bluuebunny/crossref_metadata_2025_split huggingface dataset:
Complementation of a
Treponema denticola flgE
Mutant with a Novel Coumermycin A1-Resistant
T. denticola
Shuttle Vector System
Authors: Chi Bo , Limberger Ronald J. , Kuramitsu Howard K. 
Published: July 2002

ABSTRACT

By using the mutated
gyrB
gene from a spontaneous coumermycin A1-resistant
Treponema denticola
, an
Escherichia coli-T. denticola
shuttle vector that renders
T. denticola
resistant to coumermycin was constructed. The complete
T. denticola flgE
gene was cloned into the shuttle vector pKMCou, and the vector was transformed into the
T. denticola
ATCC 33520
flgE
erythromycin-resistant knockout mutant HL210. The coumermycin-resistant transformants were motile and restored FlgE activity. This complementation system should prove useful in studying the virulence factors of
T. denticola
and uncultivable pathogenic spirochetes.
Genome Sequence Survey Identifies Unique Sequences and Key Virulence Genes with Unusual Rates of Amino Acid Substitution in Bovine
Staphylococcus aureus
Authors: Herron Lisa L. , Chakravarty Rajit , Dwan Christopher , Fitzgerald J. Ross , Musser James M. , Retzel Ernest , Kapur Vivek 
Published: July 2002

ABSTRACT

Staphylococcus aureus
is a major cause of mastitis in bovine and other ruminant species. We here present the results of a comparative genomic analysis between a bovine mastitis-associated clone, RF122, and the recently sequenced human-associated clones, Mu50 and N315, of
Staphylococcus aureus
. A shotgun sequence survey of ∼10% of the RF122 genome identified numerous unique sequences and those with elevated rates of nonsynonymous substitution. Taken together, these analyses show that there are notable differences in the genomes of bovine mastitis-associated and human clones of
S. aureus
and provide a framework for the identification of specific factors associated with host specificity in this major human and animal pathogen.
Characterization of Bacteriophage Lambda Excisionase Mutants Defective in DNA Binding
Authors: Cho Eun Hee , Alcaraz Renato , Gumport Richard I. , Gardner Jeffrey F. 
Published: July 2002

ABSTRACT
The bacteriophage λ excisionase (Xis) is a sequence-specific DNA binding protein required for excisive recombination. Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA. Efficient excision is achieved through a cooperative interaction between Xis and the host-encoded factor for inversion stimulation as well as a cooperative interaction between Xis and integrase. The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28. Several mutants, defective in promoting excision in vivo, were isolated with mutations at positions encoding polar amino acids in the putative helix (T. E. Numrych, R. I. Gumport, and J. F. Gardner, EMBO J. 11:3797–3806, 1992). We substituted alanines for the polar amino acids in this region. Mutant proteins with substitutions for polar amino acids in the amino-terminal region of the putative helix exhibited decreased excision in vivo and were defective in DNA binding. In addition, an alanine substitution at glutamic acid 40 also resulted in altered DNA binding. This indicates that the hydrophilic face of the α-helix and the region containing glutamic acid 40 may form the DNA binding surfaces of the Xis protein.
Increased Expression of
Escherichia coli
Polynucleotide Phosphorylase at Low Temperatures Is Linked to a Decrease in the Efficiency of Autocontrol
Authors: Mathy N. , Jarrige A.-C. , Robert-Le Meur M. , Portier C. 
Published: July 2002

ABSTRACT

Polynucleotide phosphorylase (PNPase) synthesis is translationally autocontrolled via an RNase III-dependent mechanism, which results in a tight correlation between protein level and messenger stability. In cells grown at 18°C, the amount of PNPase is twice that found in cells grown at 30°C. To investigate whether this effect was transcriptional or posttranscriptional, the expression of a set of
pnp-lacZ
transcriptional and translational fusions was analyzed in cells grown at different temperatures. In the absence of PNPase, there was no increase in
pnp-lacZ
expression, indicating that the increase in
pnp
expression occurs at a posttranscriptional level. Other experiments clearly show that increased
pnp
expression at low temperature is only observed under conditions in which the autocontrol mechanism of PNPase is functional. At low temperature, the destabilizing effect of PNPase on its own mRNA is less efficient, leading to a decrease in repression and an increase in the expression level.
Unusual Regulatory Elements for Iron Deficiency Induction of the
idiA
Gene of
Synechococcus elongatus
PCC 7942
Authors: Michel Klaus-Peter , Pistorius Elfriede K. , Golden Susan S. 
Published: July 2002

