bluuebunny/crossref_metadata_2025_split huggingface dataset:
IncC of Broad-Host-Range Plasmid RK2 Modulates KorB Transcriptional Repressor Activity In Vivo and Operator Binding In Vitro
Authors: Jagura-Burdzy Grazyna , Kostelidou Kalliope , Pole Jessica , Khare Dheeraj , Jones Anthony , Williams D. Ross , Thomas Christopher M. 
Published: December 2019

ABSTRACT

The
korAB
operon of broad-host-range plasmid RK2 encodes five genes, two of which,
incC
and
korB
, belong to the
parA
and
parB
families, respectively, of genome partitioning functions. Both
korB
and a third gene,
korA
, are responsible for coordinate regulation of operons encoding replication, transfer, and stable inheritance functions. Overexpression of
incC
alone caused rapid displacement of RK2. Using two different reporter systems, we show that
incC
modulates the action of KorB. Using promoter fusions to the reporter gene
xylE
, we show that
incC
potentiates the repression of transcription by
korB
. This modulation of
korB
activity was only observed with
incC1
, which encodes the full-length IncC (364 amino acids [aa]), whereas no effect was observed with
incC2
, which encodes a polypeptide of 259 aa that lacks the N-terminal 105 aa. Using bacterial extracts with IncC1 and IncC2 or IncC1 purified through the use of a His
6
tail and Ni-agarose chromatography, we showed that IncC1 potentiates the binding of KorB to DNA at representative KorB operators. The ability of IncC to stabilize KorB-DNA complexes suggests that these two proteins work together in the global regulation of many operons on the IncP-1 genomes, as well in plasmid partitioning.
Identification of Metal Ligands in the
Clostridium histolyticum
ColH Collagenase
Authors: Jung Chang-Min , Matsushita Osamu , Katayama Seiichi , Minami Junzaburo , Sakurai Jun , Okabe Akinobu 
Published: December 2019

ABSTRACT

A
Clostridium histolyticum
116-kDa collagenase has an H
415
EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases. To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues. The mutation affecting His
415
or His
419
abolished catalytic activity and zinc binding, while that affecting Glu
416
did the former but not the latter. These results suggest that the motif forms the catalytic site. We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands. The mutation affecting Glu
447
decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu
446
or Glu
451
had smaller effects on activity and zinc binding. These mutations caused a decrease in
k
cat
but no significant change in

K
m

. These results are consistent with the hypothesis that Glu
447
is the third zinc ligand. The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily. The effects of mutations affecting Glu
446
and Glu
451
suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.
Regulation of Expression of the Fructan Hydrolase Gene of
Streptococcus mutans
GS-5 by Induction and Carbon Catabolite Repression
Authors: Burne Robert A. , Wen Zezhang Thomas , Chen Yi-Ywan M. , Penders Jana E. C. 
Published: December 2019

ABSTRACT

The polymers of fructose, levan and inulin, as well as sucrose and raffinose, are substrates for the product of the
fruA
gene of
Streptococcus mutans
GS-5. The purpose of this study was to characterize the DNA immediately flanking
fruA
, to explore the regulation of expression of
fruA
by the carbohydrate source, and to begin to elucidate the molecular basis for differential expression of the gene. Located 3′ to
fruA
was an open reading frame (ORF) with similarity to β-fructosidases which was cotranscribed with
fruA
. A transcriptional initiation site, located an appropriate distance from an extended −10-like promoter, was mapped at 165 bp 5′ to the
fruA
structural gene. By the use of computer algorithms, two overlapping, stable stem-loop sequences with the potential to function as rho-independent terminators were found in the 5′ untranslated region. Catabolite response elements (CREs), which have been shown to govern carbon catabolite repression (CCR) by functioning as negative
cis
elements in gram-positive bacteria, were located close to the promoter. The levels of production of
fruA
mRNA and FruA were elevated in cells growing on levan, inulin, or sucrose as the sole carbohydrate source, and repression was observed when cells were grown on readily metabolizable hexoses. Deletion derivatives containing fusions of
fruA
promoter regions, lacking sequences 5′ or 3′ to the promoter, and a promoterless chloramphenicol acetyltransferase gene were used (i) to demonstrate the functionality of the promoter mapped by primer extension, (ii) to demonstrate that CCR of the
fru
operon requires the CRE that is located 3′ to the promoter region, and (iii) to provide preliminary evidence that supports the involvement of an antitermination mechanism in
fruA
induction.
trans
-Acting Factors Affecting Carbon Catabolite Repression of the
hut
Operon in
Bacillus subtilis
Authors: Zalieckas Jill M. , Wray Lewis V. , Fisher Susan H. 
Published: December 2019

