bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a
Pseudomonas aeruginosa
Fatty Acid Biosynthetic Gene Cluster: Purification of Acyl Carrier Protein (ACP) and Malonyl-Coenzyme A:ACP Transacylase (FabD)
Authors: Kutchma Alecksandr J. , Hoang Tung T. , Schweizer Herbert P. 
Published: December 2019

ABSTRACT

A DNA fragment containing the
Pseudomonas aeruginosa fabD
(encoding malonyl-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase),
fabG
(encoding β-ketoacyl-ACP reductase),
acpP
(encoding ACP), and
fabF
(encoding β-ketoacyl-ACP synthase II) genes was cloned and sequenced. This
fab
gene cluster is delimited by the
plsX
(encoding a poorly understood enzyme of phospholipid metabolism) and
pabC
(encoding 4-amino-4-deoxychorismate lyase) genes; the
fabF
and
pabC
genes seem to be translationally coupled. The
fabH
gene (encoding β-ketoacyl-ACP synthase III), which in most gram-negative bacteria is located between
plsX
and
fabD
, is absent from this gene cluster. A chromosomal temperature-sensitive
fabD
mutant was obtained by site-directed mutagenesis that resulted in a W258Q change. A chromosomal
fabF
insertion mutant was generated, and the resulting mutant strain contained substantially reduced levels of
cis
-vaccenic acid. Multiple attempts aimed at disruption of the chromosomal
fabG
gene were unsuccessful. We purified FabD as a hexahistidine fusion protein (H
6
-FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression and purification scheme that should be applicable to ACP from other bacteria. Matrix-assisted laser desorption–ionization spectroscopy, native polyacrylamide electrophoresis, and amino-terminal sequencing revealed that (i) most of the purified ACP was properly modified with its 4′-phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal methionine was removed. In an in vitro system, purified ACP functioned as acyl acceptor and H
6
-FabD exhibited malonyl-CoA:ACP transacylase activity.
Clustering of the Chemoreceptor Complex in
Escherichia coli
Is Independent of the Methyltransferase CheR and the Methylesterase CheB
Authors: Lybarger Suzanne R. , Maddock Janine R. 
Published: December 2019

ABSTRACT

The
Escherichia coli
chemoreceptors and their associated cytoplasmic proteins, CheA and CheW, cluster predominantly at the cell poles. The nature of the clustering remains a mystery. Recent studies suggest that CheR binding to and/or methylation of the chemoreceptors may play a role in chemoreceptor complex aggregation. In this study, we examined the intracellular distribution of the chemoreceptors by immunoelectron microscopy in strains lacking either the methyltransferase CheR or the methylesterase CheB. The localization data revealed that, in vivo, aggregation of the chemoreceptor complex was independent of either CheR or CheB.
TraC of IncN Plasmid pKM101 Associates with Membranes and Extracellular High-Molecular-Weight Structures in
Escherichia coli
Authors: Schmidt-Eisenlohr Heike , Domke Natalie , Baron Christian 
Published: December 2019

ABSTRACT

Conjugative transfer of IncN plasmid pKM101 is mediated by the TraI-TraII region-encoded transfer machinery components. Similar to the case for the related
Agrobacterium tumefaciens
T-complex transfer apparatus, this machinery is needed for assembly of pili to initiate cell-to-cell contact preceding DNA transfer. Biochemical and cell biological experiments presented here show extracellular localization of TraC, as suggested by extracellular complementation of TraC-deficient bacteria by helper cells expressing a functional plasmid transfer machinery (S. C. Winans, and G. C. Walker, J. Bacteriol. 161:402–410, 1985). Overexpression of TraC and its export in large amounts into the periplasm of
Escherichia coli
allowed purification by periplasmic extraction, ammonium sulfate precipitation, and column chromatography. Whereas TraC was soluble in overexpressing strains, it partly associated with the membranes in pKM101-carrying cells, possibly due to protein-protein interactions with other components of the TraI-TraII region-encoded transfer machinery. Membrane association of TraC was reduced in strains carrying pKM101 derivatives with transposon insertions in genes coding for other essential components of the transfer machinery,
traM
,
traB
,
traD
, and
traE
but not
eex
, coding for an entry exclusion protein not required for DNA transfer. Cross-linking identified protein-protein interactions of TraC in
E. coli
carrying pKM101 but not derivatives with transposon insertions in essential
tra
genes. Interactions with membrane-bound Tra proteins may incorporate TraC into a surface structure, suggested by its removal from the cell by shearing as part of a high-molecular-weight complex. Heterologous expression of TraC in
A. tumefaciens
partly compensated for the pilus assembly defect in strains deficient for its homolog VirB5, which further supported its role in assembly of conjugative pili. In addition to its association with high-molecular-weight structures, TraC was secreted into the extracellular milieu. Conjugation experiments showed that secreted TraC does not compensate transfer deficiency of TraC-deficient cells, suggesting that extracellular complementation may rely on cell-to-cell transfer of TraC only as part of a bona fide transfer apparatus.
HP0333, a Member of the
dprA
Family, Is Involved in Natural Transformation in
Helicobacter pylori
Authors: Ando Takafumi , Israel Dawn A. , Kusugami Kazuo , Blaser Martin J. 
Published: December 2019

