bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional and Mutational Analysis of P19, a DNA Transfer Protein with Muramidase Activity
Authors: Bayer Michaela , Iberer Robert , Bischof Karin , Rassi Edith , Stabentheiner Edith , Zellnig Günther , Koraimann Günther 
Published: July 2002

ABSTRACT

Protein P19 encoded by the conjugative resistance plasmid R1 has been identified as being one member of a large family of muramidases encoded by bacteriophages and by type III and type IV secretion systems. We carried out a mutational analysis to investigate the function of protein P19 and used in vivo complementation assays to test those of several P19 mutants. The results indicated that conserved residues present in the presumed catalytic center of P19 are absolutely essential for its function in conjugation of plasmid R1 and infection by the RNA phage R17. Overexpression of protein P19 in an early growth phase resulted in a massive lysis of
Escherichia coli
cells in liquid culture, as indicated by a rapid and distinct decrease in cell culture densities after induction. Change of the proposed catalytic glutamate at position 44 to glutamine completely abolished this effect. P19-induced cell lysis was directly shown by transmission and scanning electron microscopy. Typically, P19-overexpressing cells showed bulges protruding from the cell surfaces. Our interpretation is that these protrusions arose from a localized and spatially confined disruption of the bacterial cell wall. To our knowledge such an effect has not previously been documented for any member of the lytic transglycosylase family. From the data presented here, we conclude that protein P19 possesses the proposed localized peptidoglycan-hydrolyzing activity. This activity would be a prerequisite for efficient penetration of the cell envelope by the DNA translocation complex encoded by the conjugative plasmid.
Characterization of VPI Pathogenicity Island and CTXφ Prophage in Environmental Strains of
Vibrio cholerae
Authors: Mukhopadhyay Asish K. , Chakraborty Soumen , Takeda Yoshifumi , Nair G. Balakrish , Berg Douglas E. 
Published: July 2002

ABSTRACT

Environmental isolates of
Vibrio cholerae
of eight randomly amplified polymorphic DNA (RAPD) fingerprint types from Calcutta, India, that were unusual in containing toxin-coregulated pilus or cholera toxin genes but not O1 or O139 antigens of epidemic strains were studied by PCR and sequencing to gain insights into
V. cholerae
evolution. We found that each isolate contained a variant form of the VPI pathogenicity island. Distinguishing features included (i) four new alleles of
tcpF
(which encodes secreted virulence protein; its exact function is unknown), 20 to 70% divergent (at the protein level) from each other and canonical
tcpF
; (ii) a new allele of
toxT
(virulence regulatory gene), 36% divergent (at the protein level) in its 5′ half and nearly identical in its 3′ half to canonical
toxT
; (iii) a new
tcpA
(pilin) gene; and (iv) four variant forms of a regulatory sequence upstream of
toxT
. Also found were transpositions of an IS
903
-related element and function-unknown genes to sites in VPI. Cholera toxin (
ctx
) genes were found in isolates of two RAPD types, in each case embedded in CTXφ-like prophages. Fragments that are inferred to contain only putative repressor, replication, and integration genes were present in two other RAPD types. New possible prophage repressor and replication genes were also identified. Our results show marked genetic diversity in the virulence-associated gene clusters found in some nonepidemic
V. cholerae
strains, suggest that some of these genes contribute to fitness in nature, and emphasize the potential importance of interstrain gene exchange in the evolution of this species.
Residue Aspartate-147 from the Third Transmembrane Region of Na
+
/H
+
Antiporter NhaB of
Vibrio alginolyticus
Plays a Role in Its Activity
Authors: Nakamura Tatsunosuke , Fujisaki Yumiko , Enomoto Hiromi , Nakayama Yuji , Takabe Teruhiro , Yamaguchi Naoto , Uozumi Nobuyuki 
Published: July 2002

ABSTRACT

NhaB is a bacterial Na
+
/H
+
antiporter with unique topology. The pH dependence of NhaB from
Vibrio alginolyticus
differs from that of the
Escherichia coli
NhaB homolog. Replacement of Asp-147 with Glu made high H
+
concentrations a requirement for the NhaB activity. Replacement of Asp-147 with neutral amino acids inactivated NhaB.
Differential Effects of ReplacingEscherichiacoliRibosomal Protein L27 with Its Homologue fromAquifexaeolicus
Authors: Maguire Bruce A. , Manuilov Anton V. , Zimmermann Robert A. 
Published: July 2002

