bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nitrate and Nitrite Control of Respiratory Nitrate Reduction in Denitrifying
Pseudomonas stutzeri
by a Two-Component Regulatory System Homologous to NarXL of
Escherichia coli
Authors: Härtig Elisabeth , Schiek Ulrike , Vollack Kai-Uwe , Zumft Walter G. 
Published: December 2019

ABSTRACT

Bacterial denitrification is expressed in response to the concurrent exogenous signals of low-oxygen tension and nitrate or one of its reduction products. The mechanism by which nitrate-dependent gene activation is effected was investigated in the denitrifying bacterium
Pseudomonas stutzeri
ATCC 14405. We have identified and isolated from this organism the chromosomal region encoding the two-component sensor-regulator pair NarXL and found that it is linked with the
narG
operon for respiratory nitrate reductase. The same region encodes two putative nitrate or nitrite translocases, NarK and NarC (the latter shows the highest similarity to yeast [
Pichia
] and plant [
Nicotiana
] nitrate transporters), and the nitrate-regulated transcription factor, DnrE, of the FNR family. The roles of NarX and NarL in nitrate respiration were studied with deletion mutants. NarL activated the transcription of
narG
,
narK
, and
dnrE
but did not affect the denitrification regulons for the respiratory substrates nitrite, nitric oxide, and nitrous oxide. The promoters of
narG
,
narK
, and
dnrE
carry sequence motifs, TACYYMT, which correspond to the NarL recognition sequence established for
Escherichia coli
. The cellular response toward nitrate and nitrite was mediated by the sensor protein NarX, which discriminated weakly between these oxyanions. Our data show that the NarXL two-component regulatory system has been incorporated into the bacterial denitrification process of
P. stutzeri
for selective regulation of nitrate respiration.
Functioning of DcuC as the C
4
-Dicarboxylate Carrier during Glucose Fermentation by
Escherichia coli
Authors: Zientz Evelyn , Janausch Ingo G. , Six Stephan , Unden Gottfried 
Published: December 2019

ABSTRACT

The
dcuC
gene of
Escherichia coli
encodes an alternative C
4
-dicarboxylate carrier (DcuC) with low transport activity. The expression of
dcuC
was investigated.
dcuC
was expressed only under anaerobic conditions; nitrate and fumarate caused slight repression and stimulation of expression, respectively. Anaerobic induction depended mainly on the transcriptional regulator FNR. Fumarate stimulation was independent of the fumarate response regulator DcuR. The expression of
dcuC
was not significantly inhibited by glucose, assigning a role to DcuC during glucose fermentation. The inactivation of
dcuC
increased fumarate-succinate exchange and fumarate uptake by DcuA and DcuB, suggesting a preferential function of DcuC in succinate efflux during glucose fermentation. Upon overexpression in a
dcuC
promoter mutant (
dcuC
*), DcuC was able to compensate for DcuA and DcuB in fumarate-succinate exchange and fumarate uptake.
Bacterioferritin A Modulates Catalase A (KatA) Activity and Resistance to Hydrogen Peroxide in
Pseudomonas aeruginosa
Authors: Ma Ju-Fang , Ochsner Urs A. , Klotz Martin G. , Nanayakkara Vagira K. , Howell Michael L. , Johnson Zaiga , Posey James E. , Vasil Michael L. , Monaco John J. , Hassett Daniel J. 
Published: December 2019

ABSTRACT

We have cloned a 3.6-kb genomic DNA fragment from
Pseudomonas aeruginosa
harboring the
rpoA
,
rplQ
,
katA
, and
bfrA
genes. These loci are predicted to encode, respectively, (i) the α subunit of RNA polymerase; (ii) the L17 ribosomal protein; (iii) the major catalase, KatA; and (iv) one of two iron storage proteins called bacterioferritin A (BfrA; cytochrome
b
1
or
b
557
). Our goal was to determine the contributions of KatA and BfrA to the resistance of
P. aeruginosa
to hydrogen peroxide (H
2
O
2
). When provided on a multicopy plasmid, the
P. aeruginosa katA
gene complemented a catalase-deficient strain of
Escherichia coli
. The
katA
gene was found to contain two translational start codons encoding a heteromultimer of ∼160 to 170 kDa and having an apparent

