bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conserved Cytoplasmic Loops Are Important for both the Transport and Chemotaxis Functions of PcaK, a Protein fromPseudomonas putida with 12 Membrane-Spanning Regions
Authors: Ditty Jayna L. , Harwood Caroline S. 
Published: December 2019

ABSTRACTChemotaxis to the aromatic acid 4-hydroxybenzoate (4-HBA) byPseudomonas putidais mediated by PcaK, a membrane-bound protein that also functions as a 4-HBA transporter. PcaK belongs to the major facilitator superfamily (MFS) of transport proteins, none of which have so far been implicated in chemotaxis. Work with two well-studied MFS transporters, LacY (the lactose permease) and TetA (a tetracycline efflux protein), has revealed two stretches of amino acids located between the second and third (2-3 loop) and the eighth and ninth (8-9 loop) transmembrane regions that are required for substrate transport. These sequences are conserved among most MFS transporters, including PcaK. To determine if PcaK has functional requirements similar to those of other MFS transport proteins and to analyze the relationship between the transport and chemotaxis functions of PcaK, we generated strains with mutations in amino acid residues located in the 2-3 and 8-9 loops of PcaK. The mutant proteins were analyzed in 4-HBA transport and chemotaxis assays. Cells expressing mutant PcaK proteins had a range of phenotypes. Some transported at wild-type levels, while others were partially or completely defective in 4-HBA transport. An aspartate residue in the 8-9 loop that has no counterpart in LacY and TetA, but is conserved among members of the aromatic acid/H+symporter family of the MFS, was found to be critical for 4-HBA transport. These results indicate that conserved amino acids in the 2-3 and 8-9 loops of PcaK are required for 4-HBA transport. Amino acid changes that decreased 4-HBA transport also caused a decrease in 4-HBA chemotaxis, but the effect on chemotaxis was sometimes slightly more severe. The requirement of PcaK for both 4-HBA transport and chemotaxis demonstrates thatP. putidahas a chemoreceptor that differs from the classical chemoreceptors described forEscherichia coliandSalmonella typhimurium.
Demonstration by Mass Spectrometry that Purified Native Treponema pallidum Rare Outer Membrane Protein 1 (Tromp1) Has a Cleaved Signal Peptide
Authors: Blanco David R. , Whitelegge Julian P. , Miller James N. , Lovett Michael A. 
Published: December 2019

ABSTRACTPurified native Tromp1 was subjected to mass spectrometric analysis in order to determine conclusively whether this protein possesses a cleaved or uncleaved signal peptide. The molecular masses of Tromp1, threeTreponema pallidumlipoproteins, and a bovine serum albumin (BSA) control were determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The molecular masses of all of theT. pallidumlipoproteins and BSA were within 0.7% of their respective calculated masses. The molecular mass of Tromp1 was 31,510 Da, which is consistent with a signal-less form of Tromp1, given a calculated mass of unprocessed Tromp1 of 33,571 Da, a difference of 2,061 Da (a 6.5% difference). Purified native Tromp1 was also subjected to MALDI-TOF analysis in comparison to recombinant Tromp1 following cyanogen bromide cleavage, which further confirmed the identity of Tromp1 and showed that native Tromp1 was not degraded at the carboxy terminus. These studies confirm that Tromp1 is processed and does not contain an uncleaved signal peptide as previously reported.
Identification of a Novel Penicillin-Binding Protein from Helicobacter pylori
Authors: Krishnamurthy Partha , Parlow Mary H. , Schneider John , Burroughs Stephanie , Wickland Catherine , Vakil Nimish B. , Dunn Bruce E. , Phadnis Suhas H. 
Published: December 2019

ABSTRACTTheHelicobacter pylorigenome encodes four penicillin-binding proteins (PBPs). PBPs 1, 2, and 3 exhibit similarities to known PBPs. The sequence of PBP 4 is unique in that it displays a novel combination of two highly conserved PBP motifs and an absence of a third motif. Expression of PBP 4, but not PBP 1, 2, or 3, is significantly increased during mid- to late-log-phase growth.
In Vivo Observation of Cell Division of Anaerobic Hyperthermophiles by Using a High-Intensity Dark-Field Microscope
Authors: Horn Christian , Paulmann Bernd , Kerlen Gertraude , Junker Norbert , Huber Harald 
Published: December 2019

