o359
and
o450
. Mutants of
E. coli
possessing null mutations in
o359
were unable to synthesize ECA, and they accumulated lipid II. In addition, the in vitro incorporation of [
3
H]FucNAc from TDP-[
3
H]Fuc4NAc into lipid II was not observed in reaction mixtures using cell extracts obtained from these mutants as a source of enzyme. The ECA-negative phenotype of these mutants was complemented by plasmid constructs containing the wild-type
o359
allele, and Fuc4NAc transferase activity was demonstrated by using cell extracts obtained from the complemented mutants. Furthermore, partially purified
o359
gene product, expressed as recombinant C-terminal His-tagged protein, was able to catalyze the in vitro transfer of [
3
H]Fuc4NAc from TDP-[
3
H]Fuc4NAc to lipid II. Our data support the conclusion that
o359
of the
wec
gene cluster of
E. coli
is the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase involved in the synthesis ECA trisaccharide repeat units.
Identification and Inactivation of Three Group 2 Sigma Factor Genes in
Anabaena
sp. Strain PCC 7120
Authors: Khudyakov Ivan Y. , Golden James W. 
Published: July 2002

ABSTRACT

Three new
Anabaena
sp. strain PCC 7120 genes encoding group 2 alternative sigma factors have been cloned and characterized. Insertional inactivation of
sigD
,
sigE
, and
sigF
genes did not affect growth on nitrate under standard laboratory conditions but did transiently impair the abilities of
sigD
and
sigE
mutant strains to establish diazotrophic growth. A
sigD sigE
double mutant, though proficient in growth on nitrate and still able to differentiate into distinct proheterocysts, was unable to grow diazotrophically due to extensive fragmentation of filaments upon nitrogen deprivation. This double mutant could be complemented by wild-type copies of
sigD
or
sigE
, indicating some degree of functional redundancy that can partially mask phenotypes of single gene mutants. However, the
sigE
gene was required for lysogenic development of the temperate cyanophage A-4L. Several other combinations of double mutations, especially
sigE sigF
, caused a transient defect in establishing diazotrophic growth, manifested as a strong and prolonged bleaching response to nitrogen deprivation. We found no evidence for developmental regulation of the sigma factor genes.
luxAB
reporter fusions with
sigD
,
sigE
, and
sigF
all showed slightly reduced expression after induction of heterocyst development by nitrogen stepdown. Phylogenetic analysis of cyanobacterial group 2 sigma factor sequences revealed that they fall into several subgroups. Three morphologically and physiologically distant strains,
Anabaena
sp. strain PCC 7120,
Synechococcus
sp. strain PCC 7002, and
Synechocystis
sp. strain PCC 6803 each contain representatives of four subgroups. Unlike unicellular strains,
Anabaena
sp. strain PCC 7120 has three additional group 2 sigma factors that cluster in subgroup 2.5b, which is perhaps specific for filamentous or heterocystous cyanobacteria.
ADP-Dependent Phosphofructokinases in Mesophilic and Thermophilic Methanogenic Archaea
Authors: Verhees Corné H. , Tuininga Judith E. , Kengen Servé W. M. , Stams Alfons J. M. , van der Oost John , de Vos Willem M. 
Published: July 2002

ABSTRACT

Phosphofructokinase (PFK) is a key enzyme of the glycolytic pathway in all domains of life. Two related PFKs, ATP-dependent and PP
i
-dependent PFK, have been distinguished in bacteria and eucarya, as well as in some archaea. Hyperthermophilic archaea of the order
Thermococcales
, including
Pyrococcus
and
Thermococcus
spp., have recently been demonstrated to possess a unique ADP-dependent PFK (ADP-PFK) that appears to be phylogenetically distinct. Here, we report the presence of ADP-PFKs in glycogen-producing members of the orders
Methanococcales
and
Methanosarcinales
, including both mesophilic and thermophilic representatives. To verify the substrate specificities of the methanogenic kinases, the gene encoding the ADP-PFK from
Methanococcus jannaschii
was functionally expressed in
Escherichia coli
, and the produced enzyme was purified and characterized in detail. Compared to its counterparts from the two members of the order
Thermococcales
, the
M. jannaschii
ADP-PFK has an extremely low

