bluuebunny/crossref_metadata_2025_split huggingface dataset:
The
bspA
Locus of
Lactobacillus fermentum
BR11 Encodes an
l
-Cystine Uptake System
Authors: Turner Mark S. , Woodberry Tonia , Hafner Louise M. , Giffard Philip M. 
Published: December 2019

ABSTRACT

BspA is a basic surface-exposed protein from
Lactobacillus fermentum
BR11. Sequence comparisons have shown that it is a member of family III of the solute binding proteins. It is 89% identical to the collagen binding protein, Cnb, from
Lactobacillus reuteri
. Compared with the database of
Escherichia coli
proteins, BspA is most similar to the
l
-cystine binding protein FliY. To investigate the function of BspA, mutants depleted for BspA were generated by homologous recombination with a temperature-sensitive plasmid. These mutants were significantly impaired in their abilities to take up
l
-cystine. Uptake rates of
l
-glutamine,
l
-histidine, and
l
-lysine, which are substrates for other binding proteins with similarity to BspA, were unaffected. Evidence was obtained that BspA is necessary for maximal resistance to oxidative stress. Specifically, inactivation of BspA causes defective growth in the presence of oxygen and sensitivity to paraquat. Measurements of sulfhydryl levels showed that incubation of
L. fermentum
BR11 with
l
-cystine resulted in increased levels of sulfhydryl groups both inside and outside the cell; however, this was not the case with a BspA mutant. The role of BspA as an extracellular matrix protein adhesin was also addressed.
L. fermentum
BR11 does not bind to immobilized type I collagen or laminin above background levels but does bind immobilized fibronectin. Inactivation of BspA did not significantly affect fibronectin binding; therefore, we have not found evidence to support the notion that BspA is an extracellular matrix protein binding adhesin. As BspA is most probably not a lipoprotein, this report provides evidence that gram-positive bacterial solute binding proteins do not necessarily have to be anchored to the cytoplasmic membrane to function in solute uptake.
A Conserved Domain in
Escherichia coli
Lon Protease Is Involved in Substrate Discriminator Activity
Authors: Ebel Wolfgang , Skinner Monica M. , Dierksen Karen P. , Scott Janelle M. , Trempy Janine E. 
Published: December 2019

ABSTRACT

Lon protease of
Escherichia coli
regulates a diverse set of physiological responses including cell division, capsule production, plasmid stability, and phage replication. Little is known about the mechanism of substrate recognition by Lon. To examine the interaction of Lon with two of its substrates, RcsA and SulA, we generated point mutations in
lon
which affected its substrate specificity. The most informative
lon
mutant overproduced capsular polysaccharide (RcsA stabilized) yet was resistant to DNA-damaging agents (SulA degraded). Immunoblots revealed that RcsA protein persisted in this mutant whereas SulA protein was rapidly degraded. The mutant contains a single-base change within
lon
leading to a single amino acid change of glutamate 240 to lysine. E240 is conserved among all Lon isolates and resides in a charged domain that has a high probability of adopting a coiled-coil conformation. This conformation, implicated in mediating protein-protein interactions, appears to confer substrate discriminator activity on Lon. We propose a model suggesting that this coiled-coil domain represents the discriminator site of Lon.
Replacement of Vegetative ς
A
by Sporulation-Specific ς
F
as a Component of the RNA Polymerase Holoenzyme in Sporulating
Bacillus subtilis
Authors: Lord Matthew , Barillà Daniela , Yudkin Michael D. 
Published: December 2019

ABSTRACT

Soon after asymmetric septation in sporulating
Bacillus subtilis
cells, ς
F
is liberated in the prespore from inhibition by SpoIIAB. To initiate transcription from its cognate promoters, ς
F
must compete with ς
A
, the housekeeping sigma factor in the predivisional cell, for binding to core RNA polymerase (E). To estimate the relative affinity of E for ς
A
and ς
F
, we made separate mixtures of E with each of the two sigma factors, allowed reconstitution of the holoenzyme, and measured the concentration of free E remaining in each mixture. The affinity of E for ς
F
was found to be about 25-fold lower than that for ς
A
. We used quantitative Western blotting to estimate the concentrations of E, ς
A
, and ς
F
in sporulating cells. The cellular concentrations of E and ς
A
were both about 7.5 μM, and neither changed significantly during the first 3 h of sporulation. The concentration of ς
F
was extremely low at the beginning of sporulation, but it rose rapidly to a peak after about 2 h. At its peak, the concentration of ς
F
was some twofold higher than that of ς
A
. This difference in concentration cannot adequately account for the replacement of ς
A
holoenzyme by ς
F
holoenzyme in the prespore, and it seems that some further mechanism—perhaps the synthesis or activation of an anti-ς
A
factor—must be responsible for this replacement.
The Steady-State Internal Redox State (NADH/NAD) Reflects the External Redox State and Is Correlated with Catabolic Adaptation in
Escherichia coli
Authors: de Graef Mark R. , Alexeeva Svetlana , Snoep Jacky L. , Teixeira de Mattos M. Joost 
Published: December 2019