ABSTRACT

In
Bacillus subtilis
, CcpA-dependent carbon catabolite repression (CCR) mediated at several
cis
-acting carbon repression elements (
cre
) requires the seryl-phosphorylated form of both the HPr (
ptsH
) and Crh (
crh
) proteins. During growth in minimal medium, the
ptsH1
mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (
hut
) enzymes in cells grown in minimal medium showed that neither the
ptsH1
nor the
crh
mutation individually had any affect on
hut
CCR but that
hut
CCR was abolished in a
ptsH1 crh
double mutant. In contrast, the
ptsH1
mutation completely relieved
hut
CCR in cells grown in Luria-Bertani medium. The
ptsH1 crh
double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in
B. subtilis
mutants which synthesize low levels of active glutamine synthetase (
glnA
). In addition, these
glnA
mutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of
glnA
mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.
Ferric Enterobactin Binding and Utilization by
Neisseria gonorrhoeae
Authors: Carson Susan D. Biegel , Klebba Philip E. , Newton Salete M. C. , Sparling P. Frederick 
Published: December 2019

ABSTRACT

FetA, formerly designated FrpB, an iron-regulated, 76-kDa neisserial outer membrane protein, shows sequence homology to the TonB-dependent family of receptors that transport iron into gram-negative bacteria. Although FetA is commonly expressed by most neisserial strains and is a potential vaccine candidate for both
Neisseria gonorrhoeae
and
Neisseria meningitidis
, its function in cell physiology was previously undefined. We now report that FetA functions as an enterobactin receptor.
N. gonorrhoeae
FA1090 utilized ferric enterobactin as the sole iron source when supplied with ferric enterobactin at approximately 10 μM, but growth stimulation was abolished when an omega (Ω) cassette was inserted within
fetA
or when
tonB
was insertionally interrupted. FA1090 FetA specifically bound
59
Fe-enterobactin, with a

K
d

of approximately 5 μM. Monoclonal antibodies raised against the
Escherichia coli
enterobactin receptor, FepA, recognized FetA in Western blots, and amino acid sequence comparisons revealed that residues previously implicated in ferric enterobactin binding by FepA were partially conserved in FetA. An open reading frame downstream of
fetA
, designated
fetB
, predicted a protein with sequence similarity to the family of periplasmic binding proteins necessary for transporting siderophores through the periplasmic space of gram-negative bacteria. An Ω insertion within
fetB
abolished ferric enterobactin utilization without causing a loss of ferric enterobactin binding. These data show that FetA is a functional homolog of FepA that binds ferric enterobactin and may be part of a system responsible for transporting the siderophore into the cell.
Analyses of a Polyhydroxyalkanoic Acid Granule-Associated 16-Kilodalton Protein and Its Putative Regulator in the
pha
Locus of
Paracoccus denitrificans
Authors: Maehara Akira , Ueda Shunsaku , Nakano Hideo , Yamane Tsuneo 
Published: December 2019

ABSTRACT

The polyhydroxyalkanoic acid (PHA) granule-associated 16-kDa protein (GA16 protein) of
Paracoccus denitrificans
was identified, and its corresponding gene was cloned and analyzed at the molecular level. The N-terminal amino acid sequence of GA16 protein revealed that its structural gene is located downstream from the PHA synthase gene (
phaC
Pd
) cloned recently (S. Ueda, T. Yabutani, A. Maehara, and T. Yamane, J. Bacteriol. 178:774–779, 1996). Gene walking around
phaC
Pd
revealed two new open reading frames (ORFs) possibly related to PHA synthesis, one of which was the
phaP
Pd
gene, encoding GA16 protein, and the other was the
phaR
Pd
gene, encoding a protein that is putatively involved in the regulation of the expression of
phaP
Pd
. Overproduction of PhaP
Pd
was observed in
Escherichia coli
carrying
phaP
Pd
, but the overproduction was not observed in the presence of
phaR
Pd
. Coexpression of
phaP
Pd
and PHA biosynthesis genes in
E. coli
caused increases in both the number of poly-(3-hydroxybutyric acid) (PHB) granules and PHB content and caused decreases in both the size of the granules and the molecular weight of PHB. GA16 protein was considered a phasin protein. The
phaR
Pd
gene had significant similarities to
stdC
, a possible transcriptional factor of
Comamonas testosteroni
, as well as to other ORFs of unknown function previously found in other PHA-synthetic bacteria.
Carboxy-Terminal Processing of the Large Subunit of [Fe] Hydrogenase from
Desulfovibrio desulfuricans
ATCC 7757
Authors: Hatchikian E. Claude , Magro Valérie , Forget Nicole , Nicolet Yvain , Fontecilla-Camps Juan C. 
Published: December 2019