ABSTRACTTherpmAgene, which encodes 50S ribosomal subunit protein L27, was cloned from the extreme thermophileAquifexaeolicus, and the protein was overexpressed and purified. Comparison of theA.aeolicusprotein with its homologue fromEscherichiacoliby circular dichroism analysis and proton nuclear magnetic resonance spectroscopy showed that it readily adopts some structure in solution that is very stable, whereas theE.coliprotein is unstructured under the same conditions. A mutant ofE.colithat lacks L27 was found earlier to be impaired in the assembly and function of the 50S subunit; both defects could be corrected by expression ofE.coliL27 from an extrachromosomal copy of therpmAgene. WhenA.aeolicusL27 was expressed in the same mutant, an increase in the growth rate occurred and the “foreign” L27 protein was incorporated intoE.coliribosomes. However, the presence ofA.aeolicusL27 did not promote 50S subunit assembly. Thus, while theA.aeolicusprotein can apparently replace itsE.colihomologue functionally in completed ribosomes, it does not assist in the assembly ofE.coliribosomes that otherwise lack L27. Possible explanations for this paradoxical behavior are discussed.
Characterization ofPneumocystis carinii PHR1, a pH-Regulated Gene Important for Cell Wall Integrity
Authors: Kottom Theodore J. , Thomas Charles F. , Limper Andrew H. 
Published: July 2002

ABSTRACTPneumocystis cariniiremains an important opportunistic fungal pathogen causing life-threatening pneumonia in patients with AIDS and malignancy. Currently, little is known about how the organism adapts to environmental stresses and maintains its cellular integrity. We recently discovered an open reading frame approximately 600 bp downstream of the region codingGSC-1, a gene mediating β-glucan cell wall synthesis inP. carinii. The predicted amino acid sequence of this new gene, termedP. carinii PHR1,exhibited 38% homology toSaccharomyces cerevisiae GAS1, a glycosylphosphatidylinositol-anchored protein essential to maintaining cell wall integrity, and 37% homology toCandida albicans PHR1/PHR2, pH-responsive genes encoding proteins recently implicated in cross-linking β-1,3- and β-1,6-glucans. In view of its homology to these related fungal genes, the pH-dependent expression ofP. carinii PHR1was examined. As inC. albicans,P. carinii PHR1expression was repressed under acidic conditions but induced at neutral and more alkaline pH.PHR1-related proteins have been implicated in glucan cell wall stability under various environmental conditions. Although difficulties withP. cariniiculture and transformation have traditionally limited assessment of gene function in the organism itself, we have successfully used heterologous expression ofP. cariniigenes in related fungi to address functional correlates ofP. carinii-encoded proteins. Therefore, the potential role ofP. carinii PHR1in cell wall integrity was examined by assessing its ability to rescue anS. cerevisiae gas1mutant with absent endogenous Phr1p-like activity. Interestingly,P. carinii PHR1DNA successfully restored proliferation ofS. cerevisiae gas1mutants under lethal conditions of cell wall stress. These results indicate thatP. carinii PHR1encodes a protein responsive to environmental pH and capable of mediating fungal cell wall integrity.
Characterization of
Pseudomonas aeruginosa
Chitinase, a Gradually Secreted Protein
Authors: Folders Jindra , Algra Jon , Roelofs Marc S. , van Loon Leendert C. , Tommassen Jan , Bitter Wilbert 
Published: July 2002

ABSTRACT

The gram-negative bacterium
Pseudomonas aeruginosa
secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene,
chiC
, coding for an extracellular chitinolytic enzyme, was identified. The
chiC
gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of
Serratia marcescens
, ChiA of
Vibrio harveyi,
and ChiD of
Bacillus circulans
and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and
p
-nitrophenyl-β-
d
-
N
,
N
′,
N
"-triacetylchitotriose, but not on
p
-nitrophenyl-β-
d
-
N
-acetylglucosamine, indicating that it is an endochitinase. Expression of the
chiC
gene appears to be regulated by the quorum-sensing system of
P. aeruginosa
, since this gene was not expressed in a
lasIR vsmI
mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.
Overexpression of the Response Regulator
evgA
of the Two-Component Signal Transduction System Modulates Multidrug Resistance Conferred by Multidrug Resistance Transporters
Authors: Nishino Kunihiko , Yamaguchi Akihito 
Published: July 2002

