bluuebunny/crossref_metadata_2025_split huggingface dataset:
RsaL, a Novel Repressor of Virulence Gene Expression in
Pseudomonas aeruginosa
Authors: de Kievit Teresa , Seed Patrick C. , Nezezon Jonathon , Passador Luciano , Iglewski Barbara H. 
Published: December 2019

ABSTRACT

As components of a
Pseudomonas aeruginosa
quorum-sensing system, LasR and PAI-1 globally regulate expression of multiple virulence determinants, as well as the second
P. aeruginosa
quorum-sensing system. To date, no information exists on negative regulation of the quorum-sensing cascade in
P. aeruginosa.
Here we describe a novel gene,
rsaL
, which is located downstream from
lasR
and transcribed antisense relative to
lasR.
In
P. aeruginosa,
overexpression of
rsaL
results in reduced
lasB
expression and decreased elastase activity. With the use of a six-His protein fusion system, we demonstrate that
rsaL
encodes an 11-kDa protein. Direct quantitation of PAI-1 levels in cultures and studies utilizing
Escherichia coli
lambda lysogens carrying
lacZ
transcriptional fusions reveal that RsaL specifically represses transcription of the PAI-1 autoinducer synthase gene,
lasI
. RsaL’s repressive effect on
lasI
and the associated decrease in elastase activity have important implications for the expression of all LasR–PAI-1-dependent virulence genes and the overall pathogenicity of
P. aeruginosa.
Target Joining of Duplicated Insertion Sequence IS
21
Is Assisted by IstB Protein In Vitro
Authors: Schmid Sergio , Berger Bernard , Haas Dieter 
Published: December 2019

ABSTRACT

Tandemly repeated insertion sequence IS
21
, located on a suicide plasmid, promoted replicon fusion with bacteriophage λ in vitro in the presence of ATP. This reaction was catalyzed in a cell extract containing the 45-kDa IstA protein (cointegrase) and the 30-kDa IstB helper protein of IS
21
after both proteins had been overproduced in
Escherichia coli
. Without IstB, replicon fusion was inefficient and did not produce the 4-bp target duplications typical of IS
21
.
RecD Function Is Required for High-Pressure Growth of a Deep-Sea Bacterium
Authors: Bidle Kelly A. , Bartlett Douglas H. 
Published: December 2019

ABSTRACT

A genomic library derived from the deep-sea bacterium
Photobacterium profundum
SS9 was conjugally delivered into a previously isolated pressure-sensitive SS9 mutant, designated EC1002 (E. Chi and D. H. Bartlett, J. Bacteriol. 175:7533–7540, 1993), and exconjugants were screened for the ability to grow at 280-atm hydrostatic pressure. Several clones were identified that had restored high-pressure growth. The complementing DNA was localized and in all cases found to possess strong homology to
recD
, a DNA recombination and repair gene. EC1002 was found to be deficient in plasmid stability, a phenotype also seen in
Escherichia coli recD
mutants. The defect in EC1002 was localized to a point mutation that created a stop codon within the
recD
gene. Two additional
recD
mutants were constructed by gene disruption and were both found to possess a pressure-sensitive growth phenotype, although the magnitude of the defect depended on the extent of 3′ truncation of the
recD
coding sequence. Surprisingly, the introduction of the SS9
recD
gene into an
E. coli recD
mutant had two dramatic effects. At high pressure, SS9
recD
enabled growth in the
E. coli
mutant strain under conditions of plasmid antibiotic resistance selection and prevented cell filamentation. Both of these effects were recessive to wild-type
E. coli recD
. These results suggest that the SS9
recD
gene plays an essential role in SS9 growth at high pressure and that it may be possible to identify additional aspects of RecD function through the characterization of this activity.
mrp
, a Multigene, Multifunctional Locus in
Bacillus subtilis
with Roles in Resistance to Cholate and to Na
+
and in pH Homeostasis
Authors: Ito Masahiro , Guffanti Arthur A. , Oudega Bauke , Krulwich Terry A. 
Published: December 2019

