bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Hsc66-Hsc20 Chaperone System in
Escherichia coli
: Chaperone Activity and Interactions with the DnaK-DnaJ-GrpE System
Authors: Silberg Jonathan J. , Hoff Kevin G. , Vickery Larry E. 
Published: December 2019

ABSTRACT

Hsc66, a stress-70 protein, and Hsc20, a J-type accessory protein, comprise a newly described Hsp70-type chaperone system in addition to DnaK-DnaJ-GrpE in
Escherichia coli
. Because endogenous substrates for the Hsc66-Hsc20 system have not yet been identified, we investigated chaperone-like activities of Hsc66 and Hsc20 by their ability to suppress aggregation of denatured model substrate proteins, such as rhodanese, citrate synthase, and luciferase. Hsc66 suppressed aggregation of rhodanese and citrate synthase, and ATP caused effects consistent with complex destabilization typical of other Hsp70-type chaperones. Differences in the activities of Hsc66 and DnaK, however, suggest that these chaperones have dissimilar substrate specificity profiles. Hsc20, unlike DnaJ, did not exhibit intrinsic chaperone activity and appears to function solely as a regulatory cochaperone protein for Hsc66. Possible interactions between the Hsc66-Hsc20 and DnaK-DnaJ-GrpE chaperone systems were also investigated by measuring the effects of cochaperone proteins on Hsp70 ATPase activities. The nucleotide exchange factor GrpE did not stimulate the ATPase activity of Hsc66 and thus appears to function specifically with DnaK. Cross-stimulation by the cochaperones Hsc20 and DnaJ was observed, but the requirement for supraphysiological concentrations makes it unlikely that these interactions occur significantly in vivo. Together these results suggest that Hsc66-Hsc20 and DnaK-DnaJ-GrpE comprise separate molecular chaperone systems with distinct, nonoverlapping cellular functions.
Topological Analysis of the Aerobic Membrane-Bound Formate Dehydrogenase of
Escherichia coli
Authors: Benoit Stéphane , Abaibou Hafid , Mandrand-Berthelot Marie-Andrée 
Published: December 2019

ABSTRACT

Besides formate dehydrogenase N (FDH-N), which is involved in the major anaerobic respiratory pathway in the presence of nitrate,
Escherichia coli
synthesizes a second isoenzyme, called FDH-O, whose physiological role is to ensure rapid adaptation during a shift from aerobiosis to anaerobiosis. FDH-O is a membrane-bound enzyme complex composed of three subunits, α (FdoG), β (FdoH), and γ (FdoI), which exhibit high sequence similarity to the equivalent polypeptides of FDH-N. The topology of these three subunits has been studied by using
blaM
(β-lactamase) gene fusions. A collection of 47 different randomly generated Fdo-BlaM fusions, 4 site-specific fusions, and 3 sandwich fusions were isolated along the entire sequence of the three subunits. In contrast to previously reported predictions from sequence analysis, our data suggested that the αβ catalytic dimer is located in the cytoplasm, with a C-terminal anchor for β protruding into the periplasm. As expected, the γ subunit, which specifies cytochrome
b
, was shown to cross the cytoplasmic membrane four times, with the N and C termini exposed to the cytoplasm. Protease digestion studies of the
35
S-labelled FDH-O heterotrimer in spheroplasts add further support to this model. Consistently, prior studies regarding the bioenergetic function of formate dehydrogenase provided evidence for a mechanism in which formate is oxidized in the cytoplasm.
Physiological Role for the GlnK Protein of Enteric Bacteria: Relief of NifL Inhibition under Nitrogen-Limiting Conditions
Authors: He Luhong , Soupene Eric , Ninfa Alexander , Kustu Sydney 
Published: December 2019