ABSTRACT

Escherichia coli
MC4100 was grown in anaerobic glucose-limited chemostat cultures, either in the presence of an electron acceptor (fumarate, nitrate, or oxygen) or fully fermentatively. The steady-state NADH/NAD ratio depended on the nature of the electron acceptor. Anaerobically, the ratio was highest, and it decreased progressively with increasing midpoint potential of the electron acceptor. Similarly, decreasing the dissolved oxygen tension resulted in an increased NADH/NAD ratio. As pyruvate catabolism is a major switch point between fermentative and respiratory behavior, the fluxes through the different pyruvate-consuming enzymes were calculated. Although pyruvate formate lyase (PFL) is inactivated by oxygen, it was inferred that the in vivo activity of the enzyme occurred at low dissolved oxygen tensions (DOT ≤ 1%). A simultaneous flux from pyruvate through both PFL and the pyruvate dehydrogenase complex (PDHc) was observed. In anaerobic cultures with fumarate or nitrate as an electron acceptor, a significant flux through the PDHc was calculated on the basis of the redox balance, the measured products, and the known biochemistry. This result calls into question the common assumption that the complex cannot be active under these conditions. In vitro activity measurements of PDHc showed that the cellular content of the enzyme varied with the internal redox state and revealed an activity for dissolved oxygen tension of below 1%. Whereas Western blots showed that the E3 subunit of PDHc (dihydrolipoamide dehydrogenase) did not vary to a large extent under the conditions tested, the E2 subunit (dihydrolipoamide acetyltransferase) amount followed the trend that was found for the in vitro PDHc activity. From this it is concluded that regulation of the PDHc is exerted at the E1/E2 operon (
aceEF
). We propose that the external redox state (measured as the midpoint potentials of those terminal acceptors with which the cell has sufficient capacity to react) is reflected by the internal redox state. The latter may subsequently govern both the expression and the activity of the two pyruvate-catabolizing enzymes.
Transcriptional Organization of the
czc
Heavy-Metal Homeostasis Determinant from
Alcaligenes eutrophus
Authors: Große Cornelia , Grass Gregor , Anton Andreas , Franke Sylvia , Santos Alexander Navarrete , Lawley Blair , Brown Nigel L. , Nies Dietrich H. 
Published: December 2019

ABSTRACT

The Czc system of
Alcaligenes eutrophus
mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB
2
A cation-proton antiporter. DNA sequencing of the region upstream of the
czcNICBADRS
determinant located on megaplasmid pMOL30 revealed the 5′ end of
czcN
and a gene for a MgtC-like protein which is transcribed in the orientation opposite that of
czc
. Additional open reading frames upstream of
czc
had no homologs in the current databases. Using oligonucleotide-probed Northern blotting experiments, a 500-nucleotide
czcN
message and a 400-nucleotide
czcI
message were found, and the presence of 6,200-nucleotide
czcCBA
message (D. Van der Lelie et al., Mol. Microbiol. 23:493–503, 1997) was confirmed. Induction of
czcN
,
czcI
,
czcCBA
, and
czcDRS
followed a similar pattern: transcription was induced best by 300 μM zinc, less by 300 μM cobalt, and only slightly by 300 μM cadmium. Reverse transcription-PCR gave evidence for additional continuous transcription from
czcN
to
czcC
and from
czcD
to
czcS
, but not between
czcA
and
czcD
nor between
czcS
and a 131-amino-acid open reading frame following
czcS
. The CzcR putative response regulator was purified and shown to bind in the 5′ region of
czcN
. A reporter strain carrying a
czcNIC-lacZ-czcBADRS
determinant on plasmid pMOL30 was constructed, as were Δ
czcR
and Δ
czcS
mutants of this strain and of AE128(pMOL30) wild type. Experiments on (i) growth of these strains in liquid culture containing 5 mM Zn
2+
, (ii) induction of the β-galactosidase in the reporter strains by zinc, cobalt, and cadmium, and (iii) cDNA analysis of
czcCBA
mRNA synthesis under inducing and noninducing conditions showed that the CzcRS two-component regulatory system is involved in Czc regulation.
Distinct Affinity of Binding Sites for S-Layer Homologous Domains in
Clostridium thermocellum
and
Bacillus anthracis
Cell Envelopes
Authors: Chauvaux Sylvie , Matuschek Markus , Beguin Pierre 
Published: December 2019