ABSTRACT

A 5.9-kb region of the
Bacillus subtilis
chromosome is transcribed as a single transcript that is predicted to encode seven membrane-spanning proteins. Homologues of the first gene of this operon, for which the designation
mrp
(multiple resistance and pH adaptation) is proposed here, have been suggested to encode an Na
+
/H
+
antiporter or a K
+
/H
+
antiporter. In the present studies of the
B. subtilis mrp
operon, both polar and nonpolar mutations in
mrpA
were generated. Growth of these mutants was completely inhibited by concentrations of added Na
+
as low as 0.3 M at pH 7.0 and 0.03 M at pH 8.3; there was no comparable inhibition by added K
+
. A null mutant that was constructed by full replacement of the
mrp
operon was even more Na
+
sensitive. A double mutant with mutations in both
mrpA
and the multifunctional antiporter-encoding
tetA
(L) gene was no more sensitive than the
mrpA
mutants to Na
+
, consistent with a major role for
mrpA
in Na
+
resistance. Expression of
mrpA
from an inducible promoter, upon insertion into the
amyE
locus, restored significant Na
+
resistance in both the polar and nonpolar
mrpA
mutants but did not restore resistance in the null mutant. The
mrpA
disruption also resulted in an impairment of cytoplasmic pH regulation upon a sudden shift in external pH from 7.5 to 8.5 in the presence of Na
+
and, to some extent, K
+
in the range from 10 to 25 mM. By contrast, the
mrpA tetA
(L) double mutant, like the
tetA
(L) single mutant, completely lost its capacity for both Na
+
- and K
+
-dependent cytoplasmic pH regulation upon this kind of shift at cation concentrations ranging from 10 to 100 mM; thus,
tetA
(L) has a more pronounced involvement than
mrpA
in pH regulation. Measurements of Na
+
efflux from the wild-type strain, the nonpolar
mrpA
mutant, and the complemented mutant indicated that inducible expression of
mrpA
increased the rate of protonophore- and cyanide-sensitive Na
+
efflux over that in the wild-type in cells preloaded with 5 mM Na
+
. The
mrpA
and null mutants showed no such efflux in that concentration range. This is consistent with MrpA encoding a secondary, proton motive force-energized Na
+
/H
+
antiporter. Studies of a polar mutant that leads to loss of
mrpFG
and its complementation in
trans
by
mrpF
or
mrpFG
support a role for MrpF as an efflux system for Na
+
and cholate. Part of the Na
+
efflux capacity of the whole
mrp
operon products is attributable to
mrpF
. Neither
mrpF
nor
mrpFG
expression in
trans
enhanced the cholate or Na
+
resistance of the null mutant. Thus, one or more other
mrp
gene products must be present, but not at stoichiometric levels, for stability, assembly, or function of both MrpF and MrpA expressed in
trans
. Also, phenotypic differences among the
mrp
mutants suggest that functions in addition to Na
+
and cholate resistance and pH homeostasis will be found among the remaining
mrp
genes.
kdgR
Ecc

Negatively Regulates Genes for Pectinases, Cellulase, Protease, Harpin
Ecc
, and a Global RNA Regulator in
Erwinia carotovora
subsp.
carotovora
Authors: Liu Yang , Jiang Guoqiao , Cui Yaya , Mukherjee Asita , Ma Wei Lei , Chatterjee Arun K. 
Published: December 2019