ABSTRACT

Helicobacter pylori
is naturally competent for DNA transformation, but the mechanism by which transformation occurs is not known. For
Haemophilus influenzae
,
dprA
is required for transformation by chromosomal but not plasmid DNA, and the complete genomic sequence of
H. pylori
26695 revealed a
dprA
homolog (HP0333). Examination of genetic databases indicates that DprA homologs are present in a wide variety of bacterial species. To examine whether HP0333 has a function similar to
dprA
of
H. influenzae
, HP0333, present in each of 11 strains studied, was disrupted in two
H. pylori
isolates. For both mutants, the frequency of transformation by
H. pylori
chromosomal DNA was markedly reduced, but not eliminated, compared to their wild-type parental strains. Mutation of HP0333 also resulted in a marked decrease in transformation frequency by a shuttle plasmid (pHP1), which differs from the phenotype described in
H. influenzae
. Complementation of the mutant with HP0333 inserted in
trans
in the chromosomal
ureAB
locus completely restored the frequency of transformation to that of the wild-type strain. Thus, while
dprA
is required for high-frequency transformation, transformation also may occur independently of DprA. The presence of DprA homologs in bacteria known not to be naturally competent suggests a broad function in DNA processing.
Insertional Inactivation of Methylmalonyl Coenzyme A (CoA) Mutase and Isobutyryl-CoA Mutase Genes in
Streptomyces cinnamonensis
: Influence on Polyketide Antibiotic Biosynthesis
Authors: Vrijbloed Jan W. , Zerbe-Burkhardt Katja , Ratnatilleke Ananda , Grubelnik-Leiser Andreas , Robinson John A. 
Published: December 2019

ABSTRACT

The coenzyme B
12
-dependent isobutyryl coenzyme A (CoA) mutase (ICM) and methylmalonyl-CoA mutase (MCM) catalyze the isomerization of
n
-butyryl-CoA to isobutyryl-CoA and of methylmalonyl-CoA to succinyl-CoA, respectively. The influence that both mutases have on the conversion of
n
- and isobutyryl-CoA to methylmalonyl-CoA and the use of the latter in polyketide biosynthesis have been investigated with the polyether antibiotic (monensin) producer
Streptomyces cinnamonensis
. Mutants prepared by inserting a hygromycin resistance gene (
hygB
) into either
icmA
or
mutB
, encoding the large subunits of ICM and MCM, respectively, have been characterized. The
icmA
::
hygB
mutant was unable to grow on valine or isobutyrate as the sole carbon source but grew normally on butyrate, indicating a key role for ICM in valine and isobutyrate metabolism in minimal medium. The
mutB
::
hygB
mutant was unable to grow on propionate and grew only weakly on butyrate and isobutyrate as sole carbon sources.
13
C-labeling experiments show that in both mutants butyrate and acetoacetate may be incorporated into the propionate units in monensin A without cleavage to acetate units. Hence,
n
-butyryl-CoA may be converted into methylmalonyl-CoA through a carbon skeleton rearrangement for which neither ICM nor MCM alone is essential.
Genomic Subtraction Identifies
Salmonella typhimurium
Prophages, F-Related Plasmid Sequences, and a Novel Fimbrial Operon,
stf
, Which Are Absent in
Salmonella typhi
Authors: Emmerth Melanie , Goebel Werner , Miller Samuel I. , Hueck Christoph J. 
Published: December 2019