ABSTRACT

In
Klebsiella pneumoniae
, NifA-dependent transcription of nitrogen fixation (
nif
) genes is inhibited by a flavoprotein, NifL, in the presence of molecular oxygen and/or combined nitrogen. We recently demonstrated that the general nitrogen regulator NtrC is required to relieve NifL inhibition under nitrogen (N)-limiting conditions. We provide evidence that the sole basis for the NtrC requirement is its role as an activator of transcription for
glnK
, which encodes a P
II
-like allosteric effector. Relief of NifL inhibition is a unique physiological function for GlnK in that the structurally related GlnB protein of enteric bacteria—apparently a paralogue of GlnK—cannot substitute. Unexpectedly, although covalent modification of GlnK by uridylylation normally occurs under N-limiting conditions, several lines of evidence indicate that uridylylation is not required for relief of NifL inhibition. When GlnK was synthesized constitutively from non-NtrC-dependent promoters, it was able to relieve NifL inhibition in the absence of uridylyltransferase, the product of the
glnD
gene, and under N excess conditions. Moreover, an altered form of GlnK, GlnK
Y51N
, which cannot be uridylylated due to the absence of the requisite tyrosine, was still able to relieve NifL inhibition.
Coupled Changes in Translation and Transcription during Cobalamin-Dependent Regulation of
btuB
Expression in
Escherichia coli
Authors: Nou Xiangwu , Kadner Robert J. 
Published: December 2019

ABSTRACT

The level of the vitamin B
12
transport protein BtuB in the outer membrane of
Escherichia coli
is strongly reduced by growth in the presence of cobalamins. Previous analyses of regulatory mutants and of
btuB-lacZ
fusions indicated that the primary site of
btuB
gene regulation was at the translational level, and this required sequences throughout the 240-nucleotide (nt) leader region. Cobalamin-dependent regulation of transcriptional fusions was of a lesser magnitude but required, in addition to the leader, sequences within the first 100 nt of the coding sequence, termed the translated regulatory region (TRR). To analyze the process of transcription-level regulation of
btuB
in
E. coli
, the levels and metabolism of
btuB
RNA were analyzed by S1 nuclease protection assays, and mutations that alter the coupling of translational and transcriptional control were analyzed. Expression of transcriptional fusions was found to correlate with changes in the level of intact
btuB
RNA and was related to changes in the metabolic stability of the normally long-lived RNA. Mutational analysis showed that the
btuB
start codon and a hairpin structure that can sequester the Shine-Dalgarno sequence are necessary for cobalamin-dependent regulation and that translation of the TRR is necessary for extended RNA stability and for expression of the transcriptional fusion. The absence of regulation at the stage of transcription initiation was confirmed by the findings that several truncated
btuB
RNA fragments were expressed in a constitutive manner and that the normal regulatory response occurred even when the
btuB
promoter and upstream sequences were replaced by the heterologous
bla
and
lac
promoters. Transcription driven by phage T7 RNA polymerase was not regulated by cobalamins, although some regulation at the translational level was retained. Cobalamin-dependent changes in RNA structure were suggested from the RNase III-dependent production of a transcript fragment that is made only in the presence of cobalamin and is independent of the regulatory outcome. These results indicate that the primary control of
btuB
expression by cobalamin occurs at the level of translation initiation, which directly affects the level and stability of
btuB
RNA in a process that requires the presence of the intact translated regulatory region.
Cloning and Expression of the Gene for the Na
+
-Coupled Serine Transporter from
Escherichia coli
and Characteristics of the Transporter
Authors: Ogawa Wakano , Kim Young-Mog , Mizushima Tohru , Tsuchiya Tomofusa 
Published: December 2019

ABSTRACT

We cloned a gene (
sstT
) for the Na
+
/serine symporter from the chromosome of
Escherichia coli
by using a low-copy-number vector and sequenced it. According to the deduced amino acid sequence, the transporter (SstT) consists of 414 amino acid residues. Hydropathy analysis suggested that the SstT protein possesses 9, instead of 12, hydrophobic domains.
The
aadA
Gene of Plasmid R100 Confers Resistance to Spectinomycin and Streptomycin in
Myxococcus xanthus
Authors: Magrini Vincent , Creighton Chad , White David , Hartzell Patricia L. , Youderian Philip 
Published: December 2019