ABSTRACT

Erwinia carotovora
subsp.
carotovora
produces extracellular pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The concerted actions of these enzymes largely determine the virulence of this plant-pathogenic bacterium.
E. carotovora
subsp.
carotovora
also produces Harpin
Ecc
, the elicitor of the hypersensitive reaction. We document here that KdgR
Ecc
(Kdg, 2-keto-3-deoxygluconate; KdgR, general repressor of genes involved in pectin and galacturonate catabolism), a homolog of the
E. chrysanthemi
repressor, KdgR
Ech
and the
Escherichia coli
repressor, KdgR
Eco
, negatively controls not only the pectinases, Pel and Peh, but also Cel, Prt, and Harpin
Ecc
production in
E. carotovora
subsp.
carotovora
. The levels of
pel-1
,
peh-1
,
celV
, and

hrpN
Ecc

transcripts are markedly affected by KdgR
Ecc
. The KdgR
Ecc
−
mutant is more virulent than the KdgR
Ecc
+
parent. Thus, our data for the first time establish a global regulatory role for KdgR
Ecc
in
E. carotovora
subsp.
carotovora
. Another novel observation is the negative effect of KdgR
Ecc
on the transcription of
rsmB
(previously
aepH
), which specifies an RNA regulator controlling exoenzyme and Harpin
Ecc
production. The levels of
rsmB
RNA are higher in the KdgR
Ecc
−
mutant than in the KdgR
Ecc
+
parent. Moreover, by DNase I protection assays we determined that purified KdgR
Ecc
protected three 25-bp regions within the transcriptional unit of
rsmB
. Alignment of the protected sequences revealed the 21-mer consensus sequence of the KdgR
Ecc
-binding site as 5′-G/AA/TA/TG
AAA
[N
6
]
TTT
CAG/TG/TA-3′. Two such KdgR
Ecc
-binding sites occur in
rsmB
DNA in a close proximity to each other within nucleotides +79 and +139 and the third KdgR
Ecc
-binding site within nucleotides +207 and +231. Analysis of
lacZ
transcriptional fusions shows that the KdgR-binding sites negatively affect the expression of
rsmB
. KdgR
Ecc
also binds the operator DNAs of
pel-1
and
peh-1
genes and represses expression of a
pel1-lacZ
and a
peh1-lacZ
transcriptional fusions. We conclude that KdgR
Ecc
affects extracellular enzyme production by two ways: (i) directly, by inhibiting the transcription of exoenzyme genes; and (ii) indirectly, by preventing the production of a global RNA regulator. Our findings support the idea that KdgR
Ecc
affects transcription by promoter occlusion, i.e., preventing the initiation of transcription, and by a roadblock mechanism, i.e., by affecting the elongation of transcription.
The CtrA Response Regulator Mediates Temporal Control of Gene Expression during the
Caulobacter
Cell Cycle
Authors: Reisenauer Ann , Quon Kim , Shapiro Lucy 
Published: December 2019

ABSTRACT

In its role as a global response regulator, CtrA controls the transcription of a diverse group of genes at different times in the
Caulobacter crescentus
cell cycle. To understand the differential regulation of CtrA-controlled genes, we compared the expression of two of these genes, the
fliQ
flagellar gene and the
ccrM
DNA methyltransferase gene. Despite their similar promoter architecture, these genes are transcribed at different times in the cell cycle. P
fliQ
is activated earlier than P
ccrM
. Phosphorylated CtrA (CtrA∼P) bound to the CtrA recognition sequence in both promoters but had a 10- to 20-fold greater affinity for P
fliQ
. This difference in affinity correlates with temporal changes in the cellular levels of CtrA. Disrupting a unique inverted repeat element in P
ccrM
significantly reduced promoter activity but not the timing of transcription initiation, suggesting that the inverted repeat does not play a major role in the temporal control of
ccrM
expression. Our data indicate that differences in the affinity of CtrA∼P for P
fliQ
and P
ccrM
regulate, in part, the temporal expression of these genes. However, the timing of
fliQ
transcription but not of
ccrM
transcription was altered in cells expressing a stable CtrA derivative, indicating that changes in CtrA∼P levels alone cannot govern the cell cycle transcription of these genes. We propose that changes in the cellular concentration of CtrA∼P and its interaction with accessory proteins influence the temporal expression of
fliQ
,
ccrM
, and other key cell cycle genes and ultimately the regulation of the cell cycle.
Saturation Mutagenesis of the TATA Box and Upstream Activator Sequence in the Haloarchaeal
bop
Gene Promoter
Authors: Baliga Nitin S. , DasSarma Shiladitya 
Published: December 2019