ABSTRACT

Binding parameters were determined for the SLH (S-layer homologous) domains from the
Clostridium thermocellum
outer layer protein OlpB, from the
C. thermocellum
S-layer protein SlpA, and from the
Bacillus anthracis
S-layer proteins EA1 and Sap, using cell walls from
C. thermocellum
and
B. anthracis
. Each SLH domain bound to
C. thermocellum
and
B. anthracis
cell walls with a different

K
D

, ranging between 7.1 × 10
−7
and 1.8 × 10
−8
M. Cell wall binding sites for SLH domains displayed different binding specificities in
C. thermocellum
and
B. anthracis
. SLH-binding sites were not detected in cell walls of
Bacillus subtilis
. Cell walls of
C. thermocellum
lost their affinity for SLH domains after treatment with 48% hydrofluoric acid but not after treatment with formamide or dilute acid. A soluble component, extracted from
C. thermocellum
cells by sodium dodecyl sulfate treatment, bound the SLH domains from
C. thermocellum
but not those from
B. anthracis
proteins. A corresponding component was not found in
B. anthracis
.
The Adsorption Protein Genes of
Xanthomonas campestris
Filamentous Phages Determining Host Specificity
Authors: Lin Nien-Tsung , Liu Tzu-Jun , Lee Tze-Ching , You Bih-Yuh , Yang Ming-Haw , Wen Fu-Shyan , Tseng Yi-Hsiung 
Published: December 2019

ABSTRACT

Gene III (
gIII
) of φLf, a filamentous phage specifically infecting
Xanthomonas campestris
pv. campestris, was previously shown to encode a virion-associated protein (pIII) required for phage adsorption. In this study, the transcription start site for the gene and the N-terminal sequence of the protein were determined, resulting in the revision of the translation initiation site from the one previously predicted for this gene. For comparative study, the
gIII
of φXv, a filamentous phage specifically infecting
X. campestris
pv. vesicatoria, was cloned and sequenced. The deduced amino acid sequences of these two pIIIs exhibit a high degree of identity in their C-terminal halves and possess the structural features typical of the adsorption proteins of filamentous phages: a signal sequence in the N terminus, a long glycine-rich region near the center, and a hydrophobic membrane anchorage domain in the C terminus. The regions between
gIII
and the upstream
gVIII
, 128 nucleotides in both phages, are larger than those of other filamentous phages. A hybrid phage of φXv, consisting of the φLf pIII and all the other components derived from φXv, was able to infect
X. campestris
pv. campestris but not
X. campestris
pv. vesicatoria, indicating that
gIII
is the gene specifying host specificity and demonstrating the interchangeability of the pIIIs.
Analysis of an Autoregulatory Loop Controlling ToxT, Cholera Toxin, and Toxin-Coregulated Pilus Production in
Vibrio cholerae
Authors: Yu Rosa R. , DiRita Victor J. 
Published: December 2019

ABSTRACT

Coordinate expression of many virulence genes in the human pathogen
Vibrio cholerae
is controlled by the ToxR, TcpP, and ToxT proteins. These proteins function in a regulatory cascade in which ToxR and TcpP, two inner membrane proteins, are required to activate
toxT
and ToxT is the direct activator of virulence gene expression. ToxT-activated genes include those whose products are required for the biogenesis of cholera toxin (CTX) and the toxin-coregulated pilus, the major subunit of which is TcpA. This work examined control of
toxT
transcription. We tested a model whereby activation of
toxT
by ToxR and TcpP is required to prime an autoregulatory loop in which ToxT-dependent transcription of the
tcpA
promoter reads through a proposed terminator between the
tcpF
and
toxT
genes to result in continued ToxT production. Primer extension analysis of RNA from wild-type classical strain O395 showed that there are two products encoding
toxT
, one of which is longer than the other by 105 bp. Deletion of the
toxT
promoter (

toxT
Δpro

) resulted in the abolishment of
toxT
transcription, as predicted. Deletion of the
tcpA
promoter (

tcpA
Δpro

) had no effect on subsequent detection of the smaller
toxT
primer extension product, but the larger
toxT
product was not detected, indicating that this product may be the result of transcription from the
tcpA
promoter and not of initiation directly upstream of
toxT
. Neither mutant strain produced detectable TcpA, but the CTX levels of the strains were different. The

toxT
Δpro

strain produced little detectable CTX, while the

tcpA
Δpro

strain produced CTX levels intermediate between those of the wild-type and

toxT
Δpro

strains. Dependence of
toxT
transcription on TcpP and TcpH was confirmed by analyzing RNAs from strains carrying deletions in the genes encoding these regulators. The
tcpP
defect resulted in undetectable
toxT
transcription, whereas the
tcpH
mutation led to a diminishing of
toxT
RNA but not complete abolishment. Taken together, these results suggest that
toxT
transcription is dependent on two different promoters; one is directly upstream and is activated in part by TcpP and TcpH, and the other is much further upstream and is activated by ToxT.
The Lactate-Proton Symport of
Saccharomyces cerevisiae
Is Encoded by
JEN1
Authors: Casal Margarida , Paiva Sandra , Andrade Raquel P. , Gancedo Carlos , Leão Cecília 
Published: December 2019

ABSTRACT

A mutant of
Saccharomyces cerevisiae
deficient in the lactate-proton symport was isolated. Transformation of the mutant with a yeast genomic library allowed the isolation of the gene
JEN1
that restored lactate transport. Disruption of
JEN1
abolished uptake of lactate. The results indicate that, under the experimental conditions tested, no other monocarboxylate permease is able to efficiently transport lactate in
S. cerevisiae
.
Assembly Requirements and Role of CotH during Spore Coat Formation in
Bacillus subtilis
Authors: Zilhão R. , Naclerio G. , Henriques A. O. , Baccigalupi L. , Moran C. P. , Ricca E. 
Published: December 2019

ABSTRACT

We report Western blot data showing that the 42.8-kDa product of the previously characterized
cotH
locus (8) is a structural component of the
Bacillus subtilis
spore coat. We show that the assembly of CotH requires both CotE and GerE. In agreement with these observations, the ultrastructural analysis of purified spores suggests that CotH is needed for proper formation of both inner and outer layers of the coat.
The Type 2 Capsule Locus of
Streptococcus pneumoniae
Authors: Iannelli Francesco , Pearce Barbara J. , Pozzi Gianni 
Published: December 2019

ABSTRACT

The type 2 capsule locus of
Streptococcus pneumoniae
was characterized in Avery’s strain D39, which is the parent strain of the standard transformation recipients currently used in pneumococcal research and is largely used as a virulent strain in studies on the pathogenesis of pneumococcal infections. The capsule locus was sequenced by using a 21.7-kb PCR fragment from the D39 genome as a template. Sequence data analysis showed the presence of 18 open reading frames, 17 of which have the same direction of transcription and all of which are potentially involved in capsule biosynthesis. It was also shown that R36A and R6, which are unencapsulated (rough) derivatives of D39, carry a 7,504-bp deletion involving nine capsule genes.
Benzene-Induced Uncoupling of Naphthalene Dioxygenase Activity and Enzyme Inactivation by Production of Hydrogen Peroxide
Authors: Lee Kyoung 
Published: December 2019

ABSTRACT

Naphthalene dioxygenase (NDO) is a multicomponent enzyme system that oxidizes naphthalene to (+)-
cis
-(1
R
,2
S
)-1,2-dihydroxy-1,2-dihydronaphthalene with consumption of O
2
and two electrons from NAD(P)H. In the presence of benzene, NADH oxidation and O
2
utilization were partially uncoupled from substrate oxidation. Approximately 40 to 50% of the consumed O
2
was detected as hydrogen peroxide. The rate of benzene-dependent O
2
consumption decreased with time, but it was partially increased by the addition of catalase in the course of the O
2
consumption by NDO. Detailed experiments showed that the total amount of O
2
consumed and the rate of benzene-induced O
2
consumption increased in the presence of hydrogen peroxide-scavenging agents, and further addition of the terminal oxygenase component (ISP
NAP
) of NDO. Kinetic studies showed that ISP
NAP
was irreversibly inactivated in the reaction that contained benzene, but the inactivation was relieved to a high degree in the presence of catalase and partially relieved in the presence of 0.1 mM ferrous ion. Benzene- and naphthalene-reacted ISP
NAP
gave almost identical visible absorption spectra. In addition, hydrogen peroxide added at a range of 0.1 to 0.6 mM to the reaction mixtures inactivated the reduced ISP
NAP
containing mononuclear iron. These results show that hydrogen peroxide released during the uncoupling reaction acts both as an inhibitor of benzene-dependent O
2
consumption and as an inactivator of ISP
NAP
. It is proposed that the irreversible inactivation of ISP
NAP
occurs by a Fenton-type reaction which forms a strong oxidizing agent, hydroxyl radicals (
·
OH), from the reaction of hydrogen peroxide with ferrous mononuclear iron at the active site. Furthermore, when [
14
C]benzene was used as the substrate,
cis
-benzene 1,2-dihydrodiol formed by NDO was detected. This result shows that NDO also couples a trace amount of benzene to both O
2
consumption and NADH oxidation.
The C-Terminal Fragment of the Precursor Tail Lysozyme of Bacteriophage T4 Stays as a Structural Component of the Baseplate after Cleavage
Authors: Kanamaru Shuji , Gassner Nadine C. , Ye Nanzhang , Takeda Shigeki , Arisaka Fumio 
Published: December 2019

ABSTRACT
Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in β-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.
Role of XDHC in Molybdenum Cofactor Insertion into Xanthine Dehydrogenase of
Rhodobacter capsulatus
Authors: Leimkühler Silke , Klipp Werner 
Published: December 2019

ABSTRACT

Rhodobacter capsulatus
xanthine dehydrogenase (XDH) is composed of two subunits, XDHA and XDHB. Immediately downstream of
xdhB
, a third gene was identified, designated
xdhC
, which is cotranscribed with
xdhAB
. Interposon mutagenesis revealed that the
xdhC
gene product is required for XDH activity. However, XDHC is not a subunit of active XDH, which forms an α
2
β
2
heterotetramer in
R. capsulatus
. It was shown that XDHC neither is a transcriptional regulator for
xdh
gene expression nor influences XDH stability. To analyze the function of XDHC for XDH in
R. capsulatus
, inactive XDH was purified from an
xdhC
mutant strain. Analysis of the molybdenum cofactor content of this enzyme demonstrated that in the absence of XDHC, no molybdopterin cofactor MPT is present in the XDHAB tetramer. In contrast, absorption spectra of inactive XDH isolated from the
xdhC
mutant revealed the presence of iron-sulfur clusters and flavin adenine dinucleotide, demonstrating that XDHC is not required for the insertion of these cofactors. The absence of MPT from XDH isolated from an
xdhC
mutant indicates that XDHC either acts as a specific MPT insertase or might be a specific chaperone facilitating the insertion of MPT and/or folding of XDH during or after cofactor insertion.
Evidence for an Inducible Nucleotide-Dependent Acetone Carboxylase in
Rhodococcus rhodochrous
B276
Authors: Clark Daniel D. , Ensign Scott A. 
Published: December 2019

ABSTRACT

The metabolism of acetone was investigated in the actinomycete
Rhodococcus rhodochrous
(formerly
Nocardia corallina
) B276. Suspensions of acetone- and isopropanol-grown
R. rhodochrous
readily metabolized acetone. In contrast,
R. rhodochrous
cells cultured with glucose as the carbon source lacked the ability to metabolize acetone at the onset of the assay but gained the ability to do so in a time-dependent fashion. Chloramphenicol and rifampin prevented the time-dependent increase in this activity. Acetone metabolism by
R. rhodochrous
was CO
2
dependent, and
14
CO
2
fixation occurred concomitant with this process. A nucleotide-dependent acetone carboxylase was partially purified from cell extracts of acetone-grown
R. rhodochrous
by DEAE-Sepharose chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the acetone carboxylase was composed of three subunits with apparent molecular masses of 85, 74, and 16 kDa. Acetone metabolism by the partially purified enzyme was dependent on the presence of a divalent metal and a nucleoside triphosphate. GTP and ITP supported the highest rates of acetone carboxylation, while CTP, UTP, and XTP supported carboxylation at 10 to 50% of these rates. ATP did not support acetone carboxylation. Acetoacetate was determined to be the stoichiometric product of acetone carboxylation. The longer-chain ketones butanone, 2-pentanone, 3-pentanone, and 2-hexanone were substrates. This work has identified an acetone carboxylase with a novel nucleotide usage and broader substrate specificity compared to other such enzymes studied to date. These results strengthen the proposal that carboxylation is a common strategy used for acetone catabolism in aerobic acetone-oxidizing bacteria.