ABSTRACTTo study growth and cell division of anaerobic hyperthermophilic archaea in vivo, a cultivation technique using glass capillaries was developed. At temperatures of 90 to 98°C, at least 10 successive cell divisions ofPyrodictium abyssiTAG 11 were documented. Cells divide by binary fission. Visualized under a modified dark-field microscope, the formation of cannulae, which finally connected all cells, was observed. The cannulae elongated at 1.0 to 1.5 μm/min and reached final lengths of between 30 and 150 μm. A “snapping division”-like mode of cell fission was discovered forThermoproteus tenax.
Effect of Ionic Strength on Initial Interactions of
Escherichia coli
with Surfaces, Studied On-Line by a Novel Quartz Crystal Microbalance Technique
Authors: Otto Karen , Elwing Hans , Hermansson Malte 
Published: December 2019

ABSTRACT

A novel quartz crystal microbalance (QCM) technique was used to study the adhesion of nonfimbriated and fimbriated
Escherichia coli
mutant strains to hydrophilic and hydrophobic surfaces at different ionic strengths. This technique enabled us to measure both frequency shifts (Δ
f
), i.e., the increase in mass on the surface, and dissipation shifts (Δ
D
), i.e., the viscoelastic energy losses on the surface. Changes in the parameters measured by the extended QCM technique reflect the dynamic character of the adhesion process. We were able to show clear differences in the viscoelastic behavior of fimbriated and nonfimbriated cells attached to surfaces. The interactions between bacterial cells and quartz crystal surfaces at various ionic strengths followed different trends, depending on the cell surface structures in direct contact with the surface. While Δ
f
and Δ
D
per attached cell increased for nonfimbriated cells with increasing ionic strengths (particularly on hydrophobic surfaces), the adhesion of the fimbriated strain caused only low-level frequency and dissipation shifts on both kinds of surfaces at all ionic strengths tested. We propose that nonfimbriated cells may get better contact with increasing ionic strengths due to an increased area of contact between the cell and the surface, whereas fimbriated cells seem to have a flexible contact with the surface at all ionic strengths tested. The area of contact between fimbriated cells and the surface does not increase with increasing ionic strengths, but on hydrophobic surfaces each contact point seems to contribute relatively more to the total energy loss. Independent of ionic strength, attached cells undergo time-dependent interactions with the surface leading to increased contact area and viscoelastic losses per cell, which may be due to the establishment of a more intimate contact between the cell and the surface. Hence, the extended QCM technique provides new qualitative information about the direct contact of bacterial cells to surfaces and the adhesion mechanisms involved.
Suppression of Nonsense Mutations Induced by Expression of an RNA Complementary to a Conserved Segment of 23S rRNA
Authors: Chernyaeva Natalya S. , Murgola Emanuel J. , Mankin Alexander S. 
Published: December 2019

ABSTRACT

We identified a short RNA fragment, complementary to the
Escherichia coli
23S rRNA segment comprising nucleotides 735 to 766 (in domain II), which when expressed in vivo results in the suppression of UGA nonsense mutations in two reporter genes. Neither UAA nor UAG mutations, examined at the same codon positions, were suppressed by the expression of this antisense rRNA fragment. Our results suggest that a stable phylogenetically conserved hairpin at nucleotides 736 to 760 in 23S rRNA, which is situated close to the peptidyl transferase center, may participate in one or more specific interactions during peptide chain termination.
A- and T-Tract-Mediated Intrinsic Curvature in Native DNA between the Binding Site of the Upstream Activator NtrC and the
nifLA
Promoter of
Klebsiella pneumoniae
Facilitates Transcription
Authors: Cheema Amrita Kaur , Choudhury Nirupam Roy , Das H. K. 
Published: December 2019

