bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcriptional Activation of
ydeA
, Which Encodes a Member of the Major Facilitator Superfamily, Interferes with Arabinose Accumulation and Induction of the
Escherichia coli
Arabinose P
BAD
Promoter
Authors: Bost Sandrine , Silva Filo , Belin Dominique 
Published: December 2019

ABSTRACT

Induction of genes expressed from the arabinose P
BAD
promoter is very rapid and maximal at low arabinose concentrations. We describe here two mutations that interfere with the expression of genes cloned under arabinose control. Both mutations map to the
ydeA
promoter and stimulate
ydeA
transcription; overexpression of YdeA from a multicopy plasmid confers the same phenotype. One mutation is a large deletion that creates a more efficient −35 region (ATCACA changed to TTCACA), whereas the other affects the initiation site (TTTT changed to TGTT). The
ydeA
gene is expressed at extremely low levels in exponentially growing wild-type cells and is not induced by arabinose. Disruption of
ydeA
has no detectable effect on cell growth. Thus,
ydeA
appears to be nonessential under usual laboratory growth conditions. The
ydeA
gene encodes a membrane protein with 12 putative transmembrane segments. YdeA belongs to the largest family of bacterial secondary active transporters, the major facilitator superfamily, which includes antibiotic resistance exporters, Lac permease, and the nonessential AraJ protein. Intracellular accumulation of arabinose is strongly decreased in mutant strains overexpressing YdeA, suggesting that YdeA facilitates arabinose export. Consistent with this interpretation, very high arabinose concentrations can compensate for the negative effect of
ydeA
transcriptional activation. Our studies (i) indicate that YdeA, when transcriptionally activated, contributes to the control of the arabinose regulon and (ii) demonstrate a new way to modulate the kinetics of induction of cloned genes.
Mutations That Confer Resistance to 2-Deoxyglucose Reduce the Specific Activity of Hexokinase from
Myxococcus xanthus
Authors: Youderian Philip , Lawes Matthew C. , Creighton Chad , Cook Jessica C. , Saier Milton H. 
Published: December 2019

ABSTRACT

The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of
Myxococcus xanthus
. Mutants of
M. xanthus
resistant to 2dGlc, designated
hex
mutants, arise at a low spontaneous frequency. Expression of the
Escherichia coli glk
(glucokinase) gene in
M. xanthus hex
mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate. Enzyme assays of
M. xanthus
extracts reveal a soluble hexokinase (ATP:
d
-hexose-6-phosphotransferase; EC
2.7.1.1
) activity but no phosphotransferase system activities. The
hex
mutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the
hex
mutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation. Both 2dGlc and
N
-acetylglucosamine act as inhibitors of glucose turnover by the
M. xanthus
hexokinase in vitro, consistent with the finding that glucose and
N
-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo.
Mutational Analysis of the
vacA
Promoter Provides Insight into Gene Transcription in
Helicobacter pylori
Authors: Forsyth Mark H. , Cover Timothy L. 
Published: December 2019

ABSTRACT

Analysis of 12
Helicobacter pylori
promoters indicates the existence of a consensus −10 hexamer (TAtaaT) but little conservation of −35 sequences. In this study, mutations in either the
H. pylori vacA
−10 region or the −35 region resulted in decreased
vacA
transcription and suggested that an extended −10 motif is utilized. Thus, despite the lack of a −35 consensus sequence for
H. pylori
promoters, the −35 region plays a functional role in
vacA
transcription.
Isolation of an Active Catalytic Core of
Streptococcus downei
MFe28 GTF-I Glucosyltransferase
Authors: Monchois Vincent , Arguello-Morales Martha , Russell Roy R. B. 
Published: December 2019

ABSTRACT

Truncated variants of GTF-I from
Streptococcus downei
MFe28 were purified by means of a histidine tag. Sequential deletions showed that the C-terminal domain was not directly involved in the catalytic process but was required for primer activation. A fully active catalytic core of only 100 kDa was isolated.
Regulated Expression of the
Streptococcus mutans dlt
Genes Correlates with Intracellular Polysaccharide Accumulation
Authors: Spatafora Grace A. , Sheets Megan , June Rebecca , Luyimbazi David , Howard Katherine , Hulbert Robin , Barnard Daron , el Janne Mariam , Hudson Michael C. 
Published: December 2019

