bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutations That Alter the Kinase and Phosphatase Activities of the Two-Component Sensor EnvZ
Authors: Hsing Weihong , Russo Frank D. , Bernd Karen K. , Silhavy Thomas J. 
Published: December 2019

ABSTRACT

EnvZ, a membrane receptor kinase-phosphatase, modulates porin expression in
Escherichia coli
in response to medium osmolarity. It shares its basic scheme of signal transduction with many other sensor-kinases, passing information from the amino-terminal, periplasmic, sensory domain via the transmembrane helices to the carboxy-terminal, cytoplasmic, catalytic domain. The native receptor can exist in two active but opposed signaling states, the OmpR kinase-dominant state (K
+
P
−
) and the OmpR-P phosphatase-dominant state (K
−
P
+
). The balance between the two states determines the level of intracellular OmpR-P, which in turn determines the level of porin gene transcription. To study the structural requirements for these two states of EnvZ, mutational analysis was performed. Mutations that preferentially affect either the kinase or phosphatase have been identified and characterized both in vivo and in vitro. Most of these mapped to previously identified structural motifs, suggesting an important function for each of these conserved regions. In addition, we identified a novel motif that is weakly conserved among two-component sensors. Mutations that alter this motif, which is termed the X region, alter the confirmation of EnvZ and significantly reduce the phosphatase activity.
Purification, Characterization, and Sequence Analysis of 2-Aminomuconic 6-Semialdehyde Dehydrogenase from
Pseudomonas pseudoalcaligenes
JS45
Authors: He Zhongqi , Davis John K. , Spain Jim C. 
Published: December 2019

ABSTRACT

2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by
Pseudomonas pseudoalcaligenes
JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD
+
. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.
Only the N-Terminal Domain of FtsK Functions in Cell Division
Authors: Draper G. Craig , McLennan Neil , Begg Ken , Masters Millicent , Donachie William D. 
Published: December 2019

ABSTRACT

Deletion of
ftsK
results in the inhibition of cell division, but this inhibition can be reversed by a plasmid carrying only the first ∼17% of
ftsK
. The division block can be suppressed in most mutants by deletion of
dacA
, which codes for the
d
-alanine:
d
-alanine carboxypeptidase PBP5, or in all mutants by overexpression of
ftsN
. Overexpression of
ftsK
inhibits cell division and the formation of FtsZ rings. This division block is not due to the induction of either the SOS or the heat shock regulons.
Transcriptional Activation of the
Bacillus subtilis spoIIG
Promoter by the Response Regulator Spo0A Is Independent of the C-Terminal Domain of the RNA Polymerase Alpha Subunit
Authors: Rowe-Magnus Dean A. , Mencía Mario , Rojo Fernando , Salas Margarita , Spiegelman George B. 
Published: December 2019

ABSTRACT

In vitro transcription from the
spoIIG
promoter by
Bacillus subtilis
RNA polymerase reconstituted with wild-type alpha subunits and with C-terminal deletion mutants of the alpha subunit was equally stimulated by the response regulator Spo0A. Some differences in the structure of open complexes formed by RNA polymerase containing alpha subunit mutants were noted, although the wild-type and mutant polymerases appeared to use the same initiation mechanism.
A Region in
Bacillus subtilis
ς
H
Required for Spo0A-Dependent Promoter Activity
Authors: Buckner Cindy M. , Moran Charles P. 
Published: December 2019

ABSTRACT

Spo0A activates transcription in
Bacillus subtilis
from promoters that are used by two types of RNA polymerase, RNA polymerase containing the primary sigma factor, ς
A
, and RNA polymerase containing a secondary sigma factor, known as ς
H
. The region of ς
A
near positions 356 to 359 is required for Spo0A-dependent promoter activation, possibly because Spo0A interacts with this region of ς
A
at these promoters. To determine if the amino acids in the corresponding region of ς
H
are also important in Spo0A-dependent promoter activation, we examined the effects of single alanine substitutions at 10 positions in ς
H
(201 to 210). Two alanine substitutions in ς
H
, at glutamine 201 (Q201A) and at arginine 205 (R205A), significantly decreased activity from the Spo0A-dependent, ς
H
-dependent promoter
spoIIA
but did not affect expression from the ς
H
-dependent, Spo0A-independent promoters
citGp2
and
spoVG
. Therefore, promoter activation by Spo0A requires homologous regions in ς
A
and ς
H
. A mutant form of Spo0A, S231F, that suppresses the sporulation defect caused by several amino acid substitutions in ς
A
did not suppress the sporulation defects caused by the Q201A and R205A substitutions in ς
H
. This result and others indicate that different surfaces of Spo0A probably interact with ς
A
and ς
H
RNA polymerases.
Characterization of an A-Factor-Responsive Repressor for
amfR
Essential for Onset of Aerial Mycelium Formation in
Streptomyces griseus
Authors: Ueda Kenji , Hsheh Chia-Wen , Tosaki Tsutomu , Shinkawa Hidenori , Beppu Teruhiko , Horinouchi Sueharu 
Published: December 2019