ABSTRACT

The
nif
promoters of
Klebsiella pneumoniae
must be activated by proteins bound to upstream sequences which are thought to interact with the ς
54
-RNA polymerase holoenzyme by DNA looping. NifA is the activator for most of the promoters, and integration host factor (IHF) mediates the DNA looping. While NtrC is the activator for the
nifLA
promoter, no IHF appears to be involved. There are two A tracts and one T tract between the upstream enhancer and the
nifLA
promoter. This DNA segment exhibits anomalous electrophoretic mobility, suggesting intrinsic sequence-induced curvature in the DNA. On the one hand, mutation of the A tracts or T tract individually or together, or deletion of the A tracts and the T tract reduces the anomaly; on the other hand, creation of two additional A tracts enhances the anomaly. Intrinsic curvature in the DNA has been confirmed by circular permutation analysis after cloning the DNA fragment in the vector pBend 2 and also by electron microscopy. Computer simulation with the DNA base sequence is also suggestive of intrinsic curvature. A transcriptional fusion with the
Escherichia coli lacZ
gene of the DNA fragment containing the
nifLA
promoter and the wild-type or the mutated upstream sequences was constructed, and in vivo transcription in
K. pneumoniae
and
E. coli
was monitored. There was indeed very good correlation between the extent of intrinsic curvature of the DNA and transcription from the promoter, suggesting that DNA curvature due to the A tracts and the T tract was necessary for transcription in vivo from the
nifLA
promoter of
K. pneumoniae.
Direct Probing of the Surface Ultrastructure and Molecular Interactions of Dormant and Germinating Spores of
Phanerochaete chrysosporium
Authors: Dufrêne Yves F. , Boonaert Christophe J. P. , Gerin Patrick A. , Asther Marcel , Rouxhet Paul G. 
Published: December 2019

ABSTRACT

Atomic force microscopy (AFM) has been used to probe, under physiological conditions, the surface ultrastructure and molecular interactions of spores of the filamentous fungus
Phanerochaete chrysosporium
. High-resolution images revealed that the surface of dormant spores was uniformly covered with rodlets having a periodicity of 10 ± 1 nm, which is in agreement with earlier freeze-etching measurements. In contrast, germinating spores had a very smooth surface partially covered with rough granular structures. Force-distance curve measurements demonstrated that the changes in spore surface ultrastructure during germination are correlated with profound modifications of molecular interactions: while dormant spores showed no adhesion with the AFM probe, germinating spores exhibited strong adhesion forces, of 9 ± 2 nN magnitude. These forces are attributed to polysaccharide binding and suggested to be responsible for spore aggregation. This study represents the first direct characterization of the surface ultrastructure and molecular interactions of living fungal spores at the nanometer scale and offers new prospects for mapping microbial cell surface properties under native conditions.
Role of the Sporulation Protein BofA in Regulating Activation of the
Bacillus subtilis
Developmental Transcription Factor ς
K
Authors: Resnekov Orna 
Published: December 2019

ABSTRACT

During sporulation, the
Bacillus subtilis
transcription factor ς
K
is activated by regulated proteolytic processing. I have used a system that facilitates the analysis of the contributions of a modified form of the processing enzyme, SpoIVFB-GFP, and the regulatory proteins BofA and SpoIVFA to the conversion of pro-ς
K
to ς
K
. The results show that in the presence of BofA, SpoIVFA levels increase by greater than 20-fold, SpoIVFA is substantially stabilized, and pro-ς
K
processing is inhibited. In addition, enhanced accumulation of the SpoIVFA protein in the absence of BofA (achieved through the use of an
ftsH
null mutation) substantially inhibits pro-ς
K
processing. These results suggest that during growth, increased accumulation of the SpoIVFA protein inhibits the activity of SpoIVFB-GFP and regulates the activation of ς
K
.
Molecular Analysis of the Gene Encoding a Novel Transglycosylative Enzyme from
Alteromonas
sp. Strain O-7 and Its Physiological Role in the Chitinolytic System
Authors: Tsujibo Hiroshi , Kondo Norihiko , Tanaka Keiko , Miyamoto Katsushiro , Baba Nao , Inamori Yoshihiko 
Published: December 2019