ABSTRACT

Intracellular polysaccharides (IPS) are glycogen-like storage polymers which contribute significantly to
Streptococcus mutans
-induced cariogenesis. We previously identified and cloned a locus from the
S. mutans
chromosome which is required for the accumulation of IPS. Sequencing of this locus revealed at least four contiguous open reading frames, all of which are preceded by a common promoter region and are transcribed in the same direction. Analysis of the amino acid sequence deduced from the first of these open reading frames (ORF1) revealed domains which are highly conserved among
d
-alanine-activating enzymes (DltA) in
Lactobacillus rhamnosus
(formerly
Lactobacillus casei
) and
Bacillus subtilis
. The deduced amino acid sequences derived from ORF2, -3, and -4 also exhibit extensive similarity to DltB, -C, and -D, respectively, in these microorganisms. However, Southern hybridization experiments indicate that this operon maps to a locus on the
S. mutans
chromosome which is separate from the
glgP
,
glgA
, and
glgD
genes, whose products are known mediators of bacterial IPS accumulation. We therefore assigned a new
dlt
designation to the locus which we had formerly called
glg
. We maintain that the
dlt
genes are involved in
S. mutans
IPS accumulation, however, since they complement a mutation in
trans
which otherwise renders
S. mutans
IPS deficient. In this study, we found that expression of the
S. mutans dlt
genes is growth phase dependent and is modulated by carbohydrates internalized via the phosphoenolpyruvate phosphotransferase system (PTS). We demonstrated that the
S. mutans dlt
genes are expressed constitutively when non-PTS sugars are provided as the sole source of carbohydrate. Consistent with a role for the PTS in
dlt
expression is a similar constitutive expression of the
dlt
genes in an
S. mutans
PTS mutant grown in a chemically defined medium supplemented with glucose. In summary, these findings support a novel role for the
dlt
gene products in
S. mutans
IPS accumulation and suggest that
dlt
expression in this oral pathogen is subject to complex mechanisms of control imposed by growth phase, dietary carbohydrate, and other factors present in the plaque environment.
Promoter Analysis of the
cap8
Operon, Involved in Type 8 Capsular Polysaccharide Production in
Staphylococcus aureus
Authors: Ouyang Shu , Sau Subrata , Lee Chia Y. 
Published: December 2019

ABSTRACT

The production of type 8 capsular polysaccharide (CP8) in
Staphylococcus aureus
is regulated in response to a variety of environmental factors. The
cap8
genes required for the CP8 production in strain Becker are transcribed as a single large transcript by a primary promoter located within a 0.45-kb region upstream of the first gene of the
cap8
gene cluster. In this study, we analyzed the primary
cap8
promoter region in detail. We determined the transcription initiation site of the primary transcript by primer extension and identified the potential promoter sequences. We found several inverted and direct repeats upstream of the promoter. Deletion analysis and site-directed mutagenesis showed that a 10-bp inverted repeat of one of the repeats was required for promoter activity. We showed that the distance but not the specific sequences between the inverted repeat and the promoter was critical to the promoter activity. However, insertion of a DNA sequence with two or four helix turns in this intervening region had a slight effect on promoter activity. To demonstrate the biological significance of the 10-bp inverted repeat, we constructed a strain with a mutation in the repeat in the
S. aureus
Becker chromosome and showed that the repeat affected CP8 production mostly at the transcriptional level. By gel mobility shift assay, we demonstrated that strain Becker produced at least one protein capable of specific binding to the 10-bp inverted repeat, indicating that the repeat serves as a positive regulatory protein binding site. In addition, reporter gene fusion analysis showed that the
cap8
promoter activity was influenced by various growth media and affected most by yeast extract. Our results suggest that yeast extract may exert its profound inhibitory effect on
cap8
gene expression through the 10-bp inverted repeat element.
The Extracellular Domain of the
Saccharomyces cerevisiae
Sln1p Membrane Osmolarity Sensor Is Necessary for Kinase Activity
Authors: Ostrander Darin B. , Gorman Jessica A. 
Published: December 2019

ABSTRACT

The function of the extracellular domain (ECD) of Sln1p, a plasma membrane two-transmembrane domain (TMD) sensor of the high-osmolarity glycerol (HOG) response pathway, has been studied in the yeast
Saccharomyces cerevisiae
. Truncations of
SLN1
that retain an intact kinase domain are capable of complementing the lethality of an
sln1
Δ strain. By observing levels of Hog1p phosphorylation as well as the phosphorylation state of Sln1p, the kinase activities of various
SLN1
constructions were determined. In derivatives that do not contain the first TMD, Sln1p activity was no longer dependent on medium osmolarity but appeared to be constitutively active even under conditions of high osmolarity. Removal of the first TMD (ΔTMD1 construct) gave a protein that was strongly phosphorylated whereas Hog1p was largely dephosphorylated, as expected if the active form of Sln1p is phosphorylated. When both TMDs as well as the ECD were deleted, so that the kinase domain is cytosolic, Sln1p was not phosphorylated whereas Hog1p became constitutively hyperphosphorylated. Surprisingly, this hyperactivity of the HOG mitogen-activated protein kinase signaling pathway was not sufficient to result in cell lethality. When the ECD of the ΔTMD1 construct was replaced with a leucine zipper motif, Sln1p was hyperactive, so that Hog1p became mostly unphosphorylated. In contrast, when the Sln1p/leucine zipper construct was crippled by a mutation of one of the internal leucines, the Sln1 kinase was inactive. These experiments are consistent with the hypothesis that the ECD of Sln1p functions as a dimerization and activation domain but that osmotic regulation of activity requires the presence of the first TMD.
Clathrin and Two Components of the COPII Complex, Sec23p and Sec24p, Could Be Involved in Endocytosis of the
Saccharomyces cerevisiae
Maltose Transporter
Authors: Peñalver Élida , Lucero Pilar , Moreno Eulalia , Lagunas Rosario 
Published: December 2019

ABSTRACT

The
Saccharomyces cerevisiae
maltose transporter is a 12-transmembrane segment protein that under certain physiological conditions is degraded in the vacuole after internalization by endocytosis. Previous studies showed that endocytosis of this protein is dependent on the actin network, is independent of microtubules, and requires the binding of ubiquitin. In this work, we attempted to determine which coat proteins are involved in this endocytosis. Using mutants defective in the heavy chain of clathrin and in several subunits of the COPI and the COPII complexes, we found that clathrin, as well as two cytosolic subunits of COPII, Sec23p and Sec24p, could be involved in internalization of the yeast maltose transporter. The results also indicate that endocytosis of the maltose transporter and of the α-factor receptor could have different requirements.
Bacteroides fragilis
Transfer Factor Tn
5520
: the Smallest Bacterial Mobilizable Transposon Containing Single Integrase and Mobilization Genes That Function in
Escherichia coli
Authors: Vedantam Gayatri , Novicki Thomas J. , Hecht David W. 
Published: December 2019

ABSTRACT

Many bacterial genera, including
Bacteroides
spp., harbor mobilizable transposons, a class of transfer factors that carry genes for conjugal DNA transfer and, in some cases, antibiotic resistance. Mobilizable transposons are capable of inserting into and mobilizing other, nontransferable plasmids and are implicated in the dissemination of antibiotic resistance. This paper presents the isolation and characterization of Tn
5520
, a new mobilizable transposon from
Bacteroides fragilis
LV23. At 4,692 bp, it is the smallest mobilizable transposon reported from any bacterial genus. Tn
5520
was captured from
B. fragilis
LV23 by using the transfer-deficient shuttle vector pGAT400ΔBglII. The termini of Tn
5520
contain a 22-bp imperfect inverted repeat, and transposition does not result in a target site repeat. Tn
5520
also demonstrates insertion site sequence preferences characterized by A-T-rich nucleotide sequences. Tn
5520
has been sequenced in its entirety, and two large open reading frames whose predicted protein products exhibit strong sequence similarity to recombinase-integrase enzymes and mobilization proteins, respectively, have been identified. The transfer, mobilization, and transposition properties of Tn
5520
have been studied, revealing that Tn
5520
mobilizes plasmids in both
B. fragilis
and
Escherichia coli
at high frequency and also transposes in
E. coli.
cis
-Acting Elements Responsible for Low-Temperature-Inducible Expression of the Gene Coding for the Thermolabile Isocitrate Dehydrogenase Isozyme of a Psychrophilic Bacterium,
Vibrio
sp. Strain ABE-1
Authors: Sahara Takehiko , Suzuki Masahiro , Tsuruha Jun-Ichiro , Takada Yasuhiro , Fukunaga Noriyuki 
Published: December 2019

ABSTRACT

Transcriptional control of the low-temperature-inducible
icdII
gene, encoding the thermolabile isocitrate dehydrogenase of a psychrophilic bacterium,
Vibrio
sp. strain ABE-1, was found to be mediated in part by a transcriptional silencer locating at nucleotide positions −560 to −526 upstream from the transcription start site of
icdII
. Deletion of the silencer resulted in a 20-fold-increased level of expression of the gene at low temperature (15°C) but not at high temperature (37°C). In addition, a CCAAT sequence located 2 bases upstream of the −35 region was found to be essential for the low-temperature-inducible expression of the gene. By deletion of this sequence, low-temperature-dependent expression of the gene was completely abolished. The ability of the
icdII
promoter to control the expression of other genes was confirmed by using a fusion gene containing the
icdII
promoter region and the promoterless
icdI
open reading frame, which encodes the non-cold-inducible isocitrate dehydrogenase isozyme of
Vibrio
sp. strain ABE-1.
Escherichia coli
transformants harboring
icdII
acquired an ability to grow rapidly at low temperature.
Acarbose, a Pseudooligosaccharide, Is Transported but Not Metabolized by the Maltose-Maltodextrin System of
Escherichia coli
Authors: Brunkhorst Claudia , Andersen Christian , Schneider Erwin 
Published: December 2019

ABSTRACT

The pseudooligosaccharide acarbose is a potent inhibitor of amylases, glucosidases, and cyclodextrin glycosyltransferase and is clinically used for the treatment of so-called type II or insulin-independent diabetes. The compound consists of an unsaturated aminocyclitol, a deoxyhexose, and a maltose. The unsaturated aminocyclitol moiety (also called valienamine) is primarily responsible for the inhibition of glucosidases. Due to its structural similarity to maltotetraose, we have investigated whether acarbose is recognized as a substrate by the maltose/maltodextrin system of
Escherichia coli
. Acarbose at millimolar concentrations specifically affected the growth of
E. coli
K-12 on maltose as the sole source of carbon and energy. Uptake of radiolabeled maltose was competitively inhibited by acarbose, with a

K
i

of 1.1 μM. Maltose-grown cells transported radiolabeled acarbose, indicating that the compound is recognized as a substrate. Studying the interaction of acarbose with purified maltoporin in black lipid membranes revealed that the kinetics of acarbose binding to LamB is asymmetric. The on-rate of acarbose is approximately 30 times lower when the molecule enters the pore from the extracellular side than when it enters from the periplasmic side. Acarbose could not be utilized as a carbon source since the compound alone was not a substrate of amylomaltase (MalQ) and was only poorly attacked by maltodextrin glucosidase (MalZ).
Differential Regulation of Two Divergent
Sinorhizobium meliloti
Genes for HPII-Like Catalases during Free-Living Growth and Protective Role of Both Catalases during Symbiosis
Authors: Sigaud Samuel , Becquet Vanessa , Frendo Pierre , Puppo Alain , Hérouart Didier 
Published: December 2019

ABSTRACT

Two catalases, KatA and KatB, have been detected in
Sinorhizobium meliloti
growing on rich medium. Here we characterize a new catalase gene encoding a third catalase (KatC). KatC activity was detectable only at the end of the stationary phase in
S. meliloti
growing in minimum medium, whereas KatA activity was found during the exponential phase. Analysis with a
katC-lacZ
fusion demonstrated that
katC
expression is mainly regulated at the transcription level. An increase of catalase activity correlating with KatA induction was detected in bacteroids. A dramatic decrease of nitrogen fixation capacity in a
katA katC
double mutant was observed, suggesting that these catalases are very important for the protection of the nitrogen fixation process.
A Novel Aromatic-Ring-Hydroxylating Dioxygenase from the Diterpenoid-Degrading Bacterium
Pseudomonas abietaniphila
BKME-9
Authors: Martin Vincent J. J. , Mohn William W. 
Published: December 2019

ABSTRACT

Pseudomonas abietaniphila
BKME-9 is able to degrade dehydroabietic acid (DhA) via ring hydroxylation by a novel dioxygenase. The
ditA1
,
ditA2
, and
ditA3
genes, which encode the α and β subunits of the oxygenase and the ferredoxin of the diterpenoid dioxygenase, respectively, were isolated and sequenced. The ferredoxin gene is 9.2 kb upstream of the oxygenase genes and 872 bp upstream of a putative
meta
ring cleavage dioxygenase gene,
ditC
. A Tn
5
insertion in the α subunit gene,
ditA1
, resulted in the accumulation by the mutant strain BKME-941 of the pathway intermediate, 7-oxoDhA. Disruption of the ferredoxin gene,
ditA3
, in wild-type BKME-9 by mutant-allele exchange resulted in a strain (BKME-91) with a phenotype identical to that of the mutant strain BKME-941. Sequence analysis of the putative ferredoxin indicated that it is likely to be a [4Fe-4S]- or [3Fe-4S]-type ferredoxin and not a [2Fe-2S]-type ferredoxin, as found in all previously described ring-hydroxylating dioxygenases. Expression in
Escherichia coli
of
ditA1A2A3
, encoding the diterpenoid dioxygenase without its putative reductase component, resulted in a functional enzyme. The diterpenoid dioxygenase attacks 7-oxoDhA, and not DhA, at C-11 and C-12, producing 7-oxo-11,12-dihydroxy-8,13-abietadien acid, which was identified by
1
H nuclear magnetic resonance, UV-visible light, and high-resolution mass spectrometry. The organization of the genes encoding the various components of the diterpenoid dioxygenase, the phylogenetic distinctiveness of both the α subunit and the ferredoxin component, and the unusual Fe-S cluster of the ferredoxin all suggest that this enzyme belongs to a new class of aromatic ring-hydroxylating dioxygenases.
Glycosyltransferase Domain of Penicillin-Binding Protein 2a from
Streptococcus pneumoniae
Is Membrane Associated
Authors: di Guilmi Anne Marie , Mouz Nicolas , Martin Lydie , Hoskins JoAnn , Jaskunas S. Richard , Dideberg Otto , Vernet Thierry 
Published: December 2019

ABSTRACT

Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to β-lactams in
Streptococcus pneumoniae
is caused by low-affinity PBPs.
S. pneumoniae
PBP 2a belongs to the class A high-molecular-mass PBPs having both glycosyltransferase (GT) and transpeptide (TP) activities. Structural and functional studies of both domains are required to unravel the mechanisms of resistance, a prerequisite for the development of novel antibiotics. The extracellular region of
S. pneumoniae
PBP 2a has been expressed (PBP 2a*) in
Escherichia coli
as a glutathione
S
-transferase fusion protein. The acylation kinetic parameters of PBP 2a* for β-lactams were determined by stopped-flow fluorometry. The acylation efficiency toward benzylpenicillin was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other β-lactams, but not in resistance to benzylpenicillin. The TP domain was purified following limited proteolysis. PBP 2a* required detergents for solubility and interacted with lipid vesicles, while the TP domain was water soluble. We propose that PBP 2a* interacts with the cytoplasmic membrane in a region distinct from its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain.
Requirement of NifX and Other
nif
Proteins for In Vitro Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase
Authors: Shah Vinod K. , Rangaraj Priya , Chatterjee Ranjini , Allen Ronda M. , Roll Jon T. , Roberts Gary P. , Ludden Paul W. 
Published: December 2019

ABSTRACT

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of
Azotobacter vinelandii
defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of
A. vinelandii
DJ42.48 (Δ
nifENX ΔvnfE
) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.