ABSTRACT

Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal
bop
promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized
bop
promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeon
Halobacterium
sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum
−
bop
)
Halobacterium
mutant for purple (Pum
+
bop
+
) colonies on agar plates and analyzed for
bop
mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5′-tyT(T/a)Ta-3′, corresponding to the promoter TATA box element 30 to 25 bp 5′ of the transcription start site. A putative UAS, 5′-ACCcnactagTTnG-3′, located 52 to 39 bp 5′ of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for
bop
promoter activity.
A Region Near the C-Terminal End of
Escherichia coli
DNA Helicase II Is Required for Single-Stranded DNA Binding
Authors: Mechanic Leah E. , Latta Marcy E. , Matson Steven W. 
Published: December 2019

ABSTRACT

The role of the C terminus of
Escherichia coli
DNA helicase II (UvrD), a region outside the conserved helicase motifs, was investigated by using three mutants: UvrDΔ107C (deletion of the last 107 C-terminal amino acids), UvrDΔ102C, and UvrDΔ40C. This region, which lacks sequence similarity with other helicases, may function to tailor UvrD for its specific in vivo roles. Genetic complementation assays demonstrated that mutant proteins UvrDΔ107C and UvrDΔ102C failed to substitute for the wild-type protein in methyl-directed mismatch repair and nucleotide excision repair. UvrDΔ40C protein fully complemented the loss of helicase II in both repair pathways. UvrDΔ102C and UvrDΔ40C were purified to apparent homogeneity and characterized biochemically. UvrDΔ102C was unable to bind single-stranded DNA and exhibited a greatly reduced single-stranded DNA-stimulated ATPase activity in comparison to the wild-type protein (
k
cat
= 0.01% of the wild-type level). UvrDΔ40C was slightly defective for DNA binding and was essentially indistinguishable from wild-type UvrD when single-stranded DNA-stimulated ATP hydrolysis and helicase activities were measured. These results suggest a role for a region near the C terminus of helicase II in binding to single-stranded DNA.
Genetic Analysis of the Mobilization and Leading Regions of the IncN plasmids pKM101 and pCU1
Authors: Paterson E. Suzanne , Moré Margret I. , Pillay Gansen , Cellini Christina , Woodgate Roger , Walker Graham C. , Iyer V. N. , Winans Stephen C. 
Published: December 2019

ABSTRACT

The conjugative IncN plasmids pKM101 and pCU1 have previously been shown to contain identical
oriT
sequences as well as conserved restriction endonuclease cleavage patterns within their
tra
regions. Complementation analysis and sequence data presented here indicate that these two plasmids encode essentially identical conjugal DNA-processing proteins. This region contains three genes,
traI
,
traJ
, and
traK
, transcribed in the same orientation from a promoter that probably lies within or near the conjugal transfer origin (
oriT
). Three corresponding proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and complementation analysis confirmed that this region contains three
tra
complementation groups. All three proteins resemble proteins of the IncW plasmid R388 and other plasmids thought to have roles in processing of plasmid DNA during conjugation. The hydropathy profile of TraJ suggests a transmembrane topology similar to that of several homologous proteins. Both
traK
and
traI
were required for efficient interplasmid site-specific recombination at
oriT
, while
traJ
was not required. The leading region of pKM101 contains three genes (
stbA
,
stbB
, and
stbC
), null mutations in which cause elevated levels of plasmid instability. Plasmid instability was observed only in hosts that are proficient in interplasmid recombination, suggesting that this recombination can potentially lead to plasmid loss and that Stb proteins somehow overcome this, possibly via site-specific multimer resolution.
In Vitro Synthesis of Peptidoglycan Precursors Modified with
N
-Acetylputrescine by
Cyanophora paradoxa
Cyanelle Envelope Membranes
Authors: Pfanzagl Beatrix , Löffelhardt Wolfgang 
Published: December 2019

