bluuebunny/crossref_metadata_2025_split huggingface dataset:
Archaeal Binding Protein-Dependent ABC Transporter: Molecular and Biochemical Analysis of the Trehalose/Maltose Transport System of the Hyperthermophilic Archaeon
Thermococcus litoralis
Authors: Horlacher Reinhold , Xavier Karina B. , Santos Helena , DiRuggiero Jocelyne , Kossmann Marina , Boos Winfried 
Published: December 2019

ABSTRACT

We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon
Thermococcus litoralis
that is homologous to the
malEFG
cluster encoding the
Escherichia coli
maltose transport system. The deduced amino acid sequence of the
malE
product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The
T. litoralis malE
gene was expressed in
E. coli
under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the
E. coli
MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85°C with a

K
d

of 0.16 μM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from
T. litoralis
membranes as well as the products from all other DNA constructs expressed in
E. coli
. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the
E. coli
MalF protein are missing in the
T. litoralis
MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.
Transfer of Tn
5385
, a Composite, Multiresistance Chromosomal Element from
Enterococcus faecalis
Authors: Rice Louis B. , Carias Lenore L. 
Published: December 2019

ABSTRACT

Tn
5385
is a ca. 65-kb element integrated into the chromosomes of clinical
Enterococcus faecalis
strains CH19 and CH116. It confers resistance to erythromycin, gentamicin, mercuric chloride, streptomycin, tetracycline-minocycline, and penicillin via β-lactamase production. Tn
5385
is a composite structure containing regions previously found in staphylococcal and enterococcal plasmids. Several transposons and transposon-like elements within Tn
5385
have been identified, including conjugative transposon Tn
5381
, composite transposon Tn
5384
, and elements indistinguishable from staphylococcal transposons Tn
4001
and Tn
552
. The divergent regions of Tn
5385
are linked by a series of insertion sequence (IS) elements (IS
256
, IS
257
, and IS
1216
) of staphylococcal and enterococcal origin. The ends of Tn
5385
consist of directly repeated copies of enterococcal IS
1216
. Within the chromosomes of strains CH19 and CH116, Tn
5385
has interrupted an open reading frame with substantial homology to previously described alkyl hydrogen peroxide reductase genes. Segments of this open reading frame in both CH19 and CH116 have been deleted, but the amount of deleted DNA differs for the two insertions. Transfer of Tn
5385
from both donors into
E. faecalis
recipients occurs at a low frequency. Two types of transconjugants have been identified. In one type, the target alkyl hydrogen peroxide reductase open reading frame has been deleted, and sequences flanking Tn
5385
in the respective donors are carried over to the transconjugants. These data suggest that the mechanism of Tn
5385
insertion into the recipient chromosome in these transconjugants was recombination across flanking regions in the donors and homologous sequences in the recipients. The second type of transconjugant appears to have resulted from excision of Tn
5385
from the CH19 chromosome by recombination across the terminal IS
1216
elements and insertion into the recipient chromosome by recombination across Tn
5381
(within Tn
5385
) and a previously transferred Tn
5381
copy in the recipient chromosome. These data confirm that Tn
5385
is a composite structure with genetic material from diverse genera and suggest that it is a functional transposon. They also suggest that chromosomal recombination is a mechanism of genetic exchange in enterococci.
Expression of Invasin and Motility Are Coordinately Regulated in
Yersinia enterocolitica
Authors: Badger Julie L. , Miller Virginia L. 
Published: December 2019

ABSTRACT

The
Yersinia enterocolitica inv
gene encodes the primary invasion factor invasin, which has been previously shown to be critical in the initial stages of infection. The expression of
inv
is influenced by growth phase and temperature and is maximal during late exponential-early stationary phase at 23°C. In addition, motility of
Y. enterocolitica
is regulated by temperature.
Y. enterocolitica
cells are motile when grown at lower temperatures (30°C or below), while bacteria grown at 37°C are nonmotile. This study was initiated to determine the molecular basis for the temperature regulation of
inv
expression. Two mutants were isolated that both showed a significant decrease in invasin expression but are hypermotile when grown at 23°C. The first mutant (JB1A8v) was a result of a random mTn
5
Km insertion into the
uvrC
gene. The
uvrC
mutant JB1A8v demonstrated a significant decrease in
inv
and an increase in
fleB
(encodes flagellin) expression. These results suggest that expression of
inv
and flagellin genes is coordinated at the level of transcription. The second regulatory mutant, JB16v, was a result of a targeted insertion into a locus similar to
sspA
which in
E. coli
encodes a stationary-phase regulator. The
E. coli sspA
gene was cloned and assayed for complementation in both of the regulatory mutants. It was determined that
E. coli sspA
restored invasin expression in both the
uvrC
mutant and the
sspA
mutant. In addition, the complementing clone decreased flagellin levels in these mutants.
CpxP, a Stress-Combative Member of the Cpx Regulon
Authors: Danese Paul N. , Silhavy Thomas J. 
Published: December 2019

ABSTRACT

The CpxA/R two-component signal transduction system of
Escherichia coli
can combat a variety of extracytoplasmic protein-mediated toxicities. The Cpx system performs this function, in part, by increasing the synthesis of the periplasmic protease, DegP. However, other factors are also employed by the Cpx system for this stress-combative function. In an effort to identify these remaining factors, we screened a collection of random
lacZ
operon fusions for those fusions whose transcription is regulated by CpxA/R. Through this approach, we have identified a new locus,
cpxP
, whose transcription is stimulated by activation of the Cpx pathway.
cpxP
specifies a periplasmic protein that can combat the lethal phenotype associated with the synthesis of a toxic envelope protein. In addition, we show that
cpxP
transcription is strongly induced by alkaline pH in a CpxA-dependent manner and that
cpxP
and
cpx
mutant strains display hypersensitivity to growth in alkaline conditions.
Identification of AlcR, an AraC-Type Regulator of Alcaligin Siderophore Synthesis in
Bordetella bronchiseptica
and
Bordetella pertussis
Authors: Pradel Elizabeth , Guiso Nicole , Locht Camille 
Published: December 2019

ABSTRACT

A Fur titration assay was used to isolate DNA fragments bearing putative Fur binding sites (FBS) from a partial
Bordetella bronchiseptica
genomic DNA library. A recombinant plasmid bearing a 3.5-kb DNA insert was further studied. Successive deletions in the cloned fragment enabled us to map a putative FBS at about 2 kb from one end. Sequence analysis revealed the presence of an FBS upstream from a new gene encoding an AraC-type transcriptional regulator. The deduced protein displays similarity to PchR, an activator of pyochelin siderophore and ferripyochelin receptor synthesis in
Pseudomonas aeruginosa
. Homologous genes in
Bordetella pertussis
and
Bordetella parapertussis
were PCR amplified, and sequence comparisons indicated a very high conservation in the three species. The
B. pertussis
and
B. bronchiseptica
chromosomal genes were inactivated by allelic exchange. Under low-iron growth conditions, the mutants did not secrete the alcaligin siderophore and lacked AlcC, an alcaligin biosynthetic enzyme. Alcaligin production was restored after transformation with a plasmid bearing the wild-type gene. On the basis of its role in regulation of alcaligin biosynthesis, the new gene was designated
alcR
. Additional sequence determination showed that
alcR
is located about 2 kb downstream from the
alcABC
operon and is transcribed in the same orientation. Two tightly linked open reading frames,
alcD
and
alcE
, were identified between
alcC
and
alcR
. AlcE is a putative iron-sulfur protein; AlcD shows no homology with the proteins in the database. The production of major virulence factors and colonization in the mouse respiratory infection model are AlcR independent.
Molecular Characterization of a Phage-Inducible Middle Promoter and Its Transcriptional Activator from the Lactococcal Bacteriophage φ31
Authors: Walker Shirley A. , Klaenhammer Todd R. 
Published: December 2019

ABSTRACT

An inducible middle promoter from the lactococcal bacteriophage φ31 was isolated previously by shotgun cloning an 888-bp fragment (P
15A10
) upstream of the β-galactosidase (β-Gal) gene (
lacZ.st
) from
Streptococcus thermophilus
(D. J. O’Sullivan, S. A. Walker, S. G. West, and T. R. Klaenhammer, Bio/Technology 14:82–87, 1996). The promoter showed low levels of constitutive β-Gal activity which could be induced two- to threefold over baseline levels after phage infection. During this study, the fragment was subcloned and characterized to identify a smaller, tightly regulated promoter fragment which allowed no β-Gal activity until after phage infection. This fragment, defined within nucleotides 566 to 888 (P
566–888
; also called fragment 566–888), contained tandem, phage-inducible transcription start sites at nucleotides 703 and 744 (703/744 start sites). Consensus −10 regions were present upstream of both start sites, but no consensus −35 regions were identified for either start site. A transcriptional activator, encoded by an open reading frame (ORF2) upstream of the 703/744 start sites, was identified for P
566–888
. ORF2 activated P
566–888
when provided in
trans
in
Escherichia coli
. In addition, when combined with pTRK391 (P
15A10
::
lacZ.st
) in
Lactococcus lactis
NCK203, an antisense ORF2 construct was able to retard induction of the phage-inducible promoter as measured by β-Gal activity levels. Finally, gel shift assays showed that ORF2 was able to bind to promoter fragment 566–888. Deletion analysis of the region upstream from the tandem promoters identified a possible binding site for transcriptional activation of the phage promoters. The DNA-binding ability of ORF2 was eliminated upon deletion of part of this region, which lies centered approximately 35 bp upstream of start site 703. Deletion analysis and mutagenesis studies also elucidated a critical region downstream of the 703/744 start sites, where mutagenesis resulted in a two- to threefold increase in β-Gal activity. With these improvements, the level of expression achieved by an explosive-expression strategy was elevated from 3,000 to 11,000 β-Gal units within 120 min after induction.
Identification of the Fucose Synthetase Gene in the Colanic Acid Gene Cluster of
Escherichia coli
K-12
Authors: Andrianopoulos Kanella , Wang Lei , Reeves Peter R. 
Published: December 2019

ABSTRACT

GDP–
l
-fucose, the substrate for fucosyltransferases for addition of fucose to polysaccharides or glycoproteins in both procaryotes and eucaryotes, is made from GDP–
d
-mannose.
l
-Fucose is a component of bacterial surface antigens, including the extracellular polysaccharide colanic acid produced by most
Escherichia coli
strains. We previously sequenced the
E. coli
colanic acid gene cluster and identified one of the GDP–
l
-fucose biosynthetic pathway genes,
gmd
. We report here the identification of the gene (
fcl
), located downstream of
gmd
, encoding the fucose synthetase.
Msn2p and Msn4p Control a Large Number of Genes Induced at the Diauxic Transition Which Are Repressed by Cyclic AMP in
Saccharomyces cerevisiae
Authors: Boy-Marcotte Emmanuelle , Perrot Michel , Bussereau Françoise , Boucherie Hélian , Jacquet Michel 
Published: December 2019

ABSTRACT

The multicopy suppressors of the
snf1
defect, Msn2p and Msn4p transcription factors (Msn2/4p), activate genes through the stress-responsive
cis
element (CCCCT) in response to various stresses. This
cis
element is also the target for repression by the cyclic AMP (cAMP)-signaling pathway. We analyzed the two-dimensional gel electrophoresis pattern of protein synthesis of the
msn2 msn4
double mutant and compared it with that of the wild-type strain during exponential growth phase and at the diauxic transition. Thirty-nine gene products (including those of
ALD3
,
GDH3
,
GLK1
,
GPP2
,
HSP104
,
HXK1
,
PGM2
,
SOD2
,
SSA3
,
SSA4
,
TKL2
,
TPS1
, and
YBR149W
) are dependent upon Msn2/4p for their induction at the diauxic transition. The expression of all these genes is repressed by cAMP. Thirty other genes identified during this study are still inducible in the mutant. A subset of these genes were found to be superinduced at the diauxic transition, and others were subject to cAMP repression (including
ACH1
,
ADH2
,
ALD6
,
ATP2
,
GPD1
,
ICL1
, and
KGD2
). We conclude from this analysis that Msn2/4p control a large number of genes induced at the diauxic transition but that other, as-yet-uncharacterized regulators, also contribute to this response. In addition, we show here that cAMP repression applies to both Msn2/4p-dependent and -independent control of gene expression at the diauxic shift. Furthermore, the fact that all the Msn2/4p gene targets are subject to cAMP repression suggests that these regulators could be targets for the cAMP-signaling pathway.
Characterization of a Protocatechuate Catabolic Gene Cluster from
Rhodococcus opacus
1CP: Evidence for a Merged Enzyme with 4-Carboxymuconolactone-Decarboxylating and 3-Oxoadipate Enol-Lactone-Hydrolyzing Activity
Authors: Eulberg Dirk , Lakner Silvia , Golovleva Ludmila A. , Schlömann Michael 
Published: December 2019

ABSTRACT

The catechol and protocatechuate branches of the 3-oxoadipate pathway, which are important for the bacterial degradation of aromatic compounds, converge at the common intermediate 3-oxoadipate enol-lactone. A 3-oxoadipate enol-lactone-hydrolyzing enzyme, purified from benzoate-grown cells of
Rhodococcus opacus
(
erythropolis
) 1CP, was found to have a larger molecular mass under denaturing conditions than the corresponding enzymes previously purified from γ-proteobacteria. Sequencing of the N terminus and of tryptic peptides allowed cloning of the gene coding for the 3-oxoadipate enol-lactone hydrolase by using PCR with degenerate primers. Sequencing showed that the gene belongs to a protocatechuate catabolic gene cluster. Most interestingly, the hydrolase gene, usually termed
pcaD
, was fused to a second gene, usually termed
pcaC
, which encodes the enzyme catalyzing the preceding reaction, i.e., 4-carboxymuconolactone decarboxylase. The two enzymatic activities could not be separated chromatographically. At least six genes of protocatechuate catabolism appear to be transcribed in the same direction and in the following order:
pcaH
and
pcaG
, coding for the subunits of protocatechuate 3,4-dioxygenase, as shown by N-terminal sequencing of the subunits of the purified protein; a gene termed
pcaB
due to the homology of its gene product to 3-carboxy-
cis
,
cis
-muconate cycloisomerases;
pcaL
, the fused gene coding for PcaD and PcaC activities;
pcaR
, presumably coding for a regulator of the IclR-family; and a gene designated
pcaF
because its product resembles 3-oxoadipyl coenzyme A (3-oxoadipyl-CoA) thiolases. The presumed
pcaI
, coding for a subunit of succinyl-CoA:3-oxoadipate CoA-transferase, was found to be transcribed divergently from
pcaH.
Multiple Regions on the
Escherichia coli
Heat Shock Transcription Factor ς
32
Determine Core RNA Polymerase Binding Specificity
Authors: Joo Daniel M. , Nolte Audrey , Calendar Richard , Zhou Yan Ning , Jin Ding Jun 
Published: December 2019

ABSTRACT

We have analyzed the core RNA polymerase (RNAP) binding activity of the purified products of nine defective alleles of the
rpoH
gene, which encodes ς
32
in
Escherichia coli
. All mutations studied here lie outside of the putative core RNAP binding regions 2.1 and 2.2. Based on the estimated

K
s

s for the mutant sigma and core RNAP interaction determined by in vitro transcription and by glycerol gradient sedimentation, we have divided the mutants into three classes. The class III mutants showed greatly decreased affinity for core RNAP, whereas the class II mutants’ effect on core RNAP interaction was only clearly seen in the presence of ς
70
competitor. The class I mutant behaved nearly identically to the wild type in core RNAP binding. Two point mutations in class III altered residues that were distant from one another. One was found in conserved region 4.2, and the other was in a region conserved only among heat shock sigma factors. These data suggest that there is more than one core RNAP binding region in ς
32
and that differences in contact sites probably exist among sigma factors.
Identification of Essential Glutamates in the Acetate Kinase from
Methanosarcina thermophila
Authors: Singh-Wissmann Kavita , Ingram-Smith Cheryl , Miles Rebecca D. , Ferry James G. 
Published: December 2019

ABSTRACT

Acetate kinase catalyzes the reversible phosphorylation of acetate (CH
3
COO
−
+ ATP⇄CH
3
CO
2
PO
3
2−
+ ADP). A mechanism which involves a covalent phosphoryl-enzyme intermediate has been proposed, and chemical modification studies of the enzyme from
Escherichia coli
indicate an unspecified glutamate residue is phosphorylated (J. A. Todhunter and D. L. Purich, Biochem. Biophys. Res. Commun. 60:273–280, 1974). Alignment of the amino acid sequences for the acetate kinases from
E. coli
(
Bacteria
domain),
Methanosarcina thermophila
(
Archaea
domain), and four other phylogenetically divergent microbes revealed high identity which included five glutamates. These glutamates were replaced in the
M. thermophila
enzyme to determine if any are essential for catalysis. The histidine-tagged altered enzymes were produced in
E. coli
and purified to electrophoretic homogeneity by metal affinity chromatography. Replacements of E384 resulted in either undetectable or extremely low kinase activity, suggesting E384 is essential for catalysis which supports the proposed mechanism. Replacement of E385 influenced the

K
m

values for acetate and ATP with only moderate decreases in
k
cat
, which suggests that this residue is involved in substrate binding but not catalysis. The unaltered acetate kinase was not inactivated by
N
-ethylmaleimide; however, replacement of E385 with cysteine conferred sensitivity to
N
-ethylmaleimide which was prevented by preincubation with acetate, acetyl phosphate, ATP, or ADP, suggesting that E385 is located near the active site. Replacement of E97 decreased the

K
m

value for acetate but not ATP, suggesting this residue is involved in binding acetate. Replacement of either E32 or E334 had no significant effects on the kinetic constants, which indicates that neither residue is essential for catalysis or significantly influences the binding of acetate or ATP.
Chromosomal Regions Specific to Pathogenic Isolates of
Escherichia coli
Have a Phylogenetically Clustered Distribution
Authors: Boyd E. Fidelma , Hartl Daniel L. 
Published: December 2019

ABSTRACT

We studied the ancestry of virulence-associated genes in
Escherichia coli
by examining chromosomal regions specific to pathogenic isolates. The four virulence determinants examined were the alpha-hemolysin (
hly
) loci
hlyI
and
hlyII
, the type II capsule gene cluster
kps
, and the P (
pap
) and S (
sfa
) fimbria gene clusters. All four loci were shown previously to be associated with pathogenicity islands of uropathogenic
E. coli
isolates. The
hly
,
kps
,
sfa
, and
pap
regions each have an unexpected clustered distribution among the
E. coli
collection of reference (ECOR) strains, but all these regions were absent from a collection of diarrheagenic
E. coli
isolates. Strains in the ECOR subgroup B2 typically had a combination of at least three of the four loci, and all strains in subgroup D had a copy of the
kps
and
pap
clusters. In contrast, only four strains in subgroup A had either
hly
,
kps
,
sfa
, or
pap
, and no subgroup A strains had all four together. Strains of subgroup B1 were devoid of all four virulence regions, with the exception of one isolate that had a copy of the
sfa
gene cluster. This phylogenetic distribution of strain-specific sequences corresponds to the ECOR groups with the largest genome size, namely, B2 and D. We propose that the pathogenicity islands are ancestral to subgroups B2 and D and were acquired after speciation, with subsequent horizontal transfer into some group A, B1, and E lineages. These results suggest that the
hly
,
kps
,
sfa
, and
pap
pathogenicity determinants may play a role in the evolution of enteric bacteria quite apart from, and perhaps with precedence over, their ability to cause disease.
The
nac
(Nitrogen Assimilation Control) Gene from
Escherichia coli
Authors: Muse Wilson B. , Bender Robert A. 
Published: December 2019

ABSTRACT

The nitrogen assimilation control gene,
nac
, was detected in
Escherichia coli
but not in
Salmonella typhimurium
by Southern blotting, using a probe from the
Klebsiella aerogenes nac
(

nac
K

) gene. The
E. coli nac
gene (

nac
E

) was isolated from a cosmid clone by complementation of a
nac
mutation in

K. aerogenes. nac
E

was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence between

nac
E

and

nac
K

, with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence. The total predicted size of NAC
E
is 305 amino acids, the same as for NAC
K
. A null mutation,
nac-28
, was generated by reverse genetics. Mutants bearing
nac-28
have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source. In addition to a loss of nitrogen regulation of histidase formation,
nac-28
mutants also showed a loss of a weak repression of glutamate dehydrogenase formation. This repression was unexpected because it is balanced by a NAC-independent activation of glutamate dehydrogenase formation during nitrogen-limited growth. Attempts to purify NAC
E
by using methods established for NAC
K
failed, and NAC
E
appears to be degraded with a half-life at 30°C as short as 15 min during inhibition of protein synthesis.