ABSTRACT

hydA
and
hydB
, the genes encoding the large (46-kDa) and small (13.5-kDa) subunits of the periplasmic [Fe] hydrogenase from
Desulfovibrio desulfuricans
ATCC 7757, have been cloned and sequenced. The deduced amino acid sequence of the genes product showed complete identity to the sequence of the well-characterized [Fe] hydrogenase from the closely related species
Desulfovibrio vulgaris
Hildenborough (G. Voordouw and S. Brenner, Eur. J. Biochem. 148:515–520, 1985). The data show that in addition to the well-known signal peptide preceding the NH
2
terminus of the mature small subunit, the large subunit undergoes a carboxy-terminal processing involving the cleavage of a peptide of 24 residues, in agreement with the recently reported data on the three-dimensional structure of the enzyme (Y. Nicolet, C. Piras, P. Legrand, E. C. Hatchikian, and J. C. Fontecilla-Camps, Structure 7:13–23, 1999). We suggest that this C-terminal processing is involved in the export of the protein to the periplasm.
tRNA Synthetase Mutants of
Escherichia coli
K-12 Are Resistant to the Gyrase Inhibitor Novobiocin
Authors: Milija Jovanovic , Lilic Mirjana , Janjusevic Radmila , Jovanovic Goran , Savic Dragutin J. 
Published: December 2019

ABSTRACT

In previous studies we demonstrated that mutations in the genes
cysB
,
cysE
, and
cls
(
nov
) affect resistance of
Escherichia coli
to novobiocin (J. Rakonjac, M. Milic, and D. J. Savic, Mol. Gen. Genet. 228:307–311, 1991; R. Ivanisevic, M. Milic, D. Ajdic, J. Rakonjac, and D. J. Savic, J. Bacteriol. 177:1766–1771, 1995). In this work we expand this list with mutations in
rpoN
(the gene for RNA polymerase subunit ς
54
) and the tRNA synthetase genes
alaS
,
argS
,
ileS
, and
leuS
. Similarly to resistance to the penicillin antibiotic mecillinam, resistance to novobiocin of tRNA synthetase mutants appears to depend upon the RelA-mediated stringent response. However, at this point the overlapping pathways of mecillinam and novobiocin resistance diverge. Under conditions of stringent response induction, either by the presence of tRNA synthetase mutations or by constitutive production of RelA protein, inactivation of the
cls
gene diminishes resistance to novobiocin but not to mecillinam.
Abiotrophia elegans
sp. nov., a Possible Pathogen in Patients with Culture-Negative Endocarditis
Authors: Roggenkamp Andreas , Abele-Horn Marianne , Trebesius Karl-Heinz , Tretter Ursula , Autenrieth Ingo B. , Heesemann Jürgen 
Published: December 2019

ABSTRACT

We isolated a hitherto undescribed microorganism from a patient with endocarditis. The microscopic appearance, a negative catalase reaction, and growth as satellite colonies next to
Staphylococcus epidermidis
suggested that this microorganism is a member of the genus
Abiotrophia
, formerly known as nutritionally variant streptococci. However, the clinical isolate described herein differed markedly from the known
Abiotrophia
spp.,
A. adiacens
and
A. defectiva
, in terms of its (i) biochemical properties, (ii) restricted growth temperature range, (iii) whole-cell lysate polypeptide profile, and (iv) unique nutritional requirements. In contrast to the type strains of
A. adiacens
and
A. defectiva
, which used
l
-cysteine and pyridoxal hydrochloride as growth factors, the growth of the clinical isolate was only supported by
l
-cysteine hydrochloride and not by pyridoxal hydrochloride when the organism was tested in Todd-Hewitt or casein-soy peptone broth. Comparative 16S rRNA gene sequence analysis revealed that the microorganism was a member of the genus
Abiotrophia
and was most closely related to
A. adiacens
(96.9% homology). Phenotypic and phylogenetic data are consistent with the assumption of a new species within the genus
Abiotrophia
, for which we propose the name
Abiotrophia elegans
sp. nov. The unique nutritional requirements of this strain are of importance for diagnostic laboratories. The media of blood culture systems supplemented only with pyridoxal hydrochloride as a growth factor may fail to promote the growth of
A. elegans
sp. nov., and thus, these systems might not detect this microorganism as a possible cause of endocarditis.
Etiology of Acute Gastroenteritis in Hospitalized Children in Melbourne, Australia, from April 1980 to March 1993
Authors: Barnes Graeme L. , Uren Eric , Stevens Kerrie B. , Bishop Ruth F. 
Published: December 2019