ABSTRACT

The photosynthetic organelles (cyanelles) of the protist
Cyanophora paradoxa
are surrounded by a peptidoglycan wall, modified through amidation with
N
-acetylputrescine. Cyanelle envelope membrane preparations were shown to catalyze the lipid-linked steps of peptidoglycan biosynthesis as well as the putrescinylation and subsequent acetylation, occurring at the stage of lipid I and/or lipid II.
DnaA Boxes Are Important Elements in Setting the Initiation Mass of
Escherichia coli
Authors: Christensen Bjarke Bak , Atlung Tove , Hansen Flemming G. 
Published: December 2019

ABSTRACT

The binding of DnaA protein to its DNA binding sites—DnaA boxes—in the chromosomal
oriC
region is essential for initiation of chromosome replication. In this report, we show that additional DnaA boxes affect chromosome initiation control, i.e., increase the initiation mass. The cellular DnaA box concentration was increased by introducing pBR322-derived plasmids carrying DnaA boxes from the
oriC
region into
Escherichia coli
and by growing the strains at different generation times to obtain different plasmid copy numbers. In fast-growing cells, where the DnaA box plasmid copy number per
oriC
locus was low, the presence of extra DnaA boxes caused only a moderate increase in the initiation mass. In slowly growing cells, where the DnaA box plasmid copy number per
oriC
locus was higher, we observed more pronounced increases in the initiation mass. Our data clearly show that the presence of extra DnaA boxes increases the initiation mass, supporting the idea that the initiation mass is determined by the normal complement of DnaA protein binding sites in
E. coli
cells.
Isoaspartate in Ribosomal Protein S11 ofEscherichia coli
Authors: David Cynthia L. , Keener John , Aswad Dana W. 
Published: December 2019

ABSTRACTIsoaspartyl sites, in which an aspartic acid residue is linked to its C-flanking neighbor via its β-carboxyl side chain, are generally assumed to be an abnormal modification arising as proteins age. The enzyme proteinl-isoaspartate methyltransferase (PIMT), present in many bacteria, plants, and animals, catalyzes the conversion of isoaspartate to normal α-linked aspartyl bonds and is thought to serve an important repair function in cells. Having introduced a plasmid intoEscherichia colithat allows high-level expression of rat PIMT, we explored the possibility that the rat enzyme reduces isoaspartate levels inE. coliproteins, a result predicted by the repair hypothesis. The present study demonstrates that this is indeed the case;E. colicells expressing rat PIMT had significantly lower isoaspartate levels than control cells, especially in stationary phase. Moreover, the distribution of isoaspartate-containing proteins inE. colidiffered dramatically between logarithmic- and stationary-phase cultures. In stationary-phase cells, a number of proteins in the molecular mass range of 66 to 14 kDa contained isoaspartate, whereas in logarithmic-phase cells, nearly all of the detectable isoaspartate resided in a single 14-kDa protein which we identified as ribosomal protein S11. The near stoichiometric levels of isoaspartate in S11, estimated at 0.5 mol of isoaspartate per mol of S11, suggests that this unusual modification may be important for S11 function.
Evaluation of Four DNA Typing Techniques in Epidemiological Investigations of Bovine Tuberculosis
Authors: Cousins Debby , Williams Suzette , Liébana Ernesto , Aranaz Alicia , Bunschoten Annelies , Van Embden Jan , Ellis Trevor 
Published: December 2019