ABSTRACT

Overexpression of
evgA
, a response regulator of a two-component system, increased multidrug efflux in
Escherichia coli
. Since overexpression of the
emrKY
operon, which is controlled by
evgAS
, could account only for deoxycholate resistance, the
evgAS
locus apparently controls expression of at least one other multidrug efflux operon.
The −10 Region Is a Key Promoter Specificity Determinant for the
Bacillus subtilis
Extracytoplasmic-Function ς Factors ς
X
and ς
W
Authors: Qiu Jian , Helmann John D. 
Published: July 2002

ABSTRACT

Transcriptional selectivity derives, in large part, from the sequence-specific DNA-binding properties of the ς subunit of RNA polymerase. There are 17 ς factors in
Bacillus subtilis
which, in general, recognize distinct sets of promoters. However, some ς factors have overlapping promoter selectivity. We hypothesize that the overlap between the regulons activated by the ς
X
and ς
W
factors can be explained by overlapping specificity for the −10 region: ς
X
recognizes −10 elements with the sequence CGAC and ς
W
recognizes CGTA, while both can potentially recognize CGTC. To test this model, we mutated the ς
X
-specific autoregulatory site (P
X
), containing the −10 element CGAC, to either CGTC or GCTA. Conversely, the ς
W
autoregulatory site (P
W
) was altered from CGTA to CGTC or CGAC. Transcriptional analyses, both in vitro and in vivo, indicate that changes to the −10 element are sufficient to switch a promoter from the ς
X
to the ς
W
regulon or, conversely, from the ς
W
to the ς
X
regulon, but context effects clearly play an important role in determining promoter strength. It seems likely that these subtle differences in promoter selectivity derive from amino acid differences in conserved region 2 of ς, which contacts the −10 element. However, we were unable to alter promoter selectivity by replacements of two candidate recognition residues in ς
W
.
Requirement for NusG for Transcription Antitermination In Vivo by the λ N Protein
Authors: Zhou Ying , Filter Joshua J. , Court Donald L. , Gottesman Max E. , Friedman David I. 
Published: July 2002

ABSTRACT

Transcription antitermination by the bacteriophage λ N protein is stimulated in vitro by the
Escherichia coli
NusG protein. Earlier work suggested that NusG was not required for N activity in vivo. Here we present evidence that NusG also stimulates N-mediated transcription antitermination in intact cells.
TonB Interacts with Nonreceptor Proteins in the Outer Membrane ofEscherichia coli
Authors: Higgs Penelope I. , Letain Tracy E. , Merriam Kelley K. , Burke Neal S. , Park HaJeung , Kang ChulHee , Postle Kathleen 
Published: July 2002

ABSTRACTTheEscherichia coliTonB protein serves to couple the cytoplasmic membrane proton motive force to active transport of iron-siderophore complexes and vitamin B12across the outer membrane. Consistent with this role, TonB has been demonstrated to participate in strong interactions with both the cytoplasmic and outer membranes. The cytoplasmic membrane determinants for that interaction have been previously characterized in some detail. Here we begin to examine the nature of TonB interactions with the outer membrane. Although the presence of the siderophore enterochelin (also known as enterobactin) greatly enhanced detectable cross-linking between TonB and the outer membrane receptor, FepA, the absence of enterochelin did not prevent the localization of TonB to the outer membrane. Furthermore, the absence of FepA or indeed of all the iron-responsive outer membrane receptors did not alter this association of TonB with the outer membrane. This suggested that TonB interactions with the outer membrane were not limited to the TonB-dependent outer membrane receptors. Hydrolysis of the murein layer with lysozyme did not alter the distribution of TonB, suggesting that peptidoglycan was not responsible for the outer membrane association of TonB. Conversely, the interaction of TonB with the outer membrane was disrupted by the addition of 4 M NaCl, suggesting that these interactions were proteinaceous. Subsequently, two additional contacts of TonB with the outer membrane proteins Lpp and, putatively, OmpA were identified by in vivo cross-linking. These contacts corresponded to the 43-kDa and part of the 77-kDa TonB-specific complexes described previously. Surprisingly, mutations in these proteins individually did not appear to affect TonB phenotypes. These results suggest that there may be multiple redundant sites where TonB can interact with the outer membrane prior to transducing energy to the outer membrane receptors.
Selenium Is Mobilized In Vivo from Free Selenocysteine and Is Incorporated Specifically into Formate Dehydrogenase H and tRNA Nucleosides
Authors: Lacourciere Gerard M. 
Published: July 2002

