bluuebunny/crossref_metadata_2025_split huggingface dataset:
Response of
hya
Expression to External pH in
Escherichia coli
Authors: King Paul W. , Przybyla Alan E. 
Published: December 2019

ABSTRACT

The
hya
operon of
Escherichia coli
is composed of the genes which synthesize uptake hydrogenase isoenzyme 1 (Hyd1). Although
hya
expression and Hyd1 synthesis occur only under anaerobic conditions, Hyd1 is not essential for growth. In this study we used a
hya′-′lacZ
fusion to characterize parameters of anaerobic growth that maximize
hya
expression in an attempt to further elucidate Hyd1 function. We found that the expression pattern of
hya
followed a decline of external pH. In buffered media where the pH value was set, the onset of
hya
expression initiated earlier in growth and reached a greater peak level in acidic than in alkaline medium. When cultures expressing
hya
were shifted from acidic to alkaline conditions,
hya
expression was arrested; shifting from alkaline to acidic conditions stimulated
hya
expression. Maximal expression of
hya
under all growth conditions required the sigma factor RpoS and transcriptional regulators AppY and ArcA. In the absence of RpoS or AppY, the response of
hya
expression onset to external pH was evident and maximal
hya
levels remained greater in acidic than in alkaline medium. However, the absence of ArcA led to a diminished response of expression onset to external pH and the loss of elevated expression at an acidic external pH. The fermentation end product formate slightly altered
hya
expression levels but was not required for
hya
to respond to external pH. In contrast to
hya
expression, the onset of
hyb
operon expression, encoding uptake hydrogenase isoenzyme 2, was constitutive with respect to external pH. However, external pH did affect
hyb
expression levels, which, in contrast to
hya
, were maximal in alkaline rather than acidic medium.
Regulation of
mga
Transcription in the Group A Streptococcus: Specific Binding of Mga within Its Own Promoter and Evidence for a Negative Regulator
Authors: McIver Kevin S. , Thurman Alec S. , Scott June R. 
Published: December 2019

ABSTRACT

Transcription of
mga
, encoding the multiple virulence gene regulator of the group A streptococcus, is positively autoregulated. This regulation requires a DNA region (Pmga) that contains both a promoter proximal to
mga
(P2) and a promoter located further upstream (P1). To determine if Mga has a direct role in this process, its ability to bind to specific sequences within Pmga was tested. A purified fusion of Mga to the C-terminal end of maltose-binding protein (MBP-Mga), encoded by
malE-mga
, was shown previously to bind to the promoter regions of Mga-regulated genes, including
scpA
and
emm
. We report here that MBP-Mga can function in vivo to regulate
emm
and
mga
. Electrophoretic mobility shift assays and DNase I footprinting were used to demonstrate specific binding of MBP-Mga to two ca. 59-bp binding sites in Pmga centered around bases −108 and −180 from the major P2 start of transcription. Mga binding sites from Pemm and PscpA were shown to compete for binding at the two Pmga sites, suggesting that the same domain of Mga interacts at all of these promoter targets. Deletion of the distal Pmga binding site (site I) in vivo resulted in loss of Mga-dependent transcription from the P2 start. However, the same lesion resulted in an increase in P1 transcription that was independent of Mga. This suggests the existence of a repressor of
mga
transcription with a binding site overlapping those of Mga.
Catabolism of Branched-Chain α-Keto Acids in
Enterococcus faecalis
: the
bkd
Gene Cluster, Enzymes, and Metabolic Route
Authors: Ward Donald E. , Ross R. Paul , van der Weijden Coen C. , Snoep Jacky L. , Claiborne Al 
Published: December 2019