ABSTRACT

Expression of a thylakoid membrane-associated protein called IdiA (
i
ron-
d
eficiency-
i
nduced protein
A
) is highly elevated and tightly regulated by iron limitation in
Synechococcus elongatus
PCC 6301 and PCC 7942. Although this protein is not essential for photosystem II (PSII) activity, it plays an important role in protecting the acceptor side of PSII against oxidative damage, especially under iron-limiting growth conditions, by an unknown mechanism. We defined the iron-responsive
idiA
promoter by using insertional inactivation mutagenesis and reporter gene assays. A 67-bp DNA region was sufficient for full iron deficiency-inducible
idiA
promoter activity. Within this fragment is a palindromic sequence 4 bp upstream of a putative −35 promoter element, which resembles the binding site of FNR/CAP-type helix-turn-helix transcription factors. The absence of this palindromic sequence or a 3-bp mutation in a putative −10 region eliminated promoter activity completely. A previously identified candidate for a positively acting transcription factor is the IdiB protein, whose gene lies immediately downstream of
idiA
. IdiB shows strong similarity to helix-turn-helix transcription factors of the FNR/CAP family. A His
6x
-tagged IdiB that was overexpressed in
Escherichia coli
bound to a 59-bp fragment of the
idiA
regulatory region that included the palindrome. Although the
idiA
promoter lacks a consensus binding site for the iron-sensing regulator Fur, we attempted to inactivate
fur
in order to investigate the potential role of this factor. The resulting merodiploid mutants showed constitutive partial derepression of IdiA expression under iron-sufficient growth conditions. We concluded that IdiB is a specific iron-responsive regulator of
idiA
and that Fur has an indirect role in influencing
idiA
expression.
Thermotoga maritima
Phosphofructokinases: Expression and Characterization of Two Unique Enzymes
Authors: Ding Yan-Huai R. , Ronimus Ron S. , Morgan Hugh W. 
Published: July 2002

ABSTRACT

A pyrophosphate-dependent phosphofructokinase (PP
i
-PFK) and an ATP-dependent phosphofructokinase (ATP-PFK) from
Thermotoga maritima
have been cloned and characterized. The PP
i
-PFK is unique in that the
K

m

and
V
max
values indicate that polyphosphate is the preferred substrate over pyrophosphate; the enzyme in reality is a polyphosphate-dependent PFK. The ATP-PFK was not significantly affected by common allosteric effectors (e.g., phosphoenolpyruvate) but was strongly inhibited by PP
i
and polyphosphate. The results suggest that the control of the Embden-Meyerhof pathway in this organism is likely to be modulated by pyrophosphate and/or polyphosphate.
Control of Nitrogenase Reactivation by the GlnZ Protein in
Azospirillum brasilense
Authors: Klassen Giseli , de Souza Emanuel M. , Yates M. Geoffrey , Rigo Liu Un , Inaba Juliana , de Oliveira Pedrosa Fábio 
Published: July 2002

ABSTRACT

The
glnZ
mutant of
Azospirillum brasilense
(strain 7611) showed only partial recovery (20 to 40%) after 80 min of ammonia-induced nitrogenase switch-off, whereas the wild type recovered totally within 10 min. In contrast, the two strains showed identical anoxic-induced switch-on/switch-off, indicating no cross talk between the two reactivation mechanisms.
Genetic Screen Yields Mutations in Genes Encoding All Known Components of the
Escherichia coli
Signal Recognition Particle Pathway
Authors: Tian Hongping , Beckwith Jon 
Published: July 2002