ABSTRACT

Selenophosphate synthetase (SPS), the
selD
gene product from
Escherichia coli
, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. It was recently demonstrated that selenium delivered from selenocysteine by an
E. coli
NifS-like protein could replace free selenide in the in vitro SPS assay for selenophosphate formation (G. M. Lacourciere, H. Mihara, T. Kurihara, N. Esaki, and T. C. Stadtman, J. Biol. Chem. 275:23769-23773, 2000). During growth of
E. coli
in the presence of 0.1 μM
75
SeO
3
2−
and increasing amounts of
l
-selenocysteine, a concomitant decrease in
75
Se incorporation into formate dehydrogenase H and nucleosides of bulk tRNA was observed. This is consistent with the mobilization of selenium from
l
-selenocysteine in vivo and its use in selenophosphate formation. The ability of
E. coli
to utilize selenocysteine as a selenium source for selenophosphate biosynthesis in vivo supports the participation of the NifS-like proteins in selenium metabolism.
Characterization of Newly Emerging Newcastle Disease Virus Isolates from the People's Republic of China and Taiwan
Authors: Yu Li , Wang Zhiliang , Jiang Yihai , Chang Leo , Kwang Jimmy 
Published: July 2002

ABSTRACT
Seven Newcastle disease (ND) virus (NDV) isolates which were recovered from ND outbreaks in chicken and pigeon flocks in China and Taiwan between 1996 and 2000 were genotypically and pathotypically characterized. By phylogenetic analysis of the fusion protein genes, isolates Ch-A7/96, Ch/98-3, Ch/99, Ch/2000, and TW/2000 were placed into two novel subgenotypes, VIIc and VIId. Isolate Ch/98-1 was grouped into subgenotype VIb, while Ch-W6/96 was proven to be a mixture of isolates Ch-A7/96 and Ch/98-1. These isolates were pathotyped as viscerotropic velogenic for Ch/98-3, Ch/99, Ch/2000, and TW/2000; neurotropic velogenic for Ch-A7/96; and mesogenic for Ch/98-1. Three separate, comparative, genetic analyses of the F genes, including genetic distance measurement, phylogenetic tree analysis, and residue substitution analysis, were performed with our isolates and selected NDV strains from GenBank. Results showed that the close genetic similarity provided evidence for the epidemiological linkage between the outbreaks in China and Taiwan and that the 1990s outbreaks in Asia, the Middle East, Africa, and Europe constituted the fourth panzootic of ND. In combination with epidemiological analysis, an evolutionary model of the NDV strains, representative of the direction of transmission within the NDV strains, was proposed, and epidemiology of NDV transmission was evaluated with emphasis on molecular aspects. Finally, a cross-protective experiment indicated that at least one strain (Ch-A7/96) among our NDV isolates was an antigenic variant, responsible for recent outbreaks of ND in vaccinated chicken flocks.
Use of Random Amplified Polymorphic DNA PCR To Examine Epidemiology of
Stenotrophomonas maltophilia
and
Achromobacter
(
Alcaligenes
)
xylosoxidans
from Patients with Cystic Fibrosis
Authors: Krzewinski Jay W. , Nguyen Chau D. , Foster Jessica M. , Burns Jane L. 
Published: July 2002

ABSTRACT

Stenotrophomonas maltophilia
and
Achromobacter
(
Alcaligenes
)
xylosoxidans
have been increasingly recognized as a cause of respiratory tract colonization in cystic fibrosis (CF). Although both organisms have been associated with progressive deterioration of pulmonary function, demonstration of causality is lacking. To examine the molecular epidemiology of
S. maltophilia
and
A. xylosoxidans
in CF, isolates from patients monitored for up to 2 years were fingerprinted using a PCR-based randomly amplified polymorphic DNA (RAPD-PCR) method. Sixty-one of 69 CF centers screened had 183
S. maltophilia
culture-positive patients, and 46 centers had 92
A. xylosoxidans
-positive patients. At least one isolate from each patient was genotyped, and patients with ≥10 positive cultures (12 S
. maltophilia
cultures, 15
A. xylosoxidans
cultures) had serial isolates genotyped. In addition, centers with multiple culture-positive patients were examined for evidence of shared clones. There were no instances of shared genotypes among different CF centers. Some patients demonstrated isolates with a single genotype throughout the observation period, and others had intervening or sequential genotypes. At the six centers with multiple
S. maltophilia
culture-positive patients and the seven centers with multiple
A. xylosoxidans
-positive patients, there were three and five instances of shared genotypes, respectively. The majority of shared isolates were from pairs who were siblings or otherwise epidemiologically linked. These findings suggest RAPD-PCR typing can distinguish unique CF isolates of
S. maltophilia
and
A. xylosoxidans
, person-to-person transmission may occur, there are not a small number of clones infecting CF airways, and patients with long-term colonization may either have a persistent organism or may acquire additional organisms over time.
Evaluation of the Hexaplex Assay for Detection of Respiratory Viruses in Children
Authors: Kehl Sue C. , Henrickson Kelly J. , Hua Weimin , Fan Jiang 
Published: July 2002