ABSTRACT

DNA fingerprinting techniques were used to type 273 isolates of
Mycobacterium bovis
from Australia, Canada, the Republic of Ireland, and Iran. The results of restriction fragment length polymorphism (RFLP) analysis with DNA probes from IS
6110
, the direct repeat (DR), and the polymorphic GC-rich sequence (PGRS) were compared with those of a new PCR-based method called spacer oligonucleotide typing (spoligotyping) developed for the rapid typing of
Mycobacterium tuberculosis
(J. Kamerbeek et al., J. Clin. Microbiol. 35:907–914, 1997). Eighty-five percent of the isolates harbored a single copy of IS
6110
, and 81.5% of these carried IS
6110
on the characteristic 1.9-kb restriction fragment. RFLP analysis with IS
6110
identified 23 different types, RFLP analysis with the DR probe identified 35 types, RFLP analysis with the PGRS probe identified 77 types, and the spoligotyping method identified 35 types. By combining all results, 99 different strains could be identified. Isolate clusters were frequently associated within herds or were found between herds when epidemiological evidence confirmed animal movements. RFLP analysis with IS
6110
was sufficiently sensitive for the typing of isolates with more than three copies of IS
6110
, but RFLP analysis with the PGRS probe was the most sensitive typing technique for strains with only a single copy of IS
6110
. Spoligotyping may have advantages for the rapid typing of
M. bovis
, but it needs to be made more sensitive.
Evaluation of the OptiMAL Test for Rapid Diagnosis of
Plasmodium vivax
and
Plasmodium falciparum
Malaria
Authors: Palmer Carol J. , Lindo John F. , Klaskala Winslow I. , Quesada Jose A. , Kaminsky Rina , Baum Marianna K. , Ager Arba L. 
Published: December 2019

ABSTRACT

The development of rapid and specific diagnostic tests to identify individuals infected with malaria is of paramount importance in efforts to control the severe public health impact of this disease. This study evaluated the ability of a newly developed rapid malaria diagnostic test, OptiMAL (Flow Inc., Portland, Oreg.), to detect
Plasmodium vivax
and
Plasmodium falciparum
malaria during an outbreak in Honduras. OptiMAL is a rapid (10-min) malaria detection test which utilizes a dipstick coated with monoclonal antibodies against the intracellular metabolic enzyme parasite lactate dehydrogenase (pLDH). Differentiation of malaria parasites is based on antigenic differences between the pLDH isoforms. Since pLDH is produced only by live
Plasmodium
parasites, this test has the ability to differentiate live from dead organisms. Results from the OptiMAL test were compared to those obtained by reading 100 fields of traditional Giemsa-stained thick-smear blood films. Whole-blood samples were obtained from 202 patients suspected of having malaria. A total of 96 samples (48%) were positive by blood films, while 91 (45%) were positive by the OptiMAL test. The blood films indicated that 82% (79 of 96) of the patients were positive for
P. vivax
and 18% (17 of 96) were infected with
P. falciparum
. The OptiMAL test showed that 81% (74 of 91) were positive for
P. vivax
and 19% (17 of 91) were positive for
P. falciparum
. These results demonstrated that the OptiMAL test had sensitivities of 94 and 88% and specificities of 100 and 99%, respectively, when compared to traditional blood films for the detection of
P. vivax
and
P. falciparum
malaria. Blood samples not identified by OptiMAL as malaria positive normally contained parasites at concentrations of less than 100/μl of blood. Samples found to contain
P. falciparum
were further tested by two other commercially available rapid malaria diagnostic tests, ParaSight-F (Becton Dickinson, Cockeysville, Md.) and ICT Malaria P.f. (ICT Diagnostics, Sydney, Australia), both of which detect only
P. falciparum
. Only 11 of the 17 (65%)
P. falciparum
-positive blood samples were identified by the ICT and ParaSight-F tests. Thus, OptiMAL correctly identified
P. falciparum
malaria parasites in patient blood samples more often than did the other two commercially available diagnostic tests and showed an excellent correlation with traditional blood films in the identification of both
P. vivax
malaria and
P. falciparum
malaria. We conclude that the OptiMAL test is an effective tool for the rapid diagnosis of malaria.