ABSTRACT

We purified from the culture supernatant of
Alteromonas
sp. strain O-7 and characterized a transglycosylating enzyme which synthesized β-(1→6)-(GlcNAc)
2
, 2-acetamido-6-
O
-(2-acetamido-2-deoxy-β-
d
-glucopyranosyl)-2-deoxyglucopyranose from β-(1→4)-(GlcNAc)
2
. The gene encoding a novel transglycosylating enzyme was cloned into
Escherichia coli
, and its nucleotide sequence was determined. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 99,560 Da which corresponds very closely with the molecular mass of the cloned enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the cloned enzyme was much larger than that of enzyme (70 kDa) purified from the supernatant of this strain. These results suggest that the native enzyme was the result of partial proteolysis occurring in the N-terminal region. The enzyme showed significant sequence homology with several bacterial β-
N
-acetylhexosaminidases which belong to family 20 glycosyl hydrolases. However, this novel enzyme differs from all reported β-
N
-acetylhexosaminidases in its substrate specificity. To clarify the role of the enzyme in the chitinolytic system of the strain, the effect of β-(1→6)-(GlcNAc)
2
on the induction of chitinase was investigated. β-(1→6)-(GlcNAc)
2
induced a level of production of chitinase similar to that induced by the medium containing chitin. On the other hand, GlcNAc, (GlcNAc)
2
, and (GlcNAc)
3
conversely repressed the production of chitinase to below the basal level of chitinase activity produced constitutively in medium without a carbon source.
Identification of the Ω4499 Regulatory Region Controlling Developmental Expression of a
Myxococcus xanthus
Cytochrome P-450 System
Authors: Fisseha Makda , Biran Dvora , Kroos Lee 
Published: December 2019

ABSTRACT

Ω4499 is the site of a Tn
5 lac
insertion in the
Myxococcus xanthus
chromosome that fuses
lacZ
expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C signaling, are required for normal expression of Tn
5 lac
Ω4499. The DNA upstream of the Ω4499 insertion has been cloned, and the promoter has been localized. Analysis of the DNA sequence downstream of the promoter revealed one complete open reading frame and a second partial open reading frame that is interrupted by Tn
5 lac
Ω4499. The predicted products of these open reading frames are highly similar to reductase and oxidase components of bacterial cytochrome P-450 systems, which allow catabolism or anabolism of unusual compounds. However, the function of the gene products of the Ω4499 locus remains unclear because
M. xanthus
containing Tn
5 lac
Ω4499 exhibits no apparent defect in growth, developmental aggregation, fruiting body formation, or sporulation. Deletion analysis of the Ω4499 regulatory region showed that multiple DNA elements spanning more than 500 bp upstream of the transcriptional start site contribute to developmental promoter activity. At least two DNA elements, one downstream of −49 bp and one between −49 and −218 bp, boosted activity of the promoter in response to intercellular C signaling. Three sequences in the Ω4499 promoter region, centered at −55, −33, and −1 bp, nearly match a 7-bp sequence found in other C signal-dependent promoters. We propose that these sequences, matching the consensus sequence 5′-CAYYCCY-3′, be called C box sequences, and we speculate that these sequences are
cis
-acting regulatory elements important for the expression of
M. xanthus
genes that depend upon intercellular C signaling during development.
Genomic Plasticity in Natural Populations of
Bordetella pertussis
Authors: Stibitz Scott , Yang Mei-Shin 
Published: December 2019

ABSTRACT

We determined the genomic organization of 14 clinical strains of
Bordetella pertussis
isolated over an 18-month period in Alberta, Canada. The maps of these 14 strains, while demonstrating general similarity of gene order, display a number of examples of genomic rearrangements in the form of large chromosomal inversions.
Isolation, Characterization, and Localization of a Capsule-Associated Gene,
CAP10
, of
Cryptococcus neoformans
Authors: Chang Y. C. , Kwon-Chung K. J. 
Published: December 2019

ABSTRACT

Cryptococcus neoformans
is a pathogenic fungus which most commonly affects the central nervous system and causes fatal meningoencephalitis primarily in patients with AIDS. This fungus produces a thick extracellular polysaccharide capsule which is well recognized as a virulence factor. Here, we describe the isolation and characterization of a novel gene,
CAP10
, which is required for capsule formation. Complementation of the acapsular
cap10
mutant produced an encapsulated strain and the deletion of
CAP10
from a wild strain resulted in an acapsular phenotype. The molecular mass of the hemagglutinin epitope-tagged Cap10p is about 73 kDa, which is similar to the size predicted from sequence analysis. When
CAP10
was fused with a hybrid green fluorescent protein construct, the fluorescence signals appeared as patches in the cytoplasm. Using a reporter gene construct, we found that
CAP10
was expressed at high levels in late-stationary-phase cells. In addition, we found that the expression levels of
CAP10
are modulated by the transcriptional factor
STE12
α. Deletion of
STE12
α downregulated the expression levels of
CAP10
while overexpression of
STE12
α upregulated the expression levels of
CAP10
. Animal model studies indicate that deletion of the
CAP10
gene results in the loss of virulence, and complementation of the acapsular phenotype of
cap10
restores virulence. Thus,
CAP10
is required for capsule formation and virulence.
In Vitro Studies on the Initial Reactions of Anaerobic Ethylbenzene Mineralization
Authors: Johnson Hope A. , Spormann Alfred M. 
Published: December 2019