ABSTRACT

We describe the further utilization of a genetic screen that identifies mutations defective in the assembly of proteins into the
Escherichia coli
cytoplasmic membrane. The screen yielded mutations in each of the known genes encoding components of the
E. coli
signal recognition particle pathway:
ffh
,
ffs
, and
ftsY
, which encode Ffh, 4.5S RNA, and FtsY, respectively. In addition, the screen yielded mutations in
secM
, which is involved in regulating levels of the SecA component of the bacterium’s protein export pathway. We used a sensitive assay involving biotinylation to show that all of the mutations caused defects in the membrane insertions of three topologically distinct membrane proteins, AcrB, MalF, and FtsQ. Among the mutations that resulted in membrane protein insertion defects, only the
secM
mutations also showed defects in the translocation of proteins into the
E. coli
periplasm. Genetic evidence suggests that the S382T alteration of Ffh affects the interaction between Ffh and 4.5S RNA.
In Vivo Analysis of an Essential Archaeal Signal Recognition Particle in Its Native Host
Authors: Rose R. Wesley , Pohlschröder Mechthild 
Published: July 2002

ABSTRACT

The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (
hv54h
,
hv19h
, and
hv7Sh
, respectively) of the genetically and biochemically tractable archaeon
Haloferax volcanii
were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an
hv54h
knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in
H. volcanii
, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.
Nitric Oxide-Induced Homologous Recombination in
Escherichia coli
Is Promoted by DNA Glycosylases
Authors: Spek Erik J. , Vuong Laurel N. , Matsuguchi Tetsuya , Marinus Martin G. , Engelward Bevin P. 
Published: July 2002

ABSTRACT

Nitric oxide (NO
.
) is involved in neurotransmission, inflammation, and many other biological processes. Exposure of cells to NO
.
leads to DNA damage, including formation of deaminated and oxidized bases. Apurinic/apyrimidinic (AP) endonuclease-deficient cells are sensitive to NO
.
toxicity, which indicates that base excision repair (BER) intermediates are being generated. Here, we show that AP endonuclease-deficient cells can be protected from NO
.
toxicity by inactivation of the uracil (Ung) or formamidopyrimidine (Fpg) DNA glycosylases but not by inactivation of a 3-methyladenine (AlkA) DNA glycosylase. These results suggest that Ung and Fpg remove nontoxic NO
.
-induced base damage to create BER intermediates that are toxic if they are not processed by AP endonucleases. Our next goal was to learn how Ung and Fpg affect susceptibility to homologous recombination. The RecBCD complex is critical for repair of double-strand breaks via homologous recombination. When both Ung and Fpg were inactivated in
recBCD
cells, survival was significantly enhanced. We infer that both Ung and Fpg create substrates for recombinational repair, which is consistent with the observation that disrupting
ung
and
fpg
suppressed NO
.
-induced recombination. Taken together, a picture emerges in which the action of DNA glycosylases on NO
.
-induced base damage results in the accumulation of BER intermediates, which in turn can induce homologous recombination. These studies shed light on the underlying mechanism of NO
.
-induced homologous recombination.
Identification of Isolates ofStreptococcus canisInfecting Humans
Authors: Whatmore Adrian M. , Engler Kathryn H. , Gudmundsdottir Gudny , Efstratiou Androulla 
Published: July 2002

ABSTRACTDuring a survey of Group G and C streptococcal infections of humans two epidemiologically unrelated Group G streptococcal isolates were identified, one from a case of bacteremia and one from a wound infection. These isolates were atypical among this sample in that theemmgene could not be amplified from them by PCR. Biochemical characterization identified the isolates asStreptococcus canis, an organism normally associated with animal hosts. The biochemical identification was confirmed by sequencing of the 16S rRNA gene from both isolates and comparison with sequences of theS. canistype strain and other related streptococci of animals and humans. Comparative sequencing of fragments of two other housekeeping genes,sodAandmutS, confirmed that the isolates are most closely related toS. canis. The identification of two isolates ofS. canisfrom a relatively small sample set suggests that the practice of identifying streptococci only by the Lancefield serological group may result in underestimation of the presence ofS. canisin the human population.
Phospholipase Region of
Mycobacterium tuberculosis
Is a Preferential Locus for IS
6110
Transposition
Authors: Vera-Cabrera Lucio , Hernández-Vera Marco A. , Welsh Oliverio , Johnson Wendy M. , Castro-Garza Jorge 
Published: July 2002

ABSTRACT

Enzymes with phospholipase C activity in
Mycobacterium tuberculosis
have been recently described. The three genes encoding these proteins,
plcA
,
plcB
, and
plcC
, are located at position 2351 of the genomic map of
M. tuberculosis
H37Rv and are arranged in tandem. We have previously described the presence of variations in the restriction fragment length polymorphism patterns of the
plcA
and
plcB
genes in
M. tuberculosis
clinical isolates. In the present work we investigated the origin of this polymorphism by sequence analysis of the phospholipase-encoding regions of 11 polymorphic
M. tuberculosis
clinical isolates. To do so, a long-PCR assay was used to amplify a 5,131-bp fragment that contains the
plcA
and
plcB
genes and part of the
plcC
gene. In the
M. tuberculosis
strains studied the production of an amplicon ∼1,400 bp larger than anticipated was observed. Sequence analysis of the PCR products indicated the presence of a foreign sequence that corresponded to an IS
6110
element. We observed insertion elements in the
plcA
,
plcB
, and
plcC
genes. One site in
plcB
had the highest incidence of transposition (5 out of 11 strains). In two strains the insertion element was found in
plcA
in the same nucleotide position. In all the cases, IS
6110
was transposed in the same direction. The high level of transposition in the phospholipase region can lead to the excision of fragments of genomic DNA by recombination of neighboring IS
6110
elements, as demonstrated by finding the deletion, in two strains, of a 2,837-bp fragment that included
plcA
and most of
plcB
. This can explain the negative results obtained by some authors when detecting the
mtp40
sequence (
plcA
) by PCR. Given the high polymorphism in this region, the use of the
mtp40
sequence as a genetic marker for
M. tuberculosis
sensu stricto is very restricted.
Identification of Clinical Isolates of
Actinomyces
Species by Amplified 16S Ribosomal DNA Restriction Analysis
Authors: Hall Val , Talbot P. R. , Stubbs S. L. , Duerden B. I. 
Published: July 2002

ABSTRACT

Amplified 16S ribosomal DNA (rDNA) restriction analysis (ARDRA), using enzymes
Hae
III and
Hpa
II, was applied to 176 fresh and 299 stored clinical isolates of putative
Actinomyces
spp. referred to the Anaerobe Reference Unit of the Public Health Laboratory Service for confirmation of identity. Results were compared with ARDRA results obtained previously for reference strains and with conventional phenotypic reactions. Identities of some strains were confirmed by analysis of partial 16S rDNA sequences. Of the 475 isolates, 331 (70%) were clearly assigned to recognized
Actinomyces
species, including 94 isolates assigned to six recently described species. A further 52 isolates in 12 ARDRA profiles were designated as apparently resembling recognized species, and 44 isolates, in 18 novel profiles, were confirmed as members of genera other than
Actinomyces
. The identities of 48 isolates in nine profiles remain uncertain, and they may represent novel species of
Actinomyces
. For the majority of species, phenotypic results, published reactions for the species, and ARDRA profiles concurred. However, of 113 stored isolates originally identified as
A. meyeri
or resembling
A. meyeri
by phenotypic tests, only 21 were confirmed as
A. meyeri
by ARDRA; 63 were reassigned as
A. turicensis
, 7 as other recognized species, and 22 as unidentified actinomycetes. Analyses of incidence and clinical associations of
Actinomyces
spp. add to the currently sparse knowledge of some recently described species.
Differentiation of Resistance Phenotypes among Erythromycin-Resistant Pneumococci
Authors: Montanari Maria Pia , Mingoia Marina , Giovanetti Eleonora , Varaldo Pietro Emanuele 
Published: July 2002

ABSTRACT

Laboratory differentiation of erythromycin resistance phenotypes is poorly standardized for pneumococci. In this study, 85 clinical isolates of erythromycin-resistant (MIC ≥ 1 μg/ml)
Streptococcus pneumoniae
were tested for the resistance phenotype by the erythromycin-clindamycin double-disk test (previously used to determine the macrolide resistance phenotype in
Streptococcus pyogenes
strains) and by MIC induction tests, i.e., by determining the MICs of macrolide antibiotics without and with pre-exposure to 0.05 μg of erythromycin per ml. By the double-disk test, 65 strains, all carrying the
erm
(AM) determinant, were assigned to the constitutive macrolide, lincosamide, and streptogramin B resistance (cMLS) phenotype, and the remaining 20, all carrying the
mef
(E) gene, were assigned to the recently described M phenotype; an inducible MLS resistance (iMLS) phenotype was not found. The lack of inducible resistance to clindamycin was confirmed by determining clindamycin MICs without and with pre-exposure to subinhibitory concentrations of erythromycin. In macrolide MIC and MIC-induction tests, whereas homogeneous susceptibility patterns were observed among the 20 strains assigned to the M phenotype by the double-disk test, two distinct patterns were recognized among the 65 strains assigned to the cMLS phenotype by the same test; one pattern (
n
= 10; probably that of the true cMLS isolates) was characterized by resistance to rokitamycin also without induction, and the other pattern (
n
= 55; designated the iMcLS phenotype) was characterized by full or intermediate susceptibility to rokitamycin without induction turning to resistance after induction, with an MIC increase by more than three dilutions. A triple-disk test, set up by adding a rokitamycin disk to the erythromycin and clindamycin disks of the double-disk test, allowed the easy differentiation not only of pneumococci with the M phenotype from those with MLS resistance but also, among the latter, of those of the true cMLS phenotype from those of the iMcLS phenotype. While distinguishing MLS from M resistance in pneumococci is easily and reliably achieved, the differentiation of constitutive from inducible MLS resistance is far more uncertain and is strongly affected by the antibiotic used to test inducibility.
Increasing Multidrug Resistance in
Helicobacter pylori
Strains Isolated from Children and Adults in Mexico
Authors: Torres Javier , Camorlinga-Ponce Margarita , Pérez-Pérez Guillermo , Madrazo-De la Garza Armando , Dehesa Margarita , González-Valencia Gerardo , Muñoz Onofre 
Published: July 2002

ABSTRACT

The susceptibilities to three antimicrobials of 195
Helicobacter pylori
strains isolated from Mexican patients is reported; 80% of the strains were resistant to metronidazole, 24% were resistant to clarithromycin, and 18% presented a transient resistance to amoxicillin. Resistance to two or more antimicrobials increased significantly from 1995 to 1997.
Using Chimeric Hypoviruses To Fine-Tune the Interaction between a Pathogenic Fungus and Its Plant Host
Authors: Chen Baoshan , Geletka Lynn M. , Nuss Donald L. 
Published: July 2002

ABSTRACT

Infectious cDNA clones of mild (CHV1-Euro7) and severe (CHV1-EP713) hypovirus strains responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus
Cryphonectria parasitica
were used to construct viable chimeric viruses. Differences in virus-mediated alterations of fungal colony morphology, growth rate, and canker morphology were mapped to a region of open reading frame B extending from nucleotides 2,363 to 9,904. By swapping domains within this region, it was possible to generate chimeric hypovirus-infected
C. parasitica
isolates that exhibited a spectrum of defined colony and canker morphologies. Several severe strain traits were observed to be dominant. It was also possible to uncouple the severe strain traits of small canker size and suppression of asexual sporulation. For example, fungal isolates infected with a chimera containing nucleotides 2363 through 5310 from CHV1-Euro7 in a CHV1-713 background formed small cankers that were similar in size to that caused by CHV1-EP713-infected isolates but with the capacity for producing asexual spores at levels approaching that observed for fungal isolates infected with the mild strain. These results demonstrate that hypoviruses can be engineered to fine-tune the interaction between a pathogenic fungus and its plant host. The identification of specific hypovirus domains that differentially contribute to canker morphology and sporulation levels also provides considerable utility for continuing efforts to enhance biological control potential by balancing hypovirulence and ecological fitness.