ABSTRACT

Genes encoding a branched-chain α-keto acid dehydrogenase from
Enterococcus faecalis
10C1, E1α (
bkdA
), E1β (
bkdB
), E2 (
bkdC
), and E3 (
bkdD
), were found to reside in the gene cluster
ptb-buk-bkdDABC
. The predicted products of
ptb
and
buk
exhibited significant homology to the phosphotransbutyrylase and butyrate kinase, respectively, from
Clostridium acetobutylicum
. Activity and redox properties of the purified recombinant enzyme encoded by
bkdD
indicate that
E. faecalis
has a lipoamide dehydrogenase that is distinct from the lipoamide dehydrogenase associated with the pyruvate dehydrogenase complex. Specific activity of the
ptb
gene product expressed in
Escherichia coli
was highest with the substrates valeryl-coenzyme A (CoA), isovaleryl-CoA, and isobutyryl-CoA. In cultures, a stoichiometric conversion of α-ketoisocaproate to isovalerate was observed, with a concomitant increase in biomass. We propose that α-ketoisocaproate is converted via the BKDH complex to isovaleryl-CoA and subsequently converted into isovalerate via the combined actions of the
ptb
and
buk
gene products with the concomitant phosphorylation of ADP. In contrast, an
E. faecalis bkd
mutant constructed by disruption of the
bkdA
gene did not benefit from having α-ketoisocaproate in the growth medium, and conversion to isovalerate was less than 2% of the wild-type conversion. It is concluded that the
bkd
gene cluster encodes the enzymes that constitute a catabolic pathway for branched-chain α-keto acids that was previously unidentified in
E. faecalis.
The
Escherichia coli
NadR Regulator Is Endowed with Nicotinamide Mononucleotide Adenylyltransferase Activity
Authors: Raffaelli Nadia , Lorenzi Teresa , Mariani P. Luigi , Emanuelli Monica , Amici Adolfo , Ruggieri Silverio , Magni Giulio 
Published: December 2019

ABSTRACT

The first identification and characterization of a catalytic activity associated with NadR protein is reported. A computer-aided search for sequence similarity revealed the presence in NadR of a 29-residue region highly conserved among known nicotinamide mononucleotide adenylyltransferases. The
Escherichia coli nadR
gene was cloned into a T7-based vector and overexpressed. In addition to functionally specific DNA binding properties, the homogeneous recombinant protein catalyzes NAD synthesis from nicotinamide mononucleotide and ATP.
Acetate Utilization in
Lactococcus lactis
Deficient in Lactate Dehydrogenase: a Rescue Pathway for Maintaining Redox Balance
Authors: Hols Pascal , Ramos Ana , Hugenholtz Jeroen , Delcour Jean , de Vos Willem M. , Santos Helena , Kleerebezem Michiel 
Published: December 2019

ABSTRACT

Acetate was shown to improve glucose fermentation in
Lactococcus lactis
deficient in lactate dehydrogenase.
13
C and
1
H nuclear magnetic resonance studies using [2-
13
C]glucose and [2-
13
C]acetate as substrates demonstrated that acetate was exclusively converted to ethanol. This novel pathway provides an alternative route for NAD
+
regeneration in the absence of lactate dehydrogenase.
Identification of a Plasmid-Borne Locus in
Rhizobium etli
KIM5s Involved in Lipopolysaccharide O-Chain Biosynthesis and Nodulation of
Phaseolus vulgaris
Authors: Vinuesa Pablo , Reuhs Bradley L. , Breton Christelle , Werner Dietrich 
Published: December 2019

ABSTRACT

Screening of derivatives of
Rhizobium etli
KIM5s randomly mutagenized with mTn
5
SS
gusA
30 resulted in the identification of strain KIM-G1. Its rough colony appearance, flocculation in liquid culture, and Ndv
−
Fix
−
phenotype were indicative of a lipopolysaccharide (LPS) defect. Electrophoretic analysis of cell-associated polysaccharides showed that KIM-G1 produces only rough LPS. Composition analysis of purified LPS oligosaccharides from KIM-G1 indicated that it produces an intact LPS core trisaccharide (α-
d
-GalA-1→4[α-
d
-GalA-1→5]-Kdo) and tetrasaccharide (α-
d
-Gal-1→6[α-
d
-GalA-1→4]-α-
d
-Man-1→5Kdo), strongly suggesting that the transposon insertion disrupted a locus involved in O-antigen biosynthesis. Five monosaccharides (Glc, Man, GalA, 3-
O
-Me-6-deoxytalose, and Kdo) were identified as the components of the repeating O unit of the smooth parent strain, KIM5s. Strain KIM-G1 was complemented with a 7.2-kb DNA fragment from KIM5s that, when provided in
trans
on a broad-host-range vector, restored the smooth LPS and the full capacity of nodulation and fixation on its host
Phaseolus vulgaris
. The mTn
5
insertion in KIM-G1 was located at the N terminus of a putative α-glycosyltransferase, which most likely had a polar effect on a putative β-glycosyltransferase located downstream. A third open reading frame with strong homology to sugar epimerases and dehydratases was located upstream of the insertion site. The two glycosyltransferases are strain specific, as suggested by Southern hybridization analysis, and are involved in the synthesis of the variable portion of the LPS, i.e., the O antigen. This newly identified LPS locus was mapped to a 680-kb plasmid and is linked to the
lpsβ2
gene recently reported for
R. etli
CFN42.
Positive Transcriptional Feedback Controls Hydrogenase Expression in
Alcaligenes eutrophus
H16
Authors: Schwartz Edward , Buhrke Thorsten , Gerischer Ulrike , Friedrich Bärbel 
Published: December 2019

ABSTRACT

The protein HoxA is the central regulator of the
Alcaligenes eutrophus
H16
hox
regulon, which encodes two hydrogenases, a nickel permease and several accessory proteins required for hydrogenase biosynthesis. Expression of the regulatory gene
hoxA
was analyzed. Screening of an 8-kb region upstream of
hoxA
with a promoter probe vector localized four promoter activities. One of these was found in the region immediately 5′ of
hoxA
; the others were correlated with the nickel metabolism genes
hypA1
,
hypB1
, and
hypX
. All four activities were independent of HoxA and of the minor transcription factor ς
54
. Translational fusions revealed that
hoxA
is expressed constitutively at low levels. In contrast to these findings, immunoblotting studies revealed a clear fluctuation in the HoxA pool in response to conditions which induce the
hox
regulon. Quantitative transcript assays indicated elevated levels of
hyp
mRNA under hydrogenase-derepressing conditions. Using interposon mutagenesis, we showed that the activity of a remote promoter is required for hydrogenase expression and autotrophic growth. Site-directed mutagenesis revealed that P
MBH
, which directs transcription of the structural genes of the membrane-bound hydrogenase, contributes to the expression of
hoxA
under hydrogenase-derepressing conditions. Thus, expression of the
hox
regulon is governed by a positive feedback loop mediating amplification of the regulator HoxA. These results imply the existence of an unusually large (ca. 17,000-nucleotide) transcript.
Acylhomoserine Lactone Synthase Activity of the
Vibrio fischeri
AinS Protein
Authors: Hanzelka Brian L. , Parsek Matthew R. , Val Dale L. , Dunlap Paul V. , Cronan John E. , Greenberg E. P. 
Published: December 2019

ABSTRACT

Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a
Vibrio fischeri
enzyme that catalyzes the synthesis of
N
-(3-oxohexanoyl)-
l
-homoserine lactone from an acyl-acyl carrier protein and
S
-adenosylmethionine. Another
V. fischeri
gene,
ainS
, directs the synthesis of
N
-octanoylhomoserine lactone. The AinS protein shows no significant sequence similarity with LuxI family members, but it does show sequence similarity with the
Vibrio harveyi
LuxM protein. The
luxM
gene is required for the synthesis of
N
-(3-hydroxybutyryl)-
l
-homoserine lactone. To gain insights about whether AinS and LuxM represent a second family of acylhomoserine lactone synthases, we have purified AinS as a maltose-binding protein (MBP) fusion protein. The purified MBP-AinS fusion protein catalyzed the synthesis of
N
-octanoylhomoserine lactone from
S
-adenosylmethionine and either octanoyl-acyl carrier protein or, to a lesser extent, octanoyl coenzyme A. With the exception that octanoyl coenzyme A served as an acyl substrate for the MBP-AinS fusion protein, the substrates for and reaction kinetics of the MBP-AinS fusion protein were similar to those of the several LuxI family members previously studied. We conclude that AinS is an acylhomoserine lactone synthase and that it represents a second family of such enzymes.
Replacement of the Bacteriophage Mu Strong Gyrase Site and Effect on Mu DNA Replication
Authors: Pato M. L. , Banerjee M. 
Published: December 2019

ABSTRACT

The bacteriophage Mu strong gyrase site (SGS) is required for efficient replicative transposition and functions by promoting the synapsis of prophage termini. To look for other sites which could substitute for the SGS in promoting Mu replication, we have replaced the SGS in the middle of the Mu genome with fragments of DNA from various sources. A central fragment from the transposing virus D108 allowed efficient Mu replication and was shown to contain a strong gyrase site. However, neither the strong gyrase site from the plasmid pSC101 nor the major gyrase site from pBR322 could promote efficient Mu replication, even though the pSC101 site is a stronger gyrase site than the Mu SGS as assayed by cleavage in the presence of gyrase and the quinolone enoxacin. To look for SGS-like sites in the
Escherichia coli
chromosome which might be involved in organizing nucleoid structure, fragments of
E. coli
chromosomal DNA were substituted for the SGS: first, repeat sequences associated with gyrase binding (bacterial interspersed mosaic elements), and, second, random fragments of the entire chromosome. No fragments were found that could replace the SGS in promoting efficient Mu replication. These results demonstrate that the gyrase sites from the transposing phages possess unusual properties and emphasize the need to determine the basis of these properties.
Acetyl Coenzyme A Synthetase (ADP Forming) from the Hyperthermophilic Archaeon
Pyrococcus furiosus
: Identification, Cloning, Separate Expression of the Encoding Genes,
acdAI
and
acdBI
, in
Escherichia coli
, and In Vitro Reconstitution of the Active Heterotetrameric Enzyme from Its Recombinant Subunits
Authors: Musfeldt Meike , Selig Martina , Schönheit Peter 
Published: December 2019

ABSTRACT

Acetyl-coenzyme A (acetyl-CoA) synthetase (ADP forming) represents a novel enzyme in archaea of acetate formation and energy conservation (acetyl-CoA + ADP + P
i
→ acetate + ATP + CoA). Two isoforms of the enzyme have been purified from the hyperthermophile
Pyrococcus furiosus
. Isoform I is a heterotetramer (α
2
β
2
) with an apparent molecular mass of 145 kDa, composed of two subunits, α and β, with apparent molecular masses of 47 and 25 kDa, respectively. By using N-terminal amino acid sequences of both subunits, the encoding genes, designated
acdAI
and
acdBI
, were identified in the genome of
P. furiosus
. The genes were separately overexpressed in
Escherichia coli
, and the recombinant subunits were reconstituted in vitro to the active heterotetrameric enzyme. The purified recombinant enzyme showed molecular and catalytical properties very similar to those shown by acetyl-CoA synthetase (ADP forming) purified from
P. furiosus
.
Two Family B DNA Polymerases from
Aeropyrum pernix
, an Aerobic Hyperthermophilic Crenarchaeote
Authors: Cann Isaac K. O. , Ishino Sonoko , Nomura Norimichi , Sako Yoshihiko , Ishino Yoshizumi 
Published: December 2019

ABSTRACT

DNA polymerase activities in fractionated cell extract of
Aeropyrum pernix
, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected. The activity in fraction I was more heat stable than that in fraction II. Two different genes (
polA
and
polB
) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B). The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3′→5′ exonuclease and polymerase activities. The product of
polA
gene (Pol I) was aphidicolin resistant and heat stable up to 80°C. In contrast, the product of
polB
gene (Pol II) was aphidicolin sensitive and stable at 95°C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II of
Pyrodictium occultum
. The deduced amino acid sequence of
A. pernix
Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomain
Crenarchaeota
has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family.
Cloning and Characterization of a Cryptic Haloacid Dehalogenase fromBurkholderia cepaciaMBA4
Authors: Tsang Jimmy S. H. , Sam Laiju 
Published: December 2019

ABSTRACTBurkholderia cepaciaMBA4 has been shown to produce a single dehalogenase batch culture. Moreover, other cryptic dehalogenases were also detected when the cells were grown in continuous culture. In this paper, we report the cloning and characterization of one of the cryptic dehalogenases in MBA4. This cryptic haloacid dehalogenase, designated Chd1, was expressed constitutively inEscherichia coli. This recombinant Chd1 had a relative molecular weight of 58,000 and existed predominantly as a dimer. The subunits had a relative molecular weight of 27,000. Chd1 exhibited isomer specificity, being active towards thel-isomer of 2-monochloropropionic acid only. The structural gene,chd1, was isolated on a 1.7-kbPstI fragment. This fragment contains a functional promoter, because expression ofchd1inE. coliis orientation independent. The nucleotide sequence of this fragment was determined and characterized. An open reading frame of 840 bp encoding a putative peptide of 280 amino acids was identified. This corresponds closely with the size of the subunit. The nucleotide sequence ofchd1did not show any homology with those of other dehalogenase genes. Comparison of the predicted amino acid sequence, however, shows significant homology, ranging from 42 to 50%, with the amino acid sequences of many other dehalogenases. Chd1 is unusual in having a long leader sequence, a property of periplasmic enzymes.
Nitrite and Nitrous Oxide Reductase Regulation by Nitrogen Oxides in
Rhodobacter sphaeroides
f. sp.
denitrificans
IL106
Authors: Sabaty Monique , Schwintner Carole , Cahors Sandrine , Richaud Pierre , Verméglio Andre 
Published: December 2019

ABSTRACT

We have cloned the
nap
locus encoding the periplasmic nitrate reductase in
Rhodobacter sphaeroides
f. sp.
denitrificans
IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N
2
O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that
R. sphaeroides
f. sp.
denitrificans
IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N
2
O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase.
Interactions of the Integrase Protein of the Conjugative Transposon Tn
916
with Its Specific DNA Binding Sites
Authors: Jia Yunhua , Churchward Gordon 
Published: December 2019

ABSTRACT

The binding of two chimeric proteins, consisting of the N-terminal or C-terminal DNA binding domain of Tn
916
Int fused to maltose binding protein, to specific oligonucleotide substrates was analyzed by gel mobility shift assay. The chimeric protein with the N-terminal domain formed two complexes of different electrophoretic mobilities. The faster-moving complex, whose formation displayed no cooperativity, contained two protein monomers bound to a single DNA molecule. The slower-moving complex, whose formation involved cooperative binding (Hill coefficient > 1.0), contained four protein monomers bound to a single DNA molecule. Methylation interference experiments coupled with the analysis of protein binding to mutant oligonucleotide substrates showed that formation of the faster-moving complex containing two protein monomers required the presence of two 11-bp direct repeats (called DR2) in direct orientation. Formation of the slower-moving complex required only a single DR2 repeat. Binding of the N-terminal domains in vivo could serve to position two Int monomers on the DNA near each end of the transposon and assist in bringing together the ends of the transposon so that excision can occur. The chimeric protein with the C-terminal domain of Int also formed two complexes of different electrophoretic mobilities. The major, slower-moving complex, whose formation involved cooperative binding, contained two protein molecules bound to one DNA molecule. This finding suggested that while the C-terminal domain of Int can bind DNA as a monomer, a cooperative interaction between two monomers of the C-terminal domain may help to bring the ends of the transposon together during excision.
Identification of a Regulator That Controls Stationary-Phase Expression of Catalase-Peroxidase in
Caulobacter crescentus
Authors: Rava Paul S. , Somma Laura , Steinman Howard M. 
Published: December 2019

ABSTRACT

Expression of the catalase-peroxidase of
Caulobacter crescentus
, a gram-negative member of the α subdivision of the
Proteobacteria
, is 50-fold higher in stationary-phase cultures than in exponential cultures. To identify regulators of the starvation response, Tn
5
insertion mutants were isolated with reduced expression of a
katG::lacZ
fusion on glucose starvation. One insertion interrupted an open reading frame encoding a protein with significant amino acid sequence identity to TipA, a helix-turn-helix transcriptional activator in the response of
Streptomyces lividans
to the peptide antibiotic thiostrepton, and lesser sequence similarity to other helix-turn-helix regulators in the MerR family. The
C. crescentus
orthologue of
tipA
was named
skgA
(stationary-phase regulation of
katG
). Stationary-phase expression of
katG
was reduced by 70% in the
skgA
::Tn
5
mutant, and stationary-phase resistance to hydrogen peroxide decreased by a factor of 10. Like the wild type, the
skgA
mutant exhibited starvation-induced cross-resistance to heat and acid shock, entered into the helical morphology that occurs after 9 to 12 days in stationary phase, and during exponential growth induced
katG
in response to hydrogen peroxide challenge. Expression of
skgA
increased 5- to 10-fold in late exponential phase.
skgA
is the first regulator of a starvation-induced stress response identified in
C. crescentus
. SkgA is not a global regulator of the stationary-phase stress response; its action encompasses the oxidative stress-hydrogen peroxide response but not acid or heat responses. Moreover, SkgA is not an alternative ς factor, like RpoS, which controls multiple aspects of starvation-induced cross-resistance to stress in enteric bacteria. These observations raise the possibility that regulation of stationary-phase gene expression in this member of the α subdivision of the
Proteobacteria
is different from that in
Escherichia coli
and other members of the γ subdivision.