K
m

for H
2
O
2
of 44.7 mM. Isogenic
katA
and
bfrA
mutants were hypersusceptible to H
2
O
2
, while a
katA bfrA
double mutant demonstrated the greatest sensitivity. The
katA
and
katA bfrA
mutants possessed no detectable catalase activity. Interestingly, a
bfrA
mutant expressed only ∼47% the KatA activity of wild-type organisms, despite possessing wild-type
katA
transcription and translation. Plasmids harboring
bfrA
genes encoding BfrA altered at critical amino acids essential for ferroxidase activity could not restore wild-type catalase activity in the
bfrA
mutant. RNase protection assays revealed that
katA
and
bfrA
are on different transcripts, the levels of which are increased by both iron and H
2
O
2
. Mass spectrometry analysis of whole cells revealed no significant difference in total cellular iron levels in the
bfrA
,
katA
, and
katA bfrA
mutants relative to wild-type bacteria. Our results suggest that
P. aeruginosa
BfrA may be required as one source of iron for the heme prosthetic group of KatA and thus for protection against H
2
O
2
.
Biochemical and Molecular Characterization of the
Bacillus subtilis
Acetoin Catabolic Pathway
Authors: Huang Min , Oppermann-Sanio Fred Bernd , Steinbüchel Alexander 
Published: December 2019

ABSTRACT

A recent study indicated that
Bacillus subtilis
catabolizes acetoin by enzymes encoded by the
acu
gene cluster (F. J. Grundy, D. A. Waters, T. Y. Takova, and T. M. Henkin, Mol. Microbiol. 10:259–271, 1993) that are completely different from those in the multicomponent acetoin dehydrogenase enzyme system (AoDH ES) encoded by
aco
gene clusters found before in all other bacteria capable of utilizing acetoin as the sole carbon source for growth. By hybridization with a DNA probe covering
acoA
and
acoB
of the AoDH ES from
Clostridium magnum
, genomic fragments from
B. subtilis
harboring
acoA
,
acoB
,
acoC
,
acoL
, and
acoR
homologous genes were identified, and some of them were functionally expressed in
E. coli
. Furthermore,
acoA
was inactivated in
B. subtilis
by disruptive mutagenesis; these mutants were impaired to express PP
i
-dependent AoDH E1 activity to remove acetoin from the medium and to grow with acetoin as the carbon source. Therefore, acetoin is catabolized in
B. subtilis
by the same mechanism as all other bacteria investigated so far, leaving the function of the previously described
acu
genes obscure.
Coordinate Transcriptional Control in the Hyperthermophilic Archaeon
Sulfolobus solfataricus
Authors: Haseltine Cynthia , Montalvo-Rodriguez Rafael , Bini Elisabetta , Carl Audrey , Blum Paul 
Published: December 2019

ABSTRACT

The existence of a global gene regulatory system in the hyperthermophilic archaeon
Sulfolobus solfataricus
is described. The system is responsive to carbon source quality and acts at the level of transcription to coordinate synthesis of three physically unlinked glycosyl hydrolases implicated in carbohydrate utilization. The specific activities of three enzymes, an α-glucosidase (
malA
), a β-glycosidase (
lacS
), and an α-amylase, were reduced 4-, 20-, and 10-fold, respectively, in response to the addition of supplementary carbon sources to a minimal sucrose medium. Western blot analysis using anti-α-glucosidase and anti-β-glycosidase antibodies indicated that reduced enzyme activities resulted exclusively from decreased enzyme levels. Northern blot analysis of
malA
and
lacS
mRNAs revealed that changes in enzyme abundance arose primarily from reductions in transcript concentrations. Culture conditions precipitating rapid changes in
lacS
gene expression were established to determine the response time of the regulatory system in vivo. Full induction occurred within a single generation whereas full repression occurred more slowly, requiring nearly 38 generations. Since
lacS
mRNA abundance changed much more rapidly in response to a nutrient down shift than to a nutrient up shift, transcript synthesis rather than degradation likely plays a role in the regulatory response.
Ribonucleotide Reduction in
Pseudomonas
Species: Simultaneous Presence of Active Enzymes from Different Classes
Authors: Jordan Albert , Torrents Eduard , Sala Irma , Hellman Ulf , Gibert Isidre , Reichard Peter 
Published: December 2019