ABSTRACT

Plasmids with the
aadA
gene from plasmid R100, which confers resistance to the aminoglycosides spectinomycin and streptomycin in
Escherchia coli
, can be introduced into wild-type
Myxococcus xanthus
, strain DK1622, by electroporation. Recombinant
M. xanthus
strains with integrated plasmids carrying the
aadA
gene acquire resistance to high levels of these antibiotics. Selection for
aadA
in
M. xanthus
can be carried out independently of, or simultaneously with, selection for resistance to kanamycin. The kinds and frequencies of recombination events observed between integrative plasmids with
aadA
and the
M. xanthus
chromosome are similar to those observed after the transformation of yeast. Cleavage of integrative plasmid DNA at a site adjacent to a region of homology between the plasmid and the
M. xanthus
genome favors the targeted disruption of
M. xanthus
genes by allele replacement.
Molecular Mechanism of Peptide-Specific Pheromone Signaling in
Enterococcus faecalis
: Functions of Pheromone Receptor TraA and Pheromone-Binding Protein TraC Encoded by Plasmid pPD1
Authors: Nakayama Jiro , Takanami Yuuichiro , Horii Takaaki , Sakuda Shohei , Suzuki Akinori 
Published: December 2019

ABSTRACT

Conjugative transfer of the
Enterococcus faecalis
plasmid pPD1 is activated by cPD1, one of several peptide sex pheromones secreted by plasmid-free recipient cells, and is blocked by a donor-produced peptide inhibitor, iPD1. Using a tritiated pheromone, [
3
H]cPD1, we investigated how pPD1-harboring donor cells receive these peptide signals. Donor cells rapidly incorporated [
3
H]cPD1. The cell extract but not the membrane fraction of the donor strain exhibited significant [
3
H]cPD1-binding activity. On the basis of these data and those of tracer studies, it was demonstrated that cPD1 was internalized, where it bound to a high-molecular-weight compound. The cell extract of a strain carrying the
traA
-bearing multicopy plasmid (pDLHH21) also exhibited high [
3
H]cPD1-binding activity. A recombinant TraA exhibited a dissociation constant of 0.49 ± 0.08 nM against [
3
H]cPD1. iPD1 competitively inhibited [
3
H]cPD1 binding to TraA, whereas pheromones and inhibitors relating to other plasmid systems did not. These results show that TraA is a specific intracellular receptor for cPD1 and that iPD1 acts as an antagonist for TraA. A strain carrying the
traC
-bearing multicopy plasmid (pDLES23) exhibited significant [
3
H]cPD1-binding activity. A strain carrying
traC
-disrupted pPD1 (pAM351CM) exhibited lower [
3
H]cPD1-binding activity as well as lower sensitivity to cPD1 than a wild-type donor strain. Some of the other pheromones and inhibitors inhibited [
3
H]cPD1 binding to the
traC
transformant like cPD1 and iPD1 did. These results show that TraC, as an extracellular less-specific pheromone-binding protein, supports donor cells to receive cPD1.
Identification and Characterization of Protease-Resistant SecA Fragments: SecA Has Two Membrane-Integral Forms
Authors: Chen Xianchuan , Brown Timothy , Tai Phang C. 
Published: December 2019

ABSTRACT

We have identified and characterized the protease-resistant SecA fragments (X. Chen, H. Xu, and P. C. Tai, J. Biol. Chem. 271:29698–29706, 1996) through immunodetection with region-specific antibodies, chemical extraction, and sequencing analysis. The 66-, 36-, and 27-kDa proteolytic fragments in the membranes all start at Met
1
, whereas the 48-kDa fragment starts at Glu
361
. The overlapping of the sequences of the 66- and 48-kDa fragments indicates that they are derived from different SecA molecules. These two fragments were generated differently in response to ATP hydrolysis and protein translocation. Furthermore, the presence of membrane is required for the generation of the 48-kDa fragment but not for that of the 66-kDa fragment. These data suggest that there are two different integral forms of SecA in the membrane: SecA
S
and SecA
M
. The combination of these two forms of SecA has several membrane-interacting domains. Both forms of SecA are integrated in the membrane, since both the 48- and 66-kDa fragments could be derived from urea- or Na
2
CO
3
-washed membranes. Moreover, all fragments are resistant to extraction with a high concentration of salt or with heparin, but the membrane-specific 48-kDa SecA domain is more sensitive to Na
2
CO
3
or urea extraction. This suggests that this domain may interact with other membrane proteins in an aqueous microenvironment and therefore may form a part of the protein-conducting channel.
The Yersiniabactin Biosynthetic Gene Cluster of
Yersinia enterocolitica
: Organization and Siderophore-Dependent Regulation
Authors: Pelludat C. , Rakin A. , Jacobi C. A. , Schubert S. , Heesemann J. 
Published: December 2019