ABSTRACT
The Hexaplex assay (Prodesse, Inc., Milwaukee, Wis.) is a multiplex reverse transcriptase (RT)-PCR assay for the detection of parainfluenza virus types 1, 2, and 3, respiratory syncytial virus (RSV) types A and B, and influenza virus types A and B. We evaluated the Hexaplex assay in comparison with conventional viral cell cultures and rapid enzyme immunoassays (EIAs) for RSV (Directigen; Becton Dickinson Inc., Cockeysville, Md.) and influenza A virus (Abbott Test Pack; Abbott Laboratories, Abbott Park, Ill.) for the detection of respiratory viruses from pediatric respiratory specimens obtained from children seen at Children's Hospital of Wisconsin from December 1997 through May 1998. A total of 363 respiratory specimens were evaluated. The tissue culture prevalence of parainfluenza virus during this period of time was low (1.1%). The sensitivity, specificity, and positive and negative predictive value of Hexaplex compared to tissue culture for the detection of parainfluenza virus were 100, 95.8, 19.0, and 100%, respectively. Only one specimen was determined to contain influenza B virus by Hexaplex; it was tissue culture negative. A specimen was considered to contain RSV or influenza A virus when it was either culture positive or culture negative but Hexaplex and EIA positive. Prior to the analysis of discrepant results, the sensitivity, specificity, and positive and negative predictive value for the detection of RSV were 91.2, 100, 100, and 98.0%, respectively, for tissue culture; 84.5, 100, 100, and 96.6% for EIA; and 98.5, 91.5, 72.8, and 99.6% for Hexaplex, respectively. The sensitivity, specificity, and positive and negative predictive value for the detection of influenza A virus prior to the analysis of discrepant results were 100, 100, 100, and 100%, respectively, for culture, 78.0, 100, 100, and 89.4% for EIA, respectively, and 95.1, 94.1, 67.2, and 99.3% for Hexaplex, respectively. Culture- and/or EIA-negative, Hexaplex-positive specimens were analyzed by a second RT-PCR assay which used primers specific for a different genomic region than that used in the Hexaplex assay. After analysis of these discrepant results, the sensitivity, specificity, and positive and negative predictive value for the detection of RSV were 74.3, 100, 100, and 93.5%, respectively, for tissue culture; 70.3, 100, 100, and 92.5% for EIA; and 98.6, 97.4, 91.2, and 99.6% for Hexaplex. The sensitivity, specificity, and positive and negative predictive value for the detection of influenza A virus were 83.3, 100, 100, and 97.4%, respectively, for tissue culture; 69.4, 100, 100, and 83.3% for EIA; and 95.8, 98.7, 92.0, and 99.3% for Hexaplex. Hexaplex is a rapid, sensitive, and specific method for the detection of the seven most common respiratory viruses in children.
Real-Time PCR Assay Targeting IS
481
of
Bordetella pertussis
and Molecular Basis for Detecting
Bordetella holmesii
Authors: Reischl Udo , Lehn Norbert , Sanden Gary N. , Loeffelholz Mike J. 
Published: July 2002

ABSTRACT

Detection of
Bordetella holmesii
by a real-time PCR assay targeting IS
481
of
Bordetella pertussis
is reported. Sequencing of IS
481
-specific PCR products from
B. pertussis
and
B. holmesii
isolates revealed sequence homology. Restriction fragment length polymorphism demonstrated a low copy number of IS
481
-like sequences in
B. holmesii
. These results, and culture of
B. holmesii
from patients with cough, suggest that the specificity and predictive value of IS
481
-based PCR assays for pertussis may be compromised.