ABSTRACT

Three separate classes of ribonucleotide reductases exist in nature. They differ widely in protein structure. Class I enzymes are found in aerobic bacteria and eukaryotes; class II enzymes are found in aerobic and anaerobic bacteria; class III enzymes are found in strict and facultative anaerobic bacteria. Usually, but not always, one organism contains only one or two (in facultative anaerobes) classes. Surprisingly, the genomic sequence of
Pseudomonas aeruginosa
contains sequences for each of the three classes. Here, we show by DNA hybridization that other species of
Pseudomonas
also contain the genes for three classes. Extracts from
P. aeruginosa
and
P. stutzeri
grown aerobically or microaerobically contain active class I and II enzymes, whereas we could not demonstrate class III activity. Unexpectedly, class I activity increased greatly during microaerobic conditions. The enzymes were separated, and the large proteins of the class I enzymes were obtained in close to homogeneous form. The catalytic properties of all enzymes are similar to those of other bacterial reductases. However, the
Pseudomonas
class I reductases required the continuous presence of oxygen during catalysis, unlike the corresponding
Escherichia coli
enzyme but similar to the mouse enzyme. In similarity searches, the amino acid sequence of the class I enzyme of
P. aeruginosa
was more related to that of eukaryotes than to that of
E. coli
or other proteobacteria, with the large protein showing 42% identity to that of the mouse, suggesting the possibility of a horizontal transfer of the gene. The results raise many questions concerning the physiological function and evolution of the three classes in
Pseudomonas
species.
Type 2 NADH Dehydrogenases in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803 Are Involved in Regulation Rather Than Respiration
Authors: Howitt Crispin A. , Udall Pacer K. , Vermaas Wim F. J. 
Published: December 2019

ABSTRACT

Analysis of the genome of
Synechocystis
sp. strain PCC 6803 reveals three open reading frames (
slr0851
,
slr1743
, and
sll1484
) that may code for type 2 NAD(P)H dehydrogenases (NDH-2). The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity. However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in
Synechocystis
sp. strain PCC 6803. The three open reading frames were cloned, and deletion constructs were made for each. An expression construct containing one of the three open reading frames,
slr1743
, was able to functionally complement an
Escherichia coli
mutant lacking both NDH-1s and NDH-2s. Therefore,
slr0851
,
slr1743
, and
sll1484
have been designated
ndbA
,
ndbB
, and
ndbC
, respectively. Strains that lacked one or more of the
ndb
genes were created in wild-type and photosystem (PS) I-less backgrounds. Deletion of
ndb
genes led to small changes in photoautotrophic growth rates and respiratory activities. Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids. No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels. A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity. We suggest that the Ndb proteins in
Synechocystis
sp. strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool.
Temperature-Sensitive Motility of
Sulfolobus acidocaldarius
Influences Population Distribution in Extreme Environments
Authors: Lewus Paul , Ford Roseanne M. 
Published: December 2019

ABSTRACT

A three-dimensional tracking microscope was used to quantify the effects of temperature (50 to 80°C) and pH (2 to 4) on the motility of
Sulfolobus acidocaldarius
, a thermoacidophilic archaeon. Swimming speed and run time increased with temperature but remained relatively unchanged with increasing pH. These results were consistent with reported changes in the rate of respiration of
S. acidocaldarius
as a function of temperature and pH. Cells exhibited a forward-biased turn angle distribution with a mean of 54°. Cell trajectories during a run were in the shape of right-handed helices. A cellular dynamics simulation was used to test the hypothesis that a population of
S. acidocaldarius
cells could distribute preferentially in a spatial temperature gradient due to variation in swimming speed. Simulation results showed that a population of cells could migrate from a higher to a lower temperature in the presence of sharp temperature gradients. This simulation result was achieved without incorporating the ability of cells to sense a temporal thermal gradient; thus, the response was not thermotactic. We postulate that this temperature-sensitive motility is one survival mechanism of
S. acidocaldarius
that allows this organism to move away from lethal hot spots in its hydrothermal environment.
Site-Specific Recombination of Temperate
Myxococcus xanthus
Phage Mx8: Genetic Elements Required for Integration
Authors: Magrini Vincent , Creighton Chad , Youderian Philip 
Published: December 2019

ABSTRACT

Like most temperate bacteriophages, phage Mx8 integrates into a preferred locus on the genome of its host,
Myxococcus xanthus
, by a mechanism of site-specific recombination. The Mx8
int-attP
genes required for integration map within a 2.2-kilobase-pair (kb) fragment of the phage genome. When this fragment is subcloned into a plasmid vector, it facilitates the site-specific integration of the plasmid into the 3′ ends of either of two tandem tRNA
Asp
genes,
trnD1
and
trnD2
, located within the
attB
locus of the
M. xanthus
genome. Although Int-mediated site-specific recombination occurs between
attP
and either
attB1
(within
trnD1
) or
attB2
(within
trnD2
), the
attP × attB1
reaction is highly favored and often is accompanied by a deletion between
attB1
and
attB2
. The
int
gene is the only Mx8 gene required in
trans
for
attP × attB
recombination. The
int
promoter lies within the 106-bp region immediately upstream of one of two alternate GTG start codons, GTG-5208 (GTG at bp 5208) and GTG-5085, for integrase and likely is repressed in the prophage state. All but the C-terminal 30 amino acid residues of the Int protein are required for its ability to mediate
attP × attB
recombination efficiently. The
attP
core lies within the
int
coding sequence, and the product of integration is a prophage in which the 3′ end of
int
is replaced by host sequences. The prophage
intX
gene is predicted to encode an integrase with a different C terminus.
Site-Specific Recombination of Temperate
Myxococcus xanthus
Phage Mx8: Regulation of Integrase Activity by Reversible, Covalent Modification
Authors: Magrini Vincent , Storms Michael L. , Youderian Philip 
Published: December 2019