ABSTRACT

A-factor (2-isocapryloyl-3
R
-hydroxymethyl-γ-butyrolactone) is essential for the initiation of aerial mycelium formation in
Streptomyces griseus. amfR
is one of the genes which, when cloned on a low-copy-number plasmid, suppresses the aerial mycelium-negative phenotype of an A-factor-deficient mutant of
S. griseus
. Disruption of the chromosomal
amfR
gene resulted in complete abolition of aerial mycelium formation, indicating that
amfR
is essential for the onset of morphogenesis. Cloning and nucleotide sequencing of the region upstream of
amfR
predicted an operon consisting of
orf5
,
orf4
, and
amfR
. Consistent with this idea, Northern blotting and S1 mapping analyses suggested that these three genes were cotranscribed mainly by a promoter (P
ORF5
) in front of
orf5
. Furthermore, P
ORF5
was active only in the presence of A-factor, indicating that it is A-factor dependent. Gel mobility shift assays showed the presence of a protein (AdpB) able to bind P
ORF5
in the cell extract from an A-factor-deficient mutant but not from the wild-type strain. AdpB was purified to homogeneity and found to bind specifically to the region from −72 to −44 bp with respect to the transcriptional start point. Runoff transcriptional analysis of P
ORF5
with purified AdpB and an RNA polymerase complex isolated from vegetative mycelium showed that AdpB repressed the transcription in a concentration-dependent manner. It is thus apparent that AmfR as a switch for aerial mycelium formation and AdpB as a repressor for
amfR
are members in the A-factor regulatory cascade, leading to morphogenesis.
ClpB in a Cyanobacterium: Predicted Structure, Phylogenetic Relationships, and Regulation by Light and Temperature
Authors: Celerin Martina , Gilpin Andrea A. , Schisler Nicholas J. , Ivanov Alexander G. , Miskiewicz Ewa , Krol Marianna , Laudenbach David E. 
Published: December 2019

ABSTRACT

The sequence of a genomic clone encoding a 100-kDa stress protein of
Plectonema boryanum
(
p
-ClpB) was determined. The predicted polypeptide contains two putative ATPase regions located within two highly conserved domains (N1 and N2), a spacer region that likely forms a coiled-coil domain, and a highly conserved consensus CK2 phosphorylation domain. The coiled-coil region and the putative site of phosphorylation are not unique to
p
-ClpB; they are present in all ClpB sequences examined and are absent from the ClpB paralogs ClpA, ClpC, ClpX, and ClpY. Small quantities of a 4.5-kb
p-clpB
transcript and 110-kDa cytosolic
p
-ClpB protein were detected in cells grown under optimal conditions; however, increases in the quantities of the transcript and protein were observed in cells grown under excess light and low temperature conditions. Finally, we analyzed ClpA, ClpB, and ClpC sequences from 27 organisms in order to predict phylogenetic relationships among the homologs. We have used this information, along with an identity alignment, to redefine the Clp subfamilies.
H-NS Regulates DNA Repair in
Shigella
Authors: Palchaudhuri Sunil , Tominna Brandon , Leon Myron A. 
Published: December 2019