ABSTRACT

Salmonella typhimurium
causes systemic and fatal infection in inbred mice, while the related serotype
Salmonella typhi
is avirulent for mammals other than humans. In order to identify genes from the virulent strain
S. typhimurium
ATCC 14028 that are absent in
S. typhi
Ty2, and therefore might be involved in
S. typhimurium
mouse virulence, a PCR-supported genomic subtractive hybridization procedure was employed. We have identified a novel putative fimbrial operon,
stfACDEFG
, located at centisome 5 of the
S. typhimurium
chromosome, which is absent in
S. typhi
,
Salmonella arizonae
, and
Salmonella bongori
but was detected in several other
Salmonella
serotypes. The fimbrial genes represent a genomic insertion in
S. typhimurium
compared to the respective region between
fhuB
and
hemL
in
Escherichia coli
K-12. In addition, the subtraction procedure yielded F plasmid-related sequences from the
S. typhimurium
virulence plasmid, a number of DNA fragments representing parts of lambdoid prophages and putative sugar transporters, and several fragments with unknown sequences. The majority of subtracted chromosomal sequences map to three distinct locations, around centisomes 5, 27, and 57.
Changes in Cell Size and DNA Content in
Sulfolobus
Cultures during Dilution and Temperature Shift Experiments
Authors: Hjort Karin , Bernander Rolf 
Published: December 2019

ABSTRACT

Stationary-phase cultures of different hyperthermophilic species of the archaeal genus
Sulfolobus
were diluted into fresh growth medium and analyzed by flow cytometry and phase-fluorescence microscopy. After dilution, cellular growth started rapidly but no nucleoid partition, cell division, or chromosome replication took place until the cells had been increasing in size for several hours. Initiation of chromosome replication required that the cells first go through partition and cell division, revealing a strong interdependence between these key cell cycle events. The time points at which nucleoid partition, division, and replication occurred after the dilution were used to estimate the relative lengths of the cell cycle periods. When exponentially growing cultures were diluted into fresh growth medium, there was an unexpected transient inhibition of growth and cell division, showing that the cultures did not maintain balanced growth. Furthermore, when cultures growing at 79°C were shifted to room temperature or to ice-water baths, the cells were found to “freeze” in mid-growth. After a shift back to 79°C, growth, replication, and division rapidly resumed and the mode and kinetics of the resumption differed depending upon the nature and length of the shifts. Dilution of stationary-phase cultures provides a simple protocol for the generation of partially synchronized populations that may be used to study cell cycle-specific events.
Identification of ς
B
-Dependent Genes in
Bacillus subtilis
Using a Promoter Consensus-Directed Search and Oligonucleotide Hybridization
Authors: Petersohn Anja , Bernhardt Jörg , Gerth Ulf , Höper Dirk , Koburger Torsten , Völker Uwe , Hecker Michael 
Published: December 2019

ABSTRACT

A consensus-directed search for ς
B
promoters was used to locate potential candidates for new ς
B
-dependent genes in
Bacillus subtilis
. Screening of those candidates by oligonucleotide hybridizations with total RNA from exponentially growing or ethanol-stressed cells of the wild type as well as a
sigB
mutant revealed 22 genes that required ς
B
for induction by ethanol. Although almost 50% of the proteins encoded by the newly discovered ς
B
-dependent stress genes seem to be membrane localized, biochemical functions have so far not been defined for any of the gene products. Allocation of the genes to the ς
B
-dependent stress regulon may indicate a potential function in the establishment of a multiple stress resistance. AldY and YhdF show similarities to NAD(P)-dependent dehydrogenases and YdbP to thioredoxins, supporting our suggestion that ς
B
-dependent proteins may be involved in the maintenance of the intracellular redox balance after stress.
Multiple Mechanisms Are Used for Growth Rate and Stringent Control of
leuV
Transcriptional Initiation in
Escherichia coli
Authors: Pokholok Dmitry K. , Redlak Maria , Turnbough Charles L. , Dylla Sara , Holmes Walter M. 
Published: December 2019