ABSTRACT

Temperate
Myxococcus xanthus
phage Mx8 integrates into the
attB
locus of the
M. xanthus
genome. The phage attachment site,
attP
, is required in
cis
for integration and lies within the
int
(integrase) coding sequence. Site-specific integration of Mx8 alters the 3′ end of
int
to generate the modified
intX
gene, which encodes a less active form of integrase with a different C terminus. The phage-encoded (Int) form of integrase promotes
attP × attB
recombination more efficiently than
attR × attB
,
attL × attB
, or
attB × attB
recombination. The
attP
and
attB
sites share a common core. Sequences flanking both sides of the
attP
core within the
int
gene are necessary for
attP
function. This information shows that the directionality of the integration reaction depends on arm sequences flanking both sides of the
attP
core. Expression of the
uoi
gene immediately upstream of
int
inhibits integrative (
attP × attB
) recombination, supporting the idea that
uoi
encodes the Mx8 excisionase. Integrase catalyzes a reaction that alters the primary sequence of its gene; the change in the primary amino acid sequence of Mx8 integrase resulting from the reaction that it catalyzes is a novel mechanism by which the reversible, covalent modification of an enzyme is used to regulate its specific activity. The lower specific activity of the prophage-encoded IntX integrase acts to limit excisive site-specific recombination in lysogens carrying a single Mx8 prophage, which are less immune to superinfection than lysogens carrying multiple, tandem prophages. Thus, this mechanism serves to regulate Mx8 site-specific recombination and superinfection immunity coordinately and thereby to preserve the integrity of the lysogenic state.
Nitric Oxide Is a Signal for NNR-Mediated Transcription Activation in
Paracoccus denitrificans
Authors: Van Spanning Rob J. M. , Houben Edith , Reijnders Willem N. M. , Spiro Stephen , Westerhoff Hans V. , Saunders Neil 
Published: December 2019

ABSTRACT

By using the ′
lacZ
gene, the activities of the
nirI
,
nirS
, and
norC
promoters were assayed in the wild type and in NNR-deficient mutants of
Paracoccus denitrificans
grown under various growth conditions. In addition, induction profiles of the three promoters in response to the presence of various nitrogenous oxides were determined. Transcription from the three promoters required the absence of oxygen and the presence both of the transcriptional activator NNR and of nitric oxide. The activity of the
nnr
promoter itself was halved after the cells had been switched from aerobic respiration to denitrification. This response was apparently not a result of autoregulation or of regulation by FnrP, since the
nnr
promoter was as active in the wild-type strain as it was in NNR- or FnrP-deficient mutants.
Swarming by
Pseudomonas syringae
B728a Requires
gacS
(
lemA
) and
gacA
but Not the Acyl-Homoserine Lactone Biosynthetic Gene
ahlI
Authors: Kinscherf Thomas G. , Willis David K. 
Published: December 2019

ABSTRACT

Pseudomonas syringae
pv. syringae B728a, a causal agent of bacterial brown spot on snap beans, swarms with a characteristic dendritic pattern on semisolid (0.4%) agar plates. Filamentation of swarming cells of B728a was not observed. Mutations in either the
gacS
(formerly
lemA
) or
gacA
gene of B728a eliminate the ability of this
P. syringae
isolate to swarm without obvious effects on bacterial motility. Three field isolates showed a similar dependence on
gacS
for swarming. Since
gacS
and
gacA
mutants are known to be deficient in
N
-acyl-
l
-homoserine lactone (acyl-HSL) production, a mutant was constructed by disruption of the
ahlI
gene of B728a. This mutant did not make any acyl-HSL detectable by the
Agrobacterium traG
::
lacZ
reporter system, yet was unaffected in its ability to swarm. Other phenotypes of
gacS
and
gacA
mutations were similarly unaffected in the
ahlI
mutant.
A Novel
Sinorhizobium meliloti
Operon Encodes an α-Glucosidase and a Periplasmic-Binding-Protein-Dependent Transport System for α-Glucosides
Authors: Willis Laura B. , Walker Graham C. 
Published: December 2019