ABSTRACT

Acute infectious diarrhea is common in children. Control requires knowledge of causes. Few comprehensive long-term studies of etiology have been undertaken in developed countries. This report is of a 13-year survey of 4,637 children from 0 to 14 years of age, admitted to a large children’s hospital for treatment of gastroenteritis, in which viruses, bacteria, and parasites were sought. A recognized enteric pathogen was identified in 56.6% of children. Group A rotaviruses occurred in 39.6% of children overall and in 55% of children 12 to 23 months of age. They were a frequent cause (18.7%) of acute gastroenteritis in children under 6 months and in those aged 5 to 13 years (16%). Rotaviruses were almost entirely responsible for winter admission peaks. Enteric adenovirus types 40 and 41 (6% overall) were more frequent in children under 12 months (9.4%).
Salmonella
spp. (5.8%) and
Campylobacter jejuni
(3.4%) were more common in children over 5 years (13.1% and 6.7%, respectively). The 43.5% of cases (60% in children under 6 months) where no enteric pathogen was identified are cause for concern. The involvement of small viruses (including caliciviruses and astroviruses) may be clarified when molecular biology techniques are utilized to address this gap in our knowledge. This comprehensive 13-year study of the cause of acute infectious diarrhea in children in developed countries reinforces the importance of rotavirus and highlights a large group for whom the etiology remains unknown, an issue of particular concern with babies under 6 months of age. New techniques have the potential to identify old and new pathogens causing disease in these vulnerable infants.
Multicenter Evaluation of the
Clostridium difficile
TOX A/B TEST
Authors: Lyerly D. M. , Neville L. M. , Evans D. T. , Fill J. , Allen S. , Greene W. , Sautter R. , Hnatuck P. , Torpey D. J. , Schwalbe R. 
Published: December 2019

ABSTRACT

Clostridium difficile
, the primary cause of nosocomial diarrhea in the United States and many other industrialized countries, is recognized as a major health concern because of its ability to cause severe intestinal disease leading to complications such as relapses and infections due to vancomycin-resistant enterococci. The disease results from two toxins, toxins A and B, produced by this pathogen. In this study, we evaluated the TOX A/B TEST, a new 1-h enzyme immunoassay (EIA) that detects toxins A and B. We compared the test with the tissue culture assay, which is recognized as the “gold standard” for
C. difficile
testing. Evaluations were performed in-house at TechLab, Inc. (Blacksburg, Va.) and off-site at four clinical laboratories. Of 1,152 specimens tested, 165 were positive by the TOX A/B TEST and tissue culture and 973 were negative by both tests. The sensitivity and specificity were 92.2 and 100%, respectively. The positive and negative predictive values were 100 and 98.6%, respectively, and the correlation of the TOX A/B TEST with tissue culture was 98.8%. When discrepant samples were resolved by culture, the sensitivity and specificity were 93.2 and 98.9%, respectively. The positive and negative predictive values were 100 and 98.8%, respectively, with a correlation of 99.0%. There were no specimens that were positive by the TOX A/B TEST and negative by tissue culture. Fourteen specimens were negative by the TOX A/B TEST but positive by tissue culture. Of these, two were negative by toxigenic culture, five were positive by toxigenic culture, and seven were not available for further testing. There were no indeterminate results, since the test does not have an indeterminant zone. In a separate study, 102 specimens that were positive by tissue culture and the TOX A/B TEST were examined in toxin A-specific EIAs. Two specimens that presumptively contained toxin A-negative, toxin B-positive (toxA−/toxB+) isolates were identified. One specimen was from a patient with a clinical history consistent with
C. difficile
infection. Isolates obtained from these specimens by selective culture on solid media and in broth tested toxA−/toxB+ when grown in brain heart infusion dialysis flasks, which stimulate in vitro production of both toxins. Our findings show that the TOX A/B TEST is suitable as a diagnostic aid for
C. difficile
disease because it correlates well with tissue culture and detects isolates that may be missed with toxin A-specific EIAs.
Confirmation of the Distinct Genotype Groups within the Form Species
Candida parapsilosis
Authors: Roy Basabi , Meyer Sally A. 
Published: December 2019