ABSTRACT

We report a new role for H-NS in
Shigella
spp.: suppression of repair of DNA damage after UV irradiation. H-NS-mediated suppression of virulence gene expression is thermoregulated in
Shigella
, being functional at 30°C and nonfunctional at 37 to 40°C. We find that H-NS-mediated suppression of DNA repair after UV irradiation is also thermoregulated. Thus,
Shigella flexneri
M90T, incubated at 37 or 40°C postirradiation, shows up to 30-fold higher survival than when incubated at 30°C postirradiation. The
hns
mutants BS189 and BS208, both of which lack functional H-NS, show a high rate of survival (no repression) whether incubated at 30 or 40°C postirradiation. Suppression of DNA repair by H-NS is not mediated through genes on the invasion plasmid of
S. flexneri
M90T, since BS176, cured of plasmid, behaves identically to the parental M90T. Thus, in
Shigella
the nonfunctionality of H-NS permits enhanced DNA repair at temperatures encountered in the human host. However, pathogenic
Escherichia coli
strains (enteroinvasive and enterohemorrhagic
E. coli
) show low survival whether incubated at 30 or 40°C postirradiation.
E. coli
K-12 shows markedly different behavior; high survival postirradiation at both 30 and 40°C. These K-12 strains were originally selected from
E. coli
organisms subjected to both UV and X irradiation. Therefore, our data suggest that repair processes, extensively described for laboratory strains of
E. coli
, require experimental verification in pathogenic strains which were not adapted to irradiation.
Nucleotide Sequence and Characterization of
cdrA
, a Cell Division-Related Gene of
Helicobacter pylori
Authors: Takeuchi Hiroaki , Shirai Mutsunori , Akada Junko K. , Tsuda Masataka , Nakazawa Teruko 
Published: December 2019

ABSTRACT

We identified cell division-related gene
cdrA
in
Helicobacter pylori
HPK5. The putative gene product, CdrA, is a 367-amino-acid polypeptide that exhibited a high level of homology to conserved hypothetical ATP-binding protein HP0066 of
H. pylori
26695, except in the N-terminal region, and showed some similarity to the FtsK/SpoIIIE family proteins. We isolated a
cdrA
-disrupted mutant by allelic exchange mutagenesis. Because of the low transformation frequency, the possibility that a suppressing mutation would be found in the obtained
cdrA
mutant was discussed. A repressive role for CdrA on cell division was suggested by the observations that the wild-type strain formed filamentous cells in a high-salt level medium at early stationary phase, while a
cdrA
-disrupted mutant did not show such an abnormality. In addition, the wild-type strain adopted coccoid forms in the stationary phase, whereas the
cdrA
-disrupted mutant remained mostly as short rods. Furthermore, the
cdrA
-disrupted mutant regained the filamentation phenotype when the intact
cdrA
gene was introduced by allelic exchange. Taken together, these observations show that the
cdrA
gene plays an important role in the cell growth of
H. pylori
.
Cloning and Characterization of
PRA1
, a Gene Encoding a Novel pH-Regulated Antigen of
Candida albicans
Authors: Sentandreu Maria , Elorza M. Victoria , Sentandreu Rafael , Fonzi William A. 
Published: December 2019

ABSTRACT

Candida albicans
is an opportunistic fungal pathogen of humans. The cell wall of the organism defines the interface between the pathogen and host tissues and is likely to play an essential and pivotal role in the host-pathogen interaction. The components of the cell wall critical to this interaction are undefined. Immunoscreening of a lambda expression library with sera raised against mycelial cell walls of
C. albicans
was used to identify genes encoding cell surface proteins. One of the positive clones represented a candidal gene that was differentially expressed in response to changes in the pH of the culture medium. Maximal expression occurred at neutral pH, with no expression detected below pH 6.0. On the basis of the expression pattern, the corresponding gene was designated
PRA1
, for pH-regulated antigen. The protein predicted from the nucleotide sequence was 299 amino acids long with motifs characteristic of secreted glycoproteins. The predicted surface localization and N glycosylation of the protein were directly demonstrated by cell fractionation and immunoblot analysis. Deletion of the gene imparted a temperature-dependent defect in hypha formation, indicating a role in morphogenesis. The
PRA1
protein was homologous to surface antigens of
Aspergillus
spp. which react with serum from aspergillosis patients, suggesting that the
PRA1
protein may have a role in the host-parasite interaction during candidal infection.
Expression Cloning of a
Pseudomonas
Gene Encoding a Hydroxydecanoyl-Acyl Carrier Protein-Dependent UDP-GlcNAc Acyltransferase
Authors: Dotson Garry D. , Kaltashov Igor A. , Cotter Robert J. , Raetz Christian R. H. 
Published: December 2019