ABSTRACT

The most abundant carbon source transported into legume root nodules is photosynthetically produced sucrose, yet the importance of its metabolism by rhizobia in planta is not yet known. To identify genes involved in sucrose uptake and hydrolysis, we screened a
Sinorhizobium meliloti
genomic library and discovered a segment of
S. meliloti
DNA which allows
Ralstonia eutropha
to grow on the α-glucosides sucrose, maltose, and trehalose. Tn
5
mutagenesis localized the required genes to a 6.8-kb region containing five open reading frames which were named
agl
, for α-glucoside utilization. Four of these (
aglE
,
aglF
,
aglG
, and
aglK
) appear to encode a periplasmic-binding-protein-dependent sugar transport system, and one (
aglA
) appears to encode an α-glucosidase with homology to family 13 of glycosyl hydrolases. Cosmid-borne
agl
genes permit uptake of radiolabeled sucrose into
R. eutropha
cells. Analysis of the properties of
agl
mutants suggests that
S. meliloti
possesses at least one additional α-glucosidase as well as a lower-affinity transport system for α-glucosides. It is possible that the Fix
+
phenotype of
agl
mutants on alfalfa is due to these additional functions. Loci found by DNA sequencing to be adjacent to
aglEFGAK
include a probable regulatory gene (
aglR
),
zwf
and
edd
, which encode the first two enzymes of the Entner-Doudoroff pathway,
pgl
, which shows homology to a gene encoding a putative phosphogluconolactonase, and a novel
Rhizobium
-specific repeat element.
The Genes
rubA
and
rubB
for Alkane Degradation in
Acinetobacter
sp. Strain ADP1 Are in an Operon with
estB
, Encoding an Esterase, and
oxyR
Authors: Geißdörfer Walter , Kok Ruben G. , Ratajczak Andreas , Hellingwerf Klaas J. , Hillen Wolfgang 
Published: December 2019

ABSTRACT

Alkanes are oxidized in
Acinetobacter
sp. strain ADP1 by a three-component alkane monooxygenase, composed of alkane hydroxylase, rubredoxin, and rubredoxin reductase.
rubA
and
rubB
encode rubredoxin and a NAD(P)H-dependent rubredoxin reductase. We demonstrate here that single base pair substitutions in
rubA
or
rubB
lead to defects in alkane degradation, showing that both genes are essential for alkane utilization. Differences in the degradation capacity for hexadecane and dodecane in these mutants are discussed. Two genes,
estB
and
oxyR
, are located downstream of
rubB
, but are not necessary for alkane degradation.
estB
encodes a functional esterase.
oxyR
encodes a LysR-type transcriptional regulator, conferring resistance to hydrogen peroxide.
rubA
,
rubB
,
estB
, and
oxyR
constitute an operon, which is constitutively transcribed from a ς
70
promoter, and an
estB-oxyR
containing message is also transcribed from an internal promoter.
Interaction of
Bacillus subtilis
Fur (Ferric Uptake Repressor) with the
dhb
Operator In Vitro and In Vivo
Authors: Bsat Nada , Helmann John D. 
Published: December 2019

ABSTRACT

Bacillus subtilis
contains three metalloregulatory proteins belonging to the ferric uptake repressor (Fur) family: Fur, Zur, and PerR. We have overproduced and purified Fur protein and analyzed its interaction with the operator region controlling the expression of the dihydroxybenzoate siderophore biosynthesis (
dhb
) operon. The purified protein binds with high affinity and selectivity to the
dhb
regulatory region. DNA binding does not require added iron, nor is binding reduced by dialysis of Fur against EDTA or treatment with Chelex. Fur selectively inhibits transcription from the
dhb
promoter by ς
A
RNA polymerase, even if Fur is added after RNA polymerase holoenzyme. Since neither DNA binding nor inhibition of transcription requires the addition of ferrous ion in vitro, the mechanism by which iron regulates Fur function in vivo is not obvious. Mutagenesis of the
fur
gene reveals that in vivo repression of the
dhb
operon by iron requires His97, a residue thought to be involved in iron sensing in other Fur homologs. Moreover, we identify His96 as a second likely iron ligand, since a His96Ala mutant mediates repression at 50 μM but not at 5 μM iron. Our data lead us to suggest that Fur is able to bind DNA independently of bound iron and that the in vivo role of iron is to counteract the effect of an inhibitory factor, perhaps another metal ion, that antagonizes this DNA-binding activity.