ABSTRACT

The DNA relatedness and restriction fragment length polymorphism patterns of whole-cell DNA and the general phenotypic properties of 14 isolates of the form species
Candida parapsilosis
representing three diverse genetic groups were compared. The data confirm the unrelatedness of the three groups at the species level.
Association of Borderline Oxacillin-Susceptible Strains ofStaphylococcus aureuswith Surgical Wound Infections
Authors: Kernodle Douglas S. , Classen David C. , Stratton Charles W. , Kaiser Allen B. 
Published: December 2019

ABSTRACTStaphylococcus aureusisolates which produce type A staphylococcal β-lactamase have been associated with wound infections complicating the use of cefazolin prophylaxis in surgery. To further evaluate this finding, 215 wound isolates from 14 cities in the United States were characterized by antimicrobial susceptibility and β-lactamase type and correlated with the preoperative prophylactic regimen. Borderline-susceptibleS. aureusisolates of phage group 5 (BSSA-5), which produce large amounts of type A β-lactamase and exhibit borderline susceptibility to oxacillin, comprised a greater percentage of the 120 wound isolates associated with cefazolin prophylaxis than they did of the 95 isolates associated with other prophylactic regimens (25% versus 12.6%, respectively;P< 0.05). In contrast, methicillin-resistantS. aureusisolates were distributed evenly between the two groups (8.3% versus 11.6%, respectively). In vitro assays demonstrated that cefazolin was hydrolyzed faster by BSSA-5 strains than by other β-lactamase-producing, methicillin-susceptible strains (1.54 versus 0.50 μg/min/108CFU, respectively;P< 0.0001). These data demonstrate that BSSA-5 strains are a distinct subpopulation of methicillin-susceptibleS. aureuswhich frequently cause deep surgical wound infections. Cefazolin use in prophylaxis is a risk factor for BSSA-5 infection.
Allele-Specific PCR Method Based on
pncA
and
oxyR
Sequences for Distinguishing
Mycobacterium bovis
from
Mycobacterium tuberculosis
: Intraspecific
M. bovis pncA
Sequence Polymorphism
Authors: de los Monteros Luz Elena Espinosa , Galán Juan Carlos , Gutiérrez Montserrat , Samper Sofía , García Marín Juan F. , Martín Carlos , Domínguez Lucas , de Rafael Luis , Baquero Fernando , Gómez-Mampaso Enrique , Blázquez Jesús 
Published: December 2019

ABSTRACT

An allele-specific amplification method based on two genetic polymorphisms to differentiate
Mycobacterium tuberculosis
from
Mycobacterium bovis
was tested. Based on the differences found at position 169 in the
pncA
genes from
M. tuberculosis
and
M. bovis
, a PCR system which was able to differentiate most of the 237
M. tuberculosis
complex isolates tested in one of the two species was developed. All 121
M. tuberculosis
strains showed the expected base (cytosine) at position 169. Most of the
M. bovis
isolates had a guanine at the cited position. Nevertheless, 18 of the 116
M. bovis
isolates, all of them goat isolates, showed the
pncA
polymorphism specific to
M. tuberculosis
. These results suggest that goat
M. bovis
may be the nicotinamidase-missing link at the origin of the
M. tuberculosis
species. Based on the polymorphism found at position 285 in the
oxyR
gene, the same system was used to differentiate
M. tuberculosis
from
M. bovis
. In this case, DNAs from all 121
M. tuberculosis
isolates had the expected base (guanine) at this position. In addition, all 116
M. bovis
isolates, including those from goats, showed the identical polymorphism (adenine). The
oxyR
allele-specific amplification method can differentiate
M. bovis
from
M. tuberculosis
, is rapid (results can be obtained in less than 3 h), and is easy to perform.