K
m

for fructose 6-phosphate (9.6 μM), and it accepts both ADP and acetyl-phosphate as phosphoryl donors. Phylogenetic analysis of the ADP-PFK reveals it to be a key enzyme of the modified Embden-Meyerhof pathway of heterotrophic and chemolithoautotrophic archaea. Interestingly, uncharacterized homologs of this unusual kinase are present in several eucarya.
RNA Arbitrarily Primed PCR Survey of Genes Regulated by ToxR in the Deep-Sea Bacterium
Photobacterium profundum
Strain SS9
Authors: Bidle Kelly A. , Bartlett Douglas H. 
Published: July 2002

ABSTRACT

We are currently investigating the role of ToxR-mediated gene regulation in
Photobacterium profundum
strain SS9. SS9 is a moderately piezophilic (“pressure loving”) psychrotolerant marine bacterium belonging to the family
Vibrionaceae
. In
Vibrio cholerae
, ToxR is a transmembrane DNA binding protein involved in mediating virulence gene expression in response to various environmental signals. A homolog to
V. cholerae
ToxR that is necessary for pressure-responsive gene expression of two outer membrane protein-encoding genes was previously found in SS9. To search for additional genes regulated by ToxR in SS9, we have used RNA arbitrarily primed PCR (RAP-PCR) with wild-type and
toxR
mutant strains of SS9. Seven ToxR-activated transcripts and one ToxR-repressed transcript were identified in this analysis. The cDNAs corresponding to these partial transcripts were cloned and sequenced, and ToxR regulation of their genes was verified. The products of these genes are all predicted to fall into one or both of two functional categories, those whose products alter membrane structure and/or those that are part of a starvation response. The transcript levels of all eight newly identified genes were also characterized as a function of hydrostatic pressure. Various patterns of pressure regulation were observed, indicating that ToxR activation or repression cannot be used to predict the influence of pressure on gene expression in SS9. These results provide further information on the nature of the ToxR regulon in SS9 and indicate that RAP-PCR is a useful approach for the discovery of new genes under the control of global regulatory transcription factors.
Novel Lysophospholipase A Secreted by
Legionella pneumophila
Authors: Flieger Antje , Gong Shimei , Faigle Marion , Stevanovic Stefan , Cianciotto Nicholas P. , Neumeister Birgid 
Published: July 2002

ABSTRACT

We show that
Legionella pneumophila
possesses lysophospholipase A activity, which releases fatty acids from lysophosphatidylcholine. The NH
2
-terminal sequence of the enzyme contained FGDSLS, corresponding to a catalytic domain in a recently described group of lipolytic enzymes. Culture supernatants of a
L. pneumophila pilD
mutant lost the ability to cleave lysophosphatidylcholine.
Pseudomonas stutzeri
Has Two Closely Related
pilA
Genes (Type IV Pilus Structural Protein) with Opposite Influences on Natural Genetic Transformation
Authors: Graupner Stefan , Wackernagel Wilfried 
Published: July 2002

ABSTRACT

Pseudomonas stutzeri
has type IV pili for which the
pilA
gene (here termed
pilAI
) provides the structural protein and which are required for DNA uptake and natural genetic transformation. Downstream of
pilAI
we identified a gene, termed
pilAII
, coding for a deduced protein with a size similar to that of PilAI with 55% amino acid sequence identity and with a typical leader peptide including a leader peptidase cleavage site. Fusions to
lacZ
revealed that
pilAII
is expressed only about 10% compared to
pilAI,
although the genes are cotranscribed as shown by reverse transcription-PCR. Surprisingly, insertional inactivation of
pilAII
produced a hypertransformation phenotype giving about 16-fold-increased transformation frequencies. Hypertransformation also occurred in
pilAI pilAII
double mutants expressing heterologous
pilA
genes of nontransformable bacteria, like
Pseudomonas aeruginosa
or
Dichelobacter nodosus
. The overexpression of
pilAII
decreased transformation up to 5,000-fold compared to that of the
pilAII
mutant. However, neither inactivation of
pilAII
nor its overexpression affected the amounts of [
3
H]thymidine-labeled DNA that were competence-specifically bound and taken up by the cells. In the
pilAII
mutant, the transformation by purified single-stranded DNA (which depends on
comA
and
exbB,
as does transformation by duplex DNA) was also increased 17-fold. It is concluded that PilAII suppresses a step in transformation after the uptake of duplex DNA into the cell and perhaps before its translocation into the cytoplasm. The idea that the degree of the transformability of cells could be permanently adjusted by the expression level of an antagonistic protein is discussed.
Reciprocal Regulation of Anaerobic and Aerobic Cell Wall Mannoprotein Gene Expression in
Saccharomyces cerevisiae
Authors: Abramova Natalia , Sertil Odeniel , Mehta Sapna , Lowry Charles V. 
Published: July 2002

ABSTRACT

The
DAN/TIR
genes encode nine cell wall mannoproteins in
Saccharomyces cerevisiae
which are expressed during anaerobiosis (
DAN1
,
DAN2
,
DAN3
,
DAN4
,
TIR1
,
TIR2
,
TIR3
,
TIR4
, and
TIP1
). Most are expressed within an hour of an anaerobic shift, but
DAN2
and
DAN3
are expressed after about 3 h. At the same time,
CWP1
and
CWP2
, the genes encoding the major mannoproteins, are down-regulated, suggesting that there is a programmed remodeling of the cell wall in which Cwp1 and Cwp2 are replaced by nine anaerobic counterparts.
TIP1
,
TIR1
,
TIR2
, and
TIR4
are also induced during cold shock. Correspondingly,
CWP1
is down-regulated during cold shock. As reported elsewhere, Mox4 is a heme-inhibited activator, and Mot3 is a heme-induced repressor of the
DAN/TIR
genes (but not of
TIP1
). We show that
CWP2
(but not
CWP1
) is controlled by the same factors, but in reverse fashion—primarily by Mot3 (which can function as either an activator or repressor) but also by Mox4, accounting for the reciprocal regulation of the two groups of genes. Disruptions of
TIR1
,
TIR3
, or
TIR4
prevent anaerobic growth, indicating that each protein is essential for anaerobic adaptation. The Dan/Tir and Cwp proteins are homologous, with the greatest similarities shown within three subgroups: the Dan proteins, the Tip and Tir proteins, and, more distantly, the Cwp proteins. The clustering of homology corresponds to differences in expression: the Tip and Tir proteins are expressed during hypoxia and cold shock, the Dan proteins are more stringently repressed by oxygen and insensitive to cold shock, and the Cwp proteins are oppositely regulated by oxygen and temperature.
Competence Regulation by Oxygen Availability and by Nox Is Not Related to Specific Adjustment of Central Metabolism in
Streptococcus pneumoniae
Authors: Chapuy-Regaud Sabine , Duthoit Frédérique , Malfroy-Mastrorillo Laurence , Gourdon Pierre , Lindley Nic D. , Trombe Marie-Claude 
Published: July 2002

ABSTRACT

In
Streptococcus pneumoniae
oxygen availability is a major determinant for competence development in exponentially growing cultures. NADH oxidase activity is required for optimal competence in cultures grown aerobically. The implication of oxidative metabolism and more specifically of Nox on central metabolism has been examined. Glycolytic flux throughout exponential growth revealed homolactic fermentation with a lactate production/glucose utilization ratio close to 2, whatever the aerobiosis level of the culture. Loss-of-function mutations in
nox
, which encodes NADH oxidase, did not change this trait. Consistently, mRNA levels of glyceraldehyde-3-phosphate dehydrogenase,
l
-lactate dehydrogenase, pyruvate oxidase, and NADH oxidase remained comparable to wild-type levels, as did the specific activities of key enzymes which control central metabolism. Competence regulation by oxygen involving the NADH oxidase activity is not due to significant modification of carbon flux through glycolysis. Failure to obtain loss-of-function mutation in L-
ldh
, which encodes the
l
-lactate dehydrogenase, indicates its essential role in pneumococci whatever their growth status.
Characterization of Two CrypticHelicobacter pyloriPlasmids: a Putative Source for Horizontal Gene Transfer and Gene Shuffling
Authors: Hofreuter Dirk , Haas Rainer 
Published: July 2002

ABSTRACTManyHelicobacter pyloriisolates carry cryptic plasmids of extremely variable size. In this study we analyzed twoH. pyloriplasmids, pHel4 and pHel5, fromH. pyloristrains P8 and P29, respectively. Plasmid pHel4 consists of 10,970 bp, constituting 15 putative open reading frames (ORFs), whereas pHel5 consists of 18,291 bp, constituting 17 ORFs. The findings that both plasmids encode a conserved RepA protein and that both have an origin of replication containing an iteron place them in the group of theta plasmids. In pHel4, the products of the overlappingorf4C,orf4D,orf4E, andorf4Fsequences are homologous to MobA, MobB, MobC, and MobD, encoded by colicinogenic plasmids, suggesting that pHel4 might be mobilizable. A further putative operon consists oforf4Bandorf4A, the products of which are homologous to microcin C7 (MccC7) biosynthesis and secretion proteins MccB and MccC, respectively. Plasmid pHel5 carries putative genes encoding proteins with homology to an endonuclease and gene products of anH. pylorichromosomal plasticity zone. Both plasmids contain repeat sequences, such as the previously identified R2 repeat, which are considered preferred recombination sites. In pHel4, a new repeat sequence (R4 repeat), which seems to act as a hot spot for site-specific recombination, was identified. AllH. pyloriplasmids characterized so far have a modular structure. We suggest a model that explains the existing plasmids by insertions and deletions of genetic elements at the repeat sequences. A genetic exchange between plasmids and the bacterial chromosome, combined with plasmid mobilization, might add a novel mechanism to explain the high genetic macrodiversity within theH. pyloripopulation.
Oxygen-Mediated Regulation of Porphobilinogen Formation inRhodobacter capsulatus
Authors: Biel Alan J. , Canada Keith , Huang David , Indest Karl , Sullivan Karen 
Published: July 2002

ABSTRACTARhodobacter capsulatus hemCmutant has been isolated and used to show that oxygen regulates the intracellular levels of porphobilinogen. Experiments using ahemB-catgene fusion demonstrated that oxygen does not transcriptionally regulatehemBtranscription. Porphobilinogen synthase activity is not regulated by oxygen nor is the enzyme feedback inhibited by hemin or protoporphyrin IX. It was demonstrated that less than 20% of [14C]aminolevulinate was incorporated into bacteriochlorophyll, suggesting that the majority of the aminolevulinate is diverted from the common tetrapyrrole pathway. Porphobilinogen oxygenase activity was not observed in this organism; however, an NADPH-linked aminolevulinate dehydrogenase activity was demonstrated. The specific activity of this enzyme increased with increasing oxygen tension. The results presented here suggest that carbon flow over the common tetrapyrrole pathway is regulated by a combination of feedback inhibition of aminolevulinate synthase and diversion of aminolevulinate from the pathway by aminolevulinate dehydrogenase.
Application of Virus-Specific Immunoglobulin M (IgM), IgG, and IgA Antibody Detection with a Polyantigenic Enzyme-Linked Immunosorbent Assay for Diagnosis of Epstein-Barr Virus Infections in Childhood
Authors: Schaade Lars , Kleines Michael , Häusler Martin 
Published: July 2002