ABSTRACT

UDP-
N
-acetylglucosamine-3-
O
-acyltransferase (UDP-GlcNAc acyltransferase) catalyzes the first step of lipid A biosynthesis (M. S. Anderson and C. R. H. Raetz, J. Biol. Chem. 262:5159–5169, 1987). We here report the isolation of the
lpxA
gene of
Pseudomonas aeruginosa
from a library of
Pseudomonas
strain PAO1 expressed in
Escherichia coli
LE392 (J. Lightfoot and J. S. Lam, J. Bacteriol. 173:5624–5630, 1991).
Pseudomonas lpxA
encodes a 10-carbon-specific UDP-GlcNAc acyltransferase, whereas the
E. coli
transferase is selective for a 14-carbon acyl chain. Recombinant cosmid 1137 enabled production of a 3-hydroxydecanoyl-specific UDP-GlcNAc acyltransferase in
E. coli
. It was identified by assaying lysozyme-EDTA lysates of individual members of the library with 3-hydroxydecanoyl-acyl carrier protein (ACP) as the substrate. Cosmid 1137 contained a 20-kb insert of
P. aeruginosa
DNA. The
lpxA
gene region was localized to a 1.3-kb
Sal
I-
Pst
I fragment. Sequencing revealed that it contains one complete open reading frame (777 bp) encoding a new
lpxA
homolog. The predicted
Pseudomonas
LpxA is 258 amino acids long and contains 21 complete hexapeptide repeating units, spaced in approximately the same manner as the 24 repeats of
E. coli
LpxA. The
P. aeruginosa
UDP-GlcNAc acyltransferase is 54% identical and 67% similar to the
E. coli
enzyme. A plasmid (pGD3) containing the 1.3-kb
Sal
I-
Pst
I fragment complemented
E. coli
RO138, a temperature-sensitive mutant harboring
lpxA2
. LpxA assays of extracts of this construct indicated that it is >1,000-fold more selective for 3-hydroxydecanoyl-ACP than for 3-hydroxymyristoyl-ACP. Mass spectrometry of lipid A isolated from this strain by hydrolysis at pH 4.5 revealed [M-H]
−
1,684.5 (versus 1,796.5 for wild-type lipid A), consistent with 3-hydroxydecanoate rather than 3-hydroxymyristate at positions 3 and 3′.
Identification of therrmAGene Encoding the 23S rRNA m1G745 Methyltransferase inEscherichia coliand Characterization of an m1G745-Deficient Mutant
Authors: Gustafsson Claes , Persson Britt C. 
Published: December 2019

ABSTRACTAnEscherichia colimutant lacking the modified nucleotide m1G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83–100, 1970). In this study, we localize the position of the m1G to nucleotide 745 in 23S rRNA and characterize a mutant deficient in this modification. This mutant shows a 40% decreased growth rate in rich media, a drastic reduction in loosely coupled ribosomes, a 20% decreased polypeptide chain elongation rate, and increased resistance to the ribosome binding antibiotic viomycin. TherrmAgene encoding 23S rRNA m1G745 methyltransferase was mapped to bp 1904000 on theE. colichromosome and identified to be identical to the previously sequenced geneyebH.
Demonstration that the TyrR Protein and RNA Polymerase Complex Formed at the Divergent P3 Promoter Inhibits Binding of RNA Polymerase to the Major Promoter, P1, of the
aroP
Gene of
Escherichia coli
Authors: Wang Peixiang , Yang Ji , Ishihama Akira , Pittard A. J. 
Published: December 2019

ABSTRACT

In previous studies, we have identified three promoters (P1, P2, and P3) in the regulatory region of the
Escherichia coli aroP
gene (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206–4212, 1997). Both P1 and P2 can direct mRNA synthesis for
aroP
expression, whereas P3 is a divergent promoter which overlaps with P1. The repression of transcription from the major promoter, P1, has been postulated to involve the activation of the divergent promoter, P3, by the TyrR protein (P. Wang, J. Yang, B. Lawley, and A. J. Pittard, J. Bacteriol. 179:4213–4218, 1997). In the present study, we confirmed the proposed mechanism of P3-mediated repression of P1 transcription by studying the binding of RNA polymerase to the promoters P1 and P3 in vitro in the presence and absence of TyrR protein and its cofactors. Our results show that (i) only one RNA polymerase molecule can bind to the DNA fragment carrying the
aroP
regulatory region, (ii) RNA polymerase has a higher affinity for P1 than for either P2 or P3 and binds to P1 in the absence of TyrR protein, (iii) in the presence of TyrR protein and its cofactor, phenylalanine or tyrosine, RNA polymerase preferentially binds to P3, and (iv) RNA polymerase does not respond to the activation-defective mutant TyrR protein TyrR-RQ10 and remains bound to P1 in the presence of TyrR-RQ10 and either of the cofactors.
Relationship between Spontaneous Aminoglycoside Resistance in
Escherichia coli
and a Decrease in Oligopeptide Binding Protein
Authors: Kashiwagi Keiko , Tsuhako Maria Heloisa , Sakata Kaori , Saisho Tomoko , Igarashi Atsuko , Pinto da Costa Sérgio Olavo , Igarashi Kazuei 
Published: December 2019