ABSTRACT

The ability to synthesize and uptake the
Yersinia
siderophore yersiniabactin is a hallmark of the highly pathogenic, mouse-lethal species
Yersinia pestis
,
Y. pseudotuberculosis
, and
Y. enterocolitica
1B. We have identified four genes,
irp1
,
irp3
,
irp4
, and
irp5
, on a 13-kb chromosomal DNA fragment of
Y. enterocolitica
O8, WA-314. These genes constitute the yersiniabactin biosynthetic gene cluster together with the previously defined
irp2
. The
irp1
gene consists of 9,486 bp capable of encoding a 3,161-amino-acid high-molecular-weight protein 1 (HMWP1) polypeptide with a predicted mass of 384.6 kDa. The first 3,000 bp of
irp1
show similarity to the corresponding regions of the polyketide synthase genes of
Bacillus subtilis
and
Streptomyces antibioticus
. The remaining part of
irp1
is most similar to
irp2
, encoding HMWP2, which might be the reason for immunological cross-reactivity of the two polypeptides. Irp4 was found to have 41.7% similarity to thioesterase-like protein of the anguibactin biosynthetic genes of
Vibrio anguillarum
. Irp5 shows 41% similarity to EntE, the 2,3-dihydroxybenzoic acid-activating enzyme utilized in enterobactin synthesis of
Escherichia coli
. Irp4 and Irp5 are nearly identical to YbtT and YbtE, recently identified in
Y. pestis. irp3
has no similarity to any known gene. Inactivation of either
irp1
or
irp2
abrogates yersiniabactin synthesis. Mutations in
irp1
or
fyuA
(encoding yersiniabactin/pesticin receptor) result in downregulation of
irp2
that can be upregulated by the addition of yersiniabactin. A FyuA-green fluorescent protein translational fusion was downregulated in an
irp1
mutant. Upregulation was achieved by addition of yersiniabactin but not desferal, pesticin, or pyochelin, which indicates high specificity of the FyuA receptor and autoregulation of genes involved in synthesis and uptake of yersiniabactin.
Alanine Catabolism in
Klebsiella aerogenes
: Molecular Characterization of the
dadAB
Operon and Its Regulation by the Nitrogen Assimilation Control Protein
Authors: Janes Brian K. , Bender Robert A. 
Published: December 2019

ABSTRACT

Klebsiella aerogenes
strains with reduced levels of
d
-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in
dadA
(the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of
Klebsiella aerogenes
,
dadAB
, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of
dadA
strains. The operon has a high similarity to the
dadAB
operon of
Salmonella typhimurium
and the
dadAX
operon of
Escherichia coli
, each of which codes for the smaller subunit of
d
-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for
E. coli
and
S. typhimurium
, the
dad
operon of
K. aerogenes
is activated by the Ntr system, mediated in this case by the nitrogen assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position −44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.
Assignment of Biochemical Functions to Glycosyl Transferase Genes Which Are Essential for Biosynthesis of Exopolysaccharides in
Sphingomonas
Strain S88 and
Rhizobium leguminosarum
Authors: Pollock Thomas J. , van Workum Wilbert A. T. , Thorne Linda , Mikolajczak Marcia J. , Yamazaki Motohide , Kijne Jan W. , Armentrout Richard W. 
Published: December 2019

ABSTRACT

Glycosyl transferases which recognize identical substrates (nucleotide-sugars and lipid-linked carbohydrates) can substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes originate in different genera of bacteria. This substitution can be used to identify the substrate specificities of uncharacterized transferase genes. The
spsK
gene of
Sphingomonas
strain S88 and the
pssDE
genes of
Rhizobium leguminosarum
were identified as encoding glucuronosyl-(β1→4)-glucosyl transferases based on reciprocal genetic complementation of mutations in the
spsK
gene and the
pssDE
genes by segments of cloned DNA and by the SpsK-dependent incorporation of radioactive glucose (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells. By contrast, glycosyl transferases which form alternative sugar linkages to the same substrate caused inhibition of polysaccharide synthesis or were deleterious or lethal in a foreign host. The negative effects also suggested specific substrate requirements: we propose that
spsL
codes for a glucosyl-(β1→4)-glucuronosyl transferase in
Sphingomonas
and that
pssC
codes for a glucuronosyl-(β1→4)-glucuronosyl transferase in
R. leguminosarum
. Finally, the complementation results indicate the order of attachment of sphingan main-chain sugars to the C
55
-isoprenylphosphate carrier as -Glc-GlcA-Glc-isoprenylpyrophosphate.
Modulation of the Function of the Signal Receptor Domain of XylR, a Member of a Family of Prokaryotic Enhancer-Like Positive Regulators
Authors: Salto Rafael , Delgado Asunción , Michán Carmen , Marqués Silvia , Ramos Juan L. 
Published: December 2019