ABSTRACT

Expression of the
Escherichia coli leuV
operon, which contains three tRNA
1
Leu
genes, is regulated by several mechanisms including growth-rate-dependent control (GRDC) and stringent control (SC). Structural variants of the
leuV
promoter which differentially affect these regulatory responses have been identified, suggesting that promoter targets for GRDC and SC may be different and that GRDC of the
leuV
promoter occurs in the absence of guanosine 3′,5′-bisdiphosphate. To determine the mechanisms of the
leuV
promoter regulation, we have examined the stability of promoter open complexes and the effects of nucleotide triphosphate (NTP) concentration on the efficiency of the
leuV
promoter and its structural variants in vitro and in vivo. The
leuV
promoter open complexes were an order of magnitude more stable to heparin challenge than those of
rrnBp
1
. The major initiating nucleotide GTP as well as other NTPs increased the stability of the
leuV
promoter open complexes. When the cellular level of purine triphosphates was increased at slower growth rates by pyrimidine limitation, a 10% reduction in
leuV
promoter activity was seen. It therefore appears that transcription initiation from the
leuV
promoter is less sensitive to changes in intracellular NTP concentration than that from
rrnBp
1
. Comparative analysis of regulation of the
leuV
promoter with and without upstream activating sequences (UAS) demonstrated that the binding site for factor of inversion stimulation (FIS) located in UAS is essential for maximal GRDC. Moreover, the presence of UAS overcame the effects of
leuV
promoter mutations, which abolished GRDC of the
leuV
core promoter. However, although the presence of putative FIS binding site was essential for optimal GRDC, both mutant and wild-type
leuV
promoters containing UAS showed improved GRDC in a
fis
mutant background, suggesting that FIS protein is an important but not unique participant in the regulation of the
leuV
promoter.
Mutations Enhancing Amino Acid Catabolism Confer a Growth Advantage in Stationary Phase
Authors: Zinser Erik R. , Kolter Roberto 
Published: December 2019

ABSTRACT

Starved cultures of
Escherichia coli
undergo successive rounds of population takeovers by mutants of increasing fitness. These mutants express the growth advantage in stationary phase (GASP) phenotype. Previous work identified the
rpoS819
allele as a GASP mutation allowing cells to take over stationary-phase cultures after growth in rich media (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757–1760, 1993). Here we have identified three new GASP loci from an aged
rpoS819
strain:
sgaA
,
sgaB
, and
sgaC
. Each locus is capable of conferring GASP on the
rpoS819
parent, and they can provide successively higher fitnesses for the bacteria in the starved cultures. All four GASP mutations isolated thus far allow for faster growth on both individual and mixtures of amino acids. Each mutation confers a growth advantage on a different subset of amino acids, and these mutations act in concert to increase the overall catabolic capacity of the cell. We present a model whereby this enhanced ability to catabolize amino acids is responsible for the fitness gain during carbon starvation, as it may allow GASP mutants to outcompete the parental cells when growing on the amino acids released by dying cells.
Effect of Hook Subunit Concentration on Assembly and Control of Length of the Flagellar Hook of
Salmonella
Authors: Muramoto Kazumasa , Makishima Shigeru , Aizawa Shin-Ichi , Macnab Robert M. 
Published: December 2019

ABSTRACT

The flagellar hook of
Salmonella
is a filamentous polymer made up of subunits of the protein FlgE. Hook assembly is terminated when the length reaches about 55 nm. After our recent study of the effect of cellular levels of the hook length control protein FliK, we have now analyzed the effect of cellular levels of FlgE itself. When FlgE was overproduced in a wild-type strain, a
fliC
(flagellin) mutant, or a
fliD
(hook-associated protein 2 [HAP2], filament capping protein) mutant, the hooks remained at the wild-type length. In a
fliK
(hook length control protein) mutant, which produces long hooks (polyhooks), the overproduction of FlgE resulted in extraordinarily long hooks (superpolyhooks). In a
flgK
(HAP1, first hook-filament junction protein) mutant or a
flgL
(HAP3, second hook-filament junction protein) mutant, the overproduction of FlgE also resulted in longer than normal hooks. Thus, at elevated hook protein levels not only FliK but also FlgK and FlgL are necessary for the proper termination of hook elongation. When FlgE was severely underproduced, basal bodies without hooks were often observed. However, those hooks that were seen were of wild-type length, demonstrating that FlgE underproduction decreases the probability of the initiation of hook assembly but not the extent of hook elongation.
Halophilic 20S Proteasomes of the Archaeon
Haloferax volcanii
: Purification, Characterization, and Gene Sequence Analysis
Authors: Wilson Heather L. , Aldrich Henry C. , Maupin-Furlow Julie 
Published: December 2019