ABSTRACT

Changes in the amount of oligopeptide binding protein (OppA) in spontaneous kanamycin-resistant mutants of
Escherichia coli
were investigated. Among 20 colonies obtained from 10
8
cells cultured in the presence of 20 μg of kanamycin/ml, 1 colony had no detectable OppA and 7 colonies were mutants with reduced amounts of OppA. Sensitivity of wild-type cells to kanamycin increased slightly by transformation of the
oppA
gene, but the sensitivity of the mutants increased greatly by the transformation. A mutant with no OppA was found to be a nonsense mutant of the
oppA
gene at amino acid position 166. In a mutant having a reduced level of OppA, the reduction was due to the decrease in OppA synthesis at the translational level. These mutants were also resistant to other aminoglycoside antibiotics, including streptomycin, neomycin, and isepamicin. Isepamicin uptake activities decreased greatly in these two kinds of mutants. The results support the proposition that aminoglycoside antibiotics are transported into cells by the oligopeptide transport system, and that transport is an important factor for spontaneous resistance to aminoglycoside antibiotics.
Identification of Chlorobenzene Dioxygenase Sequence Elements Involved in Dechlorination of 1,2,4,5-Tetrachlorobenzene
Authors: Beil Stefan , Mason Jeremy R. , Timmis Kenneth N. , Pieper Dietmar H. 
Published: December 2019

ABSTRACT

The TecA chlorobenzene dioxygenase and the TodCBA toluene dioxygenase exhibit substantial sequence similarity yet have different substrate specificities.
Escherichia coli
cells producing recombinant TecA enzyme dioxygenate and simultaneously eliminate a halogen substituent from 1,2,4,5-tetrachlorobenzene but show no activity toward benzene, whereas those producing TodCBA dioxygenate benzene but not tetrachlorobenzene. A hybrid TecA dioxygenase variant containing the large α-subunit of the TodCBA dioxygenase exhibited a TodCBA dioxygenase specificity. Acquisition of dehalogenase activity was achieved by replacement of specific
todC1
α-subunit subsequences by equivalent sequences of the
tecA1
α-subunit. Substrate transformation specificities and rates by
E. coli
resting cells expressing hybrid systems were analyzed by high-performance liquid chromatography. This allowed the identification of both a single amino acid and potentially interacting regions required for dechlorination of tetrachlorobenzene. Hybrids with extended substrate ranges were generated that exhibited activity toward both benzene and tetrachlorobenzene. The regions determining substrate specificity in (chloro)benzene dioxygenases appear to be different from those previously identified in biphenyl dioxygenases.
Mechanism of Osmotic Activation of the Quaternary Ammonium Compound Transporter (QacT) of
Lactobacillus plantarum
Authors: Glaasker Erwin , Heuberger Esther H. M. L. , Konings Wil N. , Poolman Bert 
Published: December 2019

ABSTRACT

The accumulation of quaternary ammonium compounds in
Lactobacillus plantarum
is mediated via a single transport system with a high affinity for glycine betaine (apparent

K
m

of 18 μM) and carnitine and a low affinity for proline (apparent

K
m

of 950 μM) and other analogues. Mutants defective in the uptake of glycine betaine were generated by UV irradiation and selected on the basis of resistance to dehydroproline (DHP), a toxic proline analogue. Three independent DHP-resistant mutants showed reduced glycine betaine uptake rates and accumulation levels but behaved similarly to the wild type in terms of direct activation of uptake by high-osmolality conditions. Kinetic analysis of glycine betaine uptake and efflux in the wild-type and mutant cells is consistent with one uptake system for quaternary ammonium compounds in
L. plantarum
and a separate system(s) for their excretion. The mechanism of osmotic activation of the quaternary ammonium compound transport system (QacT) was studied. It was observed that the uptake rates were inhibited by the presence of internal substrate. Upon raising of the medium osmolality, the QacT system was rapidly activated (increase in maximal velocity) through a diminished inhibition by
trans
substrate as well as an effect that is independent of intracellular substrate. We also studied the effects of the cationic amphipath chlorpromazine, which inserts into the cytoplasmic membrane and thereby influences the uptake and efflux of glycine betaine. The results provide further evidence for the notion that the rapid efflux of glycine betaine upon osmotic downshock is mediated by a channel protein that is responding to membrane stretch or tension. The activation of QacT upon osmotic upshock seems to be brought about by a turgor-related parameter other than membrane stretch or tension.