ABSTRACT

The XylR protein controls expression from the
Pseudomonas putida
TOL plasmid upper pathway operon promoter (Pu) in response to aromatic effectors. XylR-dependent stimulation of transcription from a Pu::
lacZ
fusion shows different induction kinetics with different effectors. With toluene, activation followed a hyperbolic curve with an apparent
K
of 0.95 mM and a maximum β-galactosidase activity of 2,550 Miller units. With
o
-nitrotoluene, in contrast, activation followed a sigmoidal curve with an apparent
K
of 0.55 mM and a Hill coefficient of 2.65.
m
-Nitrotoluene kept the XylR regulator in an inactive transcriptional form. Therefore, upon binding of an effector, the substituent on the aromatic ring leads to productive or unproductive XylR forms. The different transcriptional states of the XylR regulator are substantiated by XylR mutants. XylRE172K is a mutant regulator that is able to stimulate transcription from the Pu promoter in the presence of
m
-nitrotoluene; however, its response to
m
-aminotoluene was negligible, in contrast with the wild-type regulator. These results illustrate the importance of the electrostatic interactions in effector recognition and in the stabilization of productive and unproductive forms by the regulator upon aromatic binding. XylRD135N and XylRD135Q are mutant regulators that are able to stimulate transcription from Pu in the absence of effectors, whereas substitution of Glu for Asp135 in XylRD135E resulted in a mutant whose ability to recognize effectors was severely impaired. Therefore, the conformation of mutant XylRD135Q as well as XylRD135N seemed to mimic that of the wild-type regulator when effector binding occurred, whereas mutant XylRD135E seemed to be blocked in a conformation similar to that of wild-type XylR and XylRE172K upon binding to an inhibitor molecule such as
m
-nitrotoluene or
m
-aminotoluene.
std1
, a Gene Involved in Glucose Transport in
Schizosaccharomyces pombe
Authors: Mehta Shwetal V. , Patil Vandana B. , Velmurugan S. , Lobo Zita , Maitra Pabitra K. 
Published: December 2019

ABSTRACT

A wild-type strain, Sp972 h
−
, of
Schizosaccharomyces pombe
was mutagenized with ethylmethanesulfonate (EMS), and 2-deoxyglucose (2-DOG)-resistant mutants were isolated. Out of 300 independent 2-DOG-resistant mutants, 2 failed to grow on glucose and fructose (mutants 3/8 and 3/23); however, their hexokinase activity was normal. They have been characterized as defective in their sugar transport properties, and the mutations have been designated as
std1-8
and
std1-23
(sugar transport defective). The mutations are allelic and segregate as part of a single gene when the mutants carrying them are crossed to a wild-type strain. We confirmed the transport deficiency of these mutants by [
14
C]glucose uptake. They also fail to grow on other monosaccharides, such as fructose, mannose, and xylulose, as well as disaccharides, such as sucrose and maltose, unlike the wild-type strain. Lack of growth of the glucose transport-deficient mutants on maltose revealed the extracellular breakdown of maltose in
S. pombe
, unlike in
Saccharomyces cerevisiae
. Both of the mutants are unable to grow on low concentrations of glucose (10 to 20 mM), while one of them, 3/23, grows on high concentrations (50 to 100 mM) as if altered in its affinity for glucose. This mutant (3/23) shows a lag period of 12 to 18 h when grown on high concentrations of glucose. The lag disappears when the culture is transferred from the log phase of its growth on high concentrations. These mutants complement phenotypically similar sugar transport mutants (YGS4 and YGS5) reported earlier by Milbradt and Hoefer (Microbiology 140:2617–2623, 1994), and the clone complementing YGS4 and YGS5 was identified as the only glucose transporter in fission yeast having 12 transmembrane domains. These mutants also demonstrate two other defects: lack of induction and repression of shunt pathway enzymes and defective mating.