ABSTRACT

A 20S proteasome, composed of α
1
and β subunits arranged in a barrel-shaped structure of four stacked rings, was purified from a halophilic archaeon
Haloferax volcanii
. The predominant peptide-hydrolyzing activity of the 600-kDa α
1
β-proteasome on synthetic substrates was cleavage carboxyl to hydrophobic residues (chymotrypsin-like [CL] activity) and was optimal at 2 M NaCl, pH 7.7 to 9.5, and 75°C. The α
1
β-proteasome also hydrolyzed insulin B-chain protein. Removal of NaCl inactivated the CL activity of the α
1
β-proteasome and dissociated the complex into monomers. Rapid equilibration of the monomers into buffer containing 2 M NaCl facilitated their reassociation into fully active α
1
β-proteasomes of 600 kDa. However, long-term incubation of the halophilic proteasome in the absence of salt resulted in hydrolysis and irreversible inactivation of the enzyme. Thus, the isolated proteasome has unusual salt requirements which distinguish it from any proteasome which has been described. Comparison of the β-subunit protein sequence with the sequence deduced from the gene revealed that a 49-residue propeptide is removed to expose a highly conserved N-terminal threonine which is proposed to serve as the catalytic nucleophile and primary proton acceptor during peptide bond hydrolysis. Consistent with this mechanism, the known proteasome inhibitors carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132) and
N
-acetyl-leucinyl-leucinyl-norleucinal (calpain inhibitor I) were found to inhibit the CL activity of the
H. volcanii
proteasome (

K
i

= 0.2 and 8 μM, respectively). In addition to the genes encoding the α
1
and β subunits, a gene encoding a second α-type proteasome protein (α
2
) was identified. All three genes coding for the proteasome subunits were mapped in the chromosome and found to be unlinked. Modification of the methods used to purify the α
1
β-proteasome resulted in the copurification of the α
2
protein with the α
1
and β subunits in nonstoichometric ratios as cylindrical particles of four stacked rings of 600 kDa with CL activity rates similar to the α
1
β-proteasome, suggesting that at least two separate 20S proteasomes are synthesized. This study is the first description of a prokaryote which produces two separate 20S proteasomes and suggests that there may be distinct physiological roles for the two different α subunits in this halophilic archaeon.
Study of the Interaction between Bacteriophage T4 asiA andEscherichia coliς70, Using the Yeast Two-Hybrid System: Neutralization of asiA Toxicity toE. coliCells by Coexpression of a Truncated ς70Fragment
Authors: Sharma Umender K. , Ravishankar Sudha , Shandil Radha Krishan , Praveen P. V. K. , Balganesh T. S. 
Published: December 2019

ABSTRACTThe interaction of T4 phage-encoded anti-sigma factor, asiA, andEscherichia coliς70was studied by using the yeast two-hybrid system. Truncation of ς70to identify the minimum region involved in the interaction showed that the fragment containing amino acid residues proximal to the C terminus (residues 547 to 603) was sufficient for complexing to asiA. Studies also indicated that some of the truncated C-terminal fragments (residues 493 to 613) had higher affinity for asiA as judged by the increased β-galactosidase activity. It is proposed that the observed higher affinity may be due to the unmasking of the binding region of asiA on the sigma protein. Advantage was taken of the increased affinity of truncated ς70fragments to asiA in designing a coexpression system wherein the toxicity of asiA expression inE. colicould be neutralized and the complex of truncated ς70and asiA could be expressed in large quantities and purified.
Synthetic Lethal Phenotypes Caused by Mutations Affecting Chromosome Partitioning in
Bacillus subtilis
Authors: Britton Robert A. , Grossman Alan D. 
Published: December 2019

ABSTRACT

We investigated the genetic interactions between mutations affecting chromosome structure and partitioning in
Bacillus subtilis
. Loss-of-function mutations in
spoIIIE
(encoding a putative DNA translocase) and
smc
(involved in chromosome structure and partitioning) caused a synthetic lethal phenotype. We constructed a conditional mutation in
smc
and found that many of the
spoIIIE smc
double-mutant cells had a chromosome bisected by a division septum. The growth defect of the double mutant was exacerbated by a null mutation in the chromosome partitioning gene
spo0J
. These results suggest that mutants defective in nucleoid structure are unable to move chromosomes out of the way of the invaginating septum and that SpoIIIE is involved in repositioning these bisected chromosomes during vegetative growth.