ABSTRACT
The Enzygnost anti-Epstein-Barr virus enzyme-linked immunosorbent assay (ELISA) system, which is based on a defined antigen mixture and on detection of antibodies of the immunoglobulin G (IgG), IgM, and IgA classes, was evaluated for its reliability in diagnosing Epstein-Barr virus infections in childhood. With samples from 66 children, the Epstein-Barr virus status and the infection phase were defined by indirect immunofluorescence and anticomplement fluorescence assays: 11 children were seronegative, 8 had a primary infection, 20 had a recent primary or past infection, and in 27 a reactivated Epstein-Barr virus infection was diagnosed. When applying the Enzygnost ELISAs, 15 serum samples (22.7%) were not interpretable due to indeterminate results in at least one of the assays used and were therefore excluded from further evaluation. The respective sensitivities and specificities for the diagnosis of seronegativity were 100 and 100%, those for the diagnosis of primary infection were 100 and 97%, those for the diagnosis of recent primary or past infection were 100 and 52%, and those for the diagnosis of reactivated infection were 10 and 100%. This poor performance of the Enzygnost system with reactivated infections is due to the prerequisite of an IgG antibody value of >650 IU/ml for the diagnosis of viral activity, which was fulfilled in only two of the children. Despite the high rate of indeterminate results, the Enzygnost system is useful in diagnosing acute and past Epstein-Barr virus infection in childhood. For serological diagnosis of viral activity in childhood, a supplementary assay is necessary.
Comparison of Various Sample Preparation Methods for PCR Diagnosis of Visceral Leishmaniasis Using Peripheral Blood
Authors: Lachaud Laurence , Chabbert Elisabeth , Dubessay Pascal , Reynes Jacques , Lamothe Jacques , Bastien Patrick 
Published: July 2002

ABSTRACT
We have compared various sample preparation methods for the PCR diagnosis of visceral leishmaniasis (VL) using peripheral blood samples and tested the influence of these protocols upon sensitivity. Four methods of lysis-DNA extraction were used with two types of blood samples: whole blood (WB) and buffy coat (BC). Comparisons were first carried out with seeded samples at various parasite concentrations. At high concentrations (≥1,000 parasites/ml), there were no significant differences in PCR sensitivity among the methods tested. At concentrations of ≤100 parasites/ml, proteinase K (PK)-based methods proved clearly superior to guanidine-EDTA-based methods. Moreover, a 10-fold increase in sensitivity was observed for BC over that for WB. Thus, the best sensitivity was obtained with the BC prepared with PK-based methods. With this combination, the PCR reliably detected 10 parasites/ml but was inconsistent when the sample contained 1 parasite/ml of blood. The methods that yielded the highest sensitivities were evaluated with seven dogs and four human VL patients. Again, the utilization of the BC prepared with PK-based methods gave the best results. The optimization of each step of the assay (sample preparation, DNA extraction, and PCR conditions) yielded a highly sensitive tool for the diagnosis of VL using patient blood, thus avoiding more invasive diagnostic procedures and allowing the detection of low parasitemia during posttherapeutic follow-up.
Inoculum Standardization for Antifungal Susceptibility Testing of Filamentous Fungi Pathogenic for Humans
Authors: Petrikkou Eva , Rodrı́guez-Tudela Juan L. , Cuenca-Estrella Manuel , Gómez Alicia , Molleja Ana , Mellado Emilia 
Published: July 2002

ABSTRACT
Two methods of inoculum preparation for filamentous fungi were compared: counting with a hematocytometer and spectrophotometric adjustment. One hundred eighty-two filamentous fungi pathogenic for humans were used. Colony counts were done for all inoculum preparations. The agreement between the hematocytometer counts and the colony counts (CFU per milliliter) was 97.2%. The reproducibility between the hematocytometer counts and the colony counts by means of an intraclass correlation coefficient was 0.70. Pearson's correlation index for hematocytometer counts versus colony counts was 0.56, whereas that for optical density versus colony counts was 0.008. Both methods can be used for inoculum size adjustment. However, the use of the spectrophotometric method requires that each species be standardized separately.