bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of the Carnitine Pathway in
Escherichia coli
: Investigation of the
cai-fix
Divergent Promoter Region
Authors: Buchet Anne , Eichler Knut , Mandrand-Berthelot Marie-Andrée 
Published: December 2019

ABSTRACT

The divergent structural operons
caiTABCDE
and
fixABCX
of
Escherichia coli
are required for anaerobic carnitine metabolism. Transcriptional monocopy
lacZ
fusion studies showed that both operons are coexpressed during anaerobic growth in the presence of carnitine, respond to common environmental stimuli (like glucose and nitrate), and are modulated positively by the same general regulators, CRP and FNR, and negatively by H-NS. Overproduction of the CaiF specific regulatory protein mediating the carnitine signal restored induction in an
fnr
mutant, corresponding to its role as the primary target for anaerobiosis. Transcript analysis identified two divergent transcription start points initiating 289 bp apart. DNase I footprinting revealed three sites with various affinities for the binding of the cAMP-CRP complex inside this regulatory region. Site-directed mutagenesis experiments indicated that previously reported perfect CRP motif 1, centered at −41.5 of the
cai
transcriptional start site, plays a direct role in the sole
cai
activation. In contrast, mutation in CRP site 2, positioned at −69.5 of the
fix
promoter, caused only a threefold reduction in
fix
expression. Thus, the role of the third CRP site, located at −126.5 of
fix
, might be to reinforce the action of site 2. A critical 50-bp
cis
-acting sequence overlapping the
fix
mRNA start site was found, by deletion analysis, to be necessary for
cai
transcription. This region is thought to be involved in transduction of the signal mediated by the CaiF regulator.
Genetic Instability of the Global Regulator
agr
Explains the Phenotype of the
xpr
Mutation in
Staphylococcus aureus
KSI9051
Authors: McNamara Peter J. , Iandolo John J. 
Published: December 2019

ABSTRACT

Staphylococcus aureus
KSI9051 has a complex mutation that was associated with the aberrant expression of cell surface and extracellular proteins (M. S. Smeltzer, M. E. Hart, and J. J. Iandolo, J. Bacteriol. 61:919–925, 1993). This mutation was named
xpr
, although no specific gene was identified. Here this mutation is referred to as Δ1058::Tn
551
. In this study, we show that in strain KSI9051, the Δ1058::Tn
551
mutation occurred coincidentally with a frameshift in
agrC
that is expected to truncate the sensor component of the known staphylococcal global regulatory locus
agr
. Remarkably, pleiotropic mutations affecting cell surface and extracellular proteins are generated at frequencies approaching 50% upon the transduction of erythromycin resistance (Em
r
) encoded by Δ1058::Tn
551
from
S. aureus
KSI905 back to its parental strain, S6C. Three independent isolates created in the manner of KSI9051 contained mutations within
agrC
. Each isolate had different mutations, suggesting that the transduction of Em
r
encoded by Δ1058::Tn
551
affects the stability of
agrC
in S6C. In similar experiments with strains from an
S. aureus
8325 genetic background, a mutant AgrC phenotype could not be isolated, implying that strain S6 has aberrant genetic behavior. A comparison of the nucleotide sequences of AgrC from several strains revealed seven errors in the GenBank entry for
agr
(
X52543
); these data were confirmed with plasmid pRN6650, the original wild-type clone of
agr.
Isolation of a Periplasmic Molecