bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Mutants Devoid of Neutral Trehalase Activity in the Fission Yeast
Schizosaccharomyces pombe
: Partial Protection from Heat Shock and High-Salt Stress
Authors: Cansado Jose , Soto Teresa , Fernandez Juana , Vicente-Soler Jero , Gacto Mariano 
Published: December 2019

ABSTRACT

Exposure of cells of
Schizosaccharomyces pombe
to heat shock or osmotic upshift results in an increased level of neutral trehalase activity, which is responsible for hydrolysis of intracellular trehalose. We constructed
S. pombe
mutants lacking neutral trehalase activity by gene replacement at the newly defined
ntp1
+
locus. Analysis of these mutants revealed that a twofold increase in trehalose accumulation, enhanced acquired thermoresistance, and marked salt tolerance characterized their ability to grow in liquid and solid media. Analysis of the expression of the trehalase gene under heat shock and osmotic upshift revealed the transcriptional activation of
ntp1
+
in response to both stresses.
The LysR-Type Transcriptional Regulator CbbR Controlling Autotrophic CO
2
Fixation by
Xanthobacter flavus
Is an NADPH Sensor
Authors: van Keulen G. , Girbal L. , van den Bergh E. R. E. , Dijkhuizen L. , Meijer W. G. 
Published: December 2019

ABSTRACT

Autotrophic growth of
Xanthobacter flavus
is dependent on the fixation of carbon dioxide via the Calvin cycle and on the oxidation of simple organic and inorganic compounds to provide the cell with energy. Maximal induction of the
cbb
and
gap-pgk
operons encoding enzymes of the Calvin cycle occurs in the absence of multicarbon substrates and the presence of methanol, formate, hydrogen, or thiosulfate. The LysR-type transcriptional regulator CbbR regulates the expression of the
cbb
and
gap-pgk
operons, but it is unknown to what cellular signal CbbR responds. In order to study the effects of low-molecular-weight compounds on the DNA-binding characteristics of CbbR, the protein was expressed in
Escherichia coli
and subsequently purified to homogeneity. CbbR of
X. flavus
is a dimer of 36-kDa subunits. DNA-binding assays suggested that two CbbR molecules bind to a 51-bp DNA fragment on which two inverted repeats containing the LysR motif are located. The addition of 200 μM NADPH, but not NADH, resulted in a threefold increase in DNA binding. The apparent
K

d
NADPH

of CbbR was determined to be 75 μM. By using circular permutated DNA fragments, it was shown that CbbR introduces a 64° bend in the DNA. The presence of NADPH in the DNA-bending assay resulted in a relaxation of the DNA bend by 9°. From the results of these in vitro experiments, we conclude that CbbR responds to NADPH. The in vivo regulation of the
cbb
and
gap-pgk
operons may therefore be regulated by the intracellular concentration of NADPH.
Altered
srf
Expression in
Bacillus subtilis
Resulting from Changes in Culture pH Is Dependent on the Spo0K Oligopeptide Permease and the ComQX System of Extracellular Control
Authors: Cosby W. Mark , Vollenbroich Dirk , Lee Oh Hyoung , Zuber Peter 
Published: December 2019

ABSTRACT

The expression of the
srf
operon of
Bacillus subtilis
, encoding surfactin synthetase and the competence regulatory protein ComS, was observed to be reduced when cells were grown in a rich glucose- and glutamine-containing medium in which late-growth culture pH was 5.0 or lower. The production of the surfactin synthetase subunits and of surfactin itself was also reduced. Raising the pH to near neutrality resulted in dramatic increases in
srf
expression and surfactin production. This apparent pH-dependent induction of
srf
expression required
spo0K
, which encodes the oligopeptide permease that functions in cell-density-dependent control of sporulation and competence, but not CSF, the competence-inducing pheromone that regulates
srf
expression in a Spo0K-dependent manner. Both ComP and ComA, the two-component regulatory pair that stimulates cell-density-dependent
srf
transcription, were required for optimal expression of
srf
at low and high pHs, but ComP was not required for pH-dependent
srf
induction. The known negative regulators of
srf
, RapC and CodY, were found not to function significantly in pH-dependent
srf
expression. Late-growth culture supernatants at low pH were not active in inducing
srf
expression in cells of low-density cultures but were rendered active when their pH was raised to near neutrality. ComQ (and very likely the
srf
-inducing pheromone ComX) and Spo0K were found to be required for the extracellular induction of
srf-lacZ
at neutral pH. The results suggest that
srf
expression, in response to changes in culture pH, requires Spo0K and another, as yet unidentified, extracellular factor. The study also provides evidence consistent with the hypothesis that ComP acts both positively and negatively in the regulation of ComA and that both activities are controlled by the ComX pheromone.
IroN, a Novel Outer Membrane Siderophore Receptor Characteristic of
Salmonella enterica
Authors: Bäumler Andreas J. , Norris Tracy L. , Lasco Todd , Voigt Wolfgang , Reissbrodt Rolf , Rabsch Wolfgang , Heffron Fred 
Published: December 2019

ABSTRACT

Speciation in enterobacteria involved horizontal gene transfer. Therefore, analysis of genes acquired by horizontal transfer that are present in one species but not its close relatives is expected to give insights into how new bacterial species were formed. In this study we characterize
iroN
, a gene located downstream of the
iroBC
operon in the
iroA
locus of
Salmonella enterica
serotype Typhi. Like
iroBC
, the
iroN
gene is present in all phylogenetic lineages of
S. enterica
but is absent from closely related species such as
Salmonella bongori
or
Escherichia coli
. Comparison of the deduced amino acid sequence of
iroN
with other proteins suggested that this gene encodes an outer membrane siderophore receptor protein. Mutational analysis in
S. enterica
and expression in
E. coli
identified a 78-kDa outer membrane protein as the
iroN
gene product. When introduced into an
E. coli fepA cir fiu aroB
mutant on a cosmid,
iroN
mediated utilization of structurally related catecholate siderophores, including
N
-(2,3-dihydroxybenzoyl)-
l
-serine, myxochelin A, benzaldehyde-2,3-dihydroxybenzhydrazone, 2-
N
,6-
N
-bis(2,3-dihydroxybenzoyl)-
l
-lysine, 2-
N
,6-
N
-bis(2,3-dihydroxybenzoyl)-
l
-lysine amide, and enterochelin. These results suggest that the
iroA
locus functions in iron acquisition in
S. enterica.
PcaU, a Transcriptional Activator of Genes for Protocatechuate Utilization in
Acinetobacter
Authors: Gerischer Ulrike , Segura Ana , Ornston L. Nicholas 
Published: December 2019

ABSTRACT

The
Acinetobacter pcaIJFBDKCHG
operon encodes the six enzymes that convert protocatechuate to citric acid cycle intermediates. Directly downstream from the operon are
qui
and
pob
genes encoding sets of enzymes that convert quinate and
p
-hydroxybenzoate, respectively, to protocatechuate. Prior to this investigation, the only known regulatory gene in the
pca-qui-pob
cluster was
pobR
, which encodes a transcriptional activator that responds to
p
-hydroxybenzoate and activates transcription of
pobA
. The
pca
and
qui
genes were known to be expressed in response to protocatechuate, but a protein that mediated this induction had not been identified. This study was initiated by characterization of a spontaneous mutation that mapped upstream from
pcaI
and prevented expression of the
pca
genes. Sequencing of wild-type DNA extending from the translational start of
pcaI
through and beyond the location of the mutation revealed a 282-bp intergenic region and a divergently transcribed open reading frame, designated
pcaU
. Downstream from
pcaU
are two open reading frames encoding proteins similar in amino acid sequence to those associated with the oxidation of acyl thioesters. Inactivation of
pcaU
reduced the induced expression of
pca
structural genes by about 90% and impeded but did not completely prevent growth of the mutant cells with protocatechuate. PcaU was expressed in
Escherichia coli
and shown to bind to a portion of the
pcaI-pcaU
intergenic region containing a sequence identical in 16 of 19 nucleotide residues to a segment of the
pob
operator. Further similarity of the two regulatory systems is indicated by 54% amino acid sequence identity in the aligned primary structures of PobR and PcaU. The
pob
and
pca
systems were shown to differ, however, in the relative orientations of transcriptional starts with respect to the site where the activator binds to DNA, the size of the intergenic region, and the tightness of transcriptional control. The spontaneous mutation blocking
pca
gene expression was located in the promoter for the
pca
operon. The 19-nucleotide residue operator sequences were shown to be parts of a consensus associated with transcriptional activation of genes associated with protocatechuate catabolism. Two different binding sites for
Pseudomonas putida
PcaR differ from the consensus in only a single nucleotide residue, and DNA directly downstream from
Acinetobacter pcaU
contains a 19-bp segment differing from the consensus in only two residues. PcaU was shown to bind to DNA containing this segment as well as to the DNA in the
pcaU-pcaI
intergenic region.
Characterization of the Gene Cassette Required for Biosynthesis of the (α1→6)-Linked
N
-Acetyl-
d
-Mannosamine-1-Phosphate Capsule of Serogroup A
Neisseria meningitidis
Authors: Swartley John S. , Liu Li-Jun , Miller Yoon K. , Martin Larry E. , Edupuganti Srilatha , Stephens David S. 
Published: December 2019

ABSTRACT

The (α1→6)-linked
N
-acetyl-
d
-mannosamine-1-phosphate meningococcal capsule of serogroup A
Neisseria meningitidis
is biochemically distinct from the sialic acid-containing capsules produced by other disease-associated meningococcal serogroups (e.g., B, C, Y, and W-135). We defined the genetic cassette responsible for expression of the serogroup A capsule. The cassette comprised a 4,701-bp nucleotide sequence located between the outer membrane capsule transporter gene,
ctrA
, and
galE
, encoding the UDP-glucose-4-epimerase. Four open reading frames (ORFs) not found in the genomes of the other meningococcal serogroups were identified. The first serogroup A ORF was separated from
ctrA
by a 218-bp intergenic region. Reverse transcriptase (RT) PCR and primer extension studies of serogroup A mRNA showed that all four ORFs were cotranscribed in the opposite orientation to
ctrA
and that transcription of the ORFs was initiated from the intergenic region by a ς-70-type promoter that overlapped the
ctrA
promoter. The first ORF exhibited 58% amino acid identity with the UDP-
N
-acetyl-
d
-glucosamine (UDP-GlcNAc) 2-epimerase of
Escherichia coli
, which is responsible for the conversion of UDP-GlcNAc into UDP-
N
-acetyl-
d
-mannosamine. Polar or nonpolar mutagenesis of each of the ORFs resulted in an abrogation of serogroup A capsule production as determined by colony immunoblots and enzyme-linked immunosorbent assay. Replacement of the serogroup A biosynthetic gene cassette with a serogroup B cassette by transformation resulted in capsule switching from a serogroup A capsule to a serogroup B capsule. These data indicate that assembly of the serogroup A capsule likely begins with monomeric UDP-GlcNAc and requires proteins encoded by three other genes found in the serogroup A
N. meningitidis
-specific operon located between
ctrA
and
galE
.
Hybrid
Bordetella pertussis-Escherichia coli
RNA Polymerases: Selectivity of Promoter Activation
Authors: Steffen Pierre , Ullmann Agnes 
Published: December 2019

ABSTRACT

We constructed hybrid
Bordetella pertussis-Escherichia coli
RNA polymerases and compared productive interactions between transcription activators and cognate RNA polymerase subunits in an in vitro transcription system. Virulence-associated genes of
B. pertussis
, in the presence of their activator BvgA, are transcribed by all variants of hybrid RNA polymerases, whereas transcription at the
E. coli lac
promoter regulated by the cyclic AMP-catabolite gene activator protein has an absolute requirement for the
E. coli
α subunit. This suggests that activator contact sites involve a high degree of selectivity.
Trehalose Is Not Relevant for In Vivo Activity of ς
S
-Containing RNA Polymerase in
Escherichia coli
Authors: Germer Jens , Muffler Andrea , Hengge-Aronis Regine 
Published: December 2019

ABSTRACT

The ς
S
- and ς
70
-associated forms of RNA polymerase core enzyme (E) of
Escherichia coli
have very similar promoter recognition specificities in vitro. Nevertheless, the in vivo expression of many stress response genes is strongly dependent on ς
S
. Based on in vitro assays, it has recently been proposed that the disaccharide trehalose specifically stimulates the formation and activity of Eς
S
and thereby contributes to promoter selectivity (S. Kusano and A. Ishihama, J. Bacteriol. 179:3649–3654, 1997). However, we demonstrate here that a trehalose-free
otsA
mutant exhibits growth phase-related and osmotic induction of various ς
S
-dependent genes which is indistinguishable from that of an otherwise isogenic wild-type strain and that stationary-phase cells do not accumulate trehalose (even though the trehalose-synthesizing enzymes are induced). We conclude that in vivo trehalose does not play a role in the expression of ς
S
-dependent genes and therefore also not in sigma factor selectivity at the promoters of these genes.
An Archaeal Aerotaxis Transducer Combines Subunit I Core Structures of Eukaryotic Cytochrome
c
Oxidase and Eubacterial Methyl-Accepting Chemotaxis Proteins
Authors: Brooun Alexei , Bell James , Freitas Tracey , Larsen Randy W. , Alam Maqsudul 
Published: December 2019

ABSTRACT

Signal transduction in the archaeon
Halobacterium salinarum
is mediated by three distinct subfamilies of transducer proteins. Here we report the complete
htrVIII
gene sequence and present analysis of the encoded primary structure and its functional features. HtrVIII is a 642-amino-acid protein and belongs to halobacterial transducer subfamily B. At the N terminus, the protein contains six transmembrane segments that exhibit homology to the heme-binding sites of the eukaryotic cytochrome
c
oxidase. The C-terminal domain has high homology with the eubacterial methyl-accepting chemotaxis protein. The HtrVIII protein mediates aerotaxis: a strain with a deletion of the
htrVIII
gene loses aerotaxis, while an overproducing strain exhibits stronger aerotaxis. We also demonstrate that HtrVIII is a methyl-accepting protein and demethylates during the aerotaxis response.
The Cell Wall-Anchored
Streptomyces reticuli
Avicel-Binding Protein (AbpS) and Its Gene
Authors: Walter Stefan , Wellmann Egbert , Schrempf Hildgund 
Published: December 2019

ABSTRACT

Streptomyces reticuli
produces a 35-kDa cellulose-binding protein (AbpS) which interacts strongly with crystalline forms of cellulose (Avicel, bacterial microcrystalline cellulose, and tunicin cellulose); other polysaccharides are recognized on weakly (chitin and Valonia cellulose) or not at all (xylan, starch, and agar). The protein could be purified to homogeneity due to its affinity to Avicel. After we sequenced internal peptides, the corresponding gene was identified by reverse genetics. In vivo labelling experiments with fluorescein isothiocyanate (FITC), FITC-labelled secondary antibodies, or proteinase K treatment revealed that the anchored AbpS protrudes from the surfaces of the hyphae. When we investigated the hydrophobicity of the deduced AbpS, one putative transmembrane segment was predicted at the C terminus. By analysis of the secondary structure, a large centrally located α-helix which has weak homology to the tropomyosin protein family was found. Physiological studies showed that AbpS is synthesized during the late logarithmic phase, independently of the carbon source.
Roles of the Catalytic Domain and Two Cellulose Binding Domains of
Thermomonospora fusca
E4 in Cellulose Hydrolysis
Authors: Irwin Diana , Shin Dong-Hoon , Zhang Sheng , Barr Brian K. , Sakon Joshua , Karplus P. Andrew , Wilson David B. 
Published: December 2019

ABSTRACT

Thermomonospora fusca
E4 is an unusual 90.4-kDa endocellulase comprised of a catalytic domain (CD), an internal family IIIc cellulose binding domain (CBD), a fibronectinlike domain, and a family II CBD. Constructs containing the CD alone (E4-51), the CD plus the family IIIc CBD (E4-68), and the CD plus the fibronectinlike domain plus the family II CBD (E4-74) were made by using recombinant DNA techniques. The activities of each purified protein on bacterial microcrystalline cellulose (BMCC), filter paper, swollen cellulose, and carboxymethyl cellulose were measured. Only the whole enzyme, E4-90, could reach the target digestion of 4.5% on filter paper. Removal of the internal family IIIc CBD (E4-51 and E4-74) decreased activity markedly on every substrate. E4-74 did bind to BMCC but had almost no hydrolytic activity, while E4-68 retained 32% of the activity on BMCC even though it did not bind. A low-activity mutant of one of the catalytic bases, E4-68 (Asp55Cys), did bind to BMCC, although E4-51 (Asp55Cys) did not. The ratios of soluble to insoluble reducing sugar produced after filter paper hydrolysis by E4-90, E4-68, E4-74, and E4-51 were 6.9, 3.5, 1.3, and 0.6, respectively, indicating that the family IIIc CBD is important for E4 processivity.
The TolQRA Proteins Are Required for Membrane Insertion of the Major Capsid Protein of the Filamentous Phage f1 during Infection
Authors: Click Eva Marie , Webster Robert E. 
Published: December 2019

ABSTRACT

Infection of
Escherichia coli
by the filamentous bacteriophage f1 is initiated by interaction of the end of the phage particle containing the gene III protein with the tip of the F conjugative pilus. This is followed by the translocation of the phage DNA into the cytoplasm and the insertion of the major phage capsid protein, pVIII, into the cytoplasmic membrane. DNA transfer requires the chromosomally encoded TolA, TolQ, and TolR cytoplasmic membrane proteins. By using radiolabeled phages, it can be shown that no pVIII is inserted into the cytoplasmic membrane when the bacteria contain null mutations in
tolQ
, -
R
and -
A
. The rate of infection can be varied by using bacteria expressing various mutant TolA proteins. Analysis of the infection process in these strains demonstrates a direct correlation between the rate of infection and the incorporation of infecting bacteriophage pVIII into the cytoplasmic membrane.
Identification and Characterization of Epoxide Carboxylase Activity in Cell Extracts of
Nocardia corallina
B276
Authors: Allen Jeffrey R. , Ensign Scott A. 
Published: December 2019

ABSTRACT

The metabolism of aliphatic epoxides (epoxyalkanes) by the alkene-utilizing actinomycete
Nocardia corallina
B276 was investigated. Suspensions of
N. corallina
cells grown with propylene as the carbon source readily degraded propylene and epoxypropane, while suspensions of glucose-grown cells did not. The addition of propylene and epoxypropane to glucose-grown cells resulted in a time-dependent increase in propylene- and epoxypropane-degrading activities that was prevented by the addition of rifampin and chloramphenicol. The expression of alkene- and epoxide-degrading activities was correlated with the high-level expression of several polypeptides not present in extracts of glucose-grown cells. Epoxypropane and epoxybutane degradation by propylene-grown cell suspensions of
N. corallina
was stimulated by the addition of CO
2
and inhibited by the depletion of CO
2
. Cell extracts catalyzed the carboxylation of epoxypropane to form acetoacetate in a reaction that was dependent on the addition of CO
2
, NAD
+
, and a reductant (NADPH or dithiothreitol). In the absence of CO
2
, epoxypropane was isomerized by cell extracts to form acetone at a rate approximately 10-fold lower than the rate of epoxypropane carboxylation. Methylepoxypropane was found to be a time-dependent, irreversible inactivator of epoxyalkane-degrading activity. These properties demonstrate that epoxyalkane metabolism in
N. corallina
occurs by a carboxylation reaction forming β-keto acids as products and provide evidence for the involvement in this reaction of an epoxide carboxylase with properties and cofactor requirements similar to those of the four-component epoxide carboxylase enzyme system of the gram-negative bacterium
Xanthobacter
strain Py2 (J. R. Allen and S. A. Ensign, J. Biol. Chem. 272:32121–32128, 1997). The addition of epoxide carboxylase component I from
Xanthobacter
strain Py2 to methylepoxypropane-inactivated
N. corallina
extracts restored epoxide carboxylase activity, and the addition of epoxide carboxylase component II from
Xanthobacter
Py2 to active
N. corallina
extracts stimulated epoxide isomerase rates to the same levels observed with the purified
Xanthobacter
system. Antibodies raised against
Xanthobacter
strain Py2 epoxide carboxylase component I cross-reacted with a polypeptide in propylene-grown
N. corallina
extracts with the same molecular weight as component I but did not cross-react with glucose-grown extracts. Together, these results suggest a common pathway of epoxyalkane metabolism for phylogenetically distinct bacteria that involves CO
2
fixation and the activity of a multicomponent epoxide carboxylase enzyme system.
Lysis Genes of the
Bacillus subtilis
Defective Prophage PBSX
Authors: Krogh Susanne , Jørgensen Steen T. , Devine Kevin M. 
Published: December 2019

ABSTRACT

Four genes identified within the late operon of PBSX show characteristics expected of a host cell lysis system; they are
xepA
, encoding an exported protein;
xhlA
, encoding a putative membrane-associated protein;
xhlB
, encoding a putative holin; and
xlyA
, encoding a putative endolysin. In this work, we have assessed the contribution of each gene to host cell lysis by expressing the four genes in different combinations under the control of their natural promoter located on the chromosome of
Bacillus subtilis
168. The results show that
xepA
is unlikely to be involved in host cell lysis. Expression of both
xhlA
and
xhlB
is necessary to effect host cell lysis of
B. subtilis
. Expression of
xhlB
(encoding the putative holin) together with
xlyA
(encoding the endolysin) cannot effect cell lysis, indicating that the PBSX lysis system differs from those identified in the phages of gram-negative bacteria. Since host cell lysis can be achieved when
xlyA
is inactivated, it is probable that PBSX encodes a second endolysin activity which also uses XhlA and XhlB for export from the cell. The chromosome-based expression system developed in this study to investigate the functions of the PBSX lysis genes should be a valuable tool for the analysis of other host cell lysis systems and for expression and functional analysis of other lethal gene products in gram-positive bacteria.