ABSTRACT

Anaerobic mineralization of ethylbenzene by the denitrifying bacterium
Azoarcus
sp. strain EB1 was recently shown to be initiated by dehydrogenation of ethylbenzene to 1-phenylethanol. 1-Phenylethanol is converted to benzoate (benzoyl coenzyme A) via acetophenone as transient intermediate. We developed in vitro assays to examine ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities in cell extracts of this strain. With
p
-benzoquinone as the electron acceptor, cell extracts of
Azoarcus
sp. strain EB1 catalyzed ethylbenzene oxidation at a specific rate of 10 nmol min
−1
[mg of protein]
−1
and an apparent

K
m

for ethylbenzene of approximately 60 μM. The membrane-associated ethylbenzene dehydrogenase activity was found to oxidize 4-fluoroethylbenzene and propylbenzene but was unable to transform 4-chloro-ethylbenzene, the ethyltoluenes, and styrene. Enzymatic ethylbenzene oxidation was stereospecific, with (
S
)-(−)-1-phenylethanol being the only enantiomer detected by chiral high-pressure liquid chromatography analysis. Moreover, cell extracts catalyzed the oxidation of (
S
)-(−)-1-phenylethanol but not of (
R
)-(+)-1-phenylethanol to acetophenone. When cell extracts were dialyzed, (
S
)-(−)-1-phenylethanol oxidation occurred only in the presence of NAD
+
, suggesting that NAD
+
is the physiological electron acceptor of 1-phenylethanol dehydrogenase. Both ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities were present in
Azoarcus
sp. strain EB1 cells that were grown anaerobically on ethylbenzene, 1-phenylethanol, and acetophenone, but these activities were absent in benzoate-grown cells.
Identification of Methylation Sites and Effects of Phototaxis Stimuli on Transducer Methylation in
Halobacterium salinarum
Authors: Perazzona Bastianella , Spudich John L. 
Published: December 2019

ABSTRACT

The two transducers in the phototaxis system of the archaeon
Halobacterium salinarum
, HtrI and HtrII, are methyl-accepting proteins homologous to the chemotaxis transducers in eubacteria. Consensus sequences predict three glutamate pairs containing potential methylation sites in HtrI and one in HtrII. Mutagenic substitution of an alanine pair for one of these, Glu265-Glu266, in HtrI and for the homologous Glu513-Glu514 in HtrII eliminated methylation of these two transducers, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autofluorography. Photostimulation of the repellent receptor sensory rhodopsin II (SRII) induced reversible demethylation of HtrII, while no detectable change in the extent of methylation of HtrI was observed in response to stimulation of its cognate sensory rhodopsin, the attractant receptor SRI. Cells containing HtrI or HtrII with all consensus sites replaced by alanine still exhibited phototaxis responses and behavioral adaptation, and methanol release assays showed that methyl group turnover was still induced in response to photostimulation of SRI or SRII. By pulse-chase experiments with in vivo
l
-[
methyl
-
3
H]methionine-labeled cells, we found that repetitive photostimulation of SRI complexed with wild-type (or nonmethylatable) HtrI induced methyl group turnover in transducers other than HtrI to the same extent as in wild-type HtrI. Both attractant and repellent stimuli cause a transient increase in the turnover rate of methyl groups in wild-type
H. salinarum
cells. This result is unlike that obtained with
Escherichia coli
, in which attractant stimuli decrease and repellent stimuli increase turnover rate, and is similar to that obtained with
Bacillus subtilis
, which also shows turnover rate increases regardless of the nature of the stimulus. We found that a CheY deletion mutant of
H. salinarum
exhibited the
E. coli
-like asymmetric pattern, as has recently also been observed in
B. subtilis
. Further, we demonstrate that the CheY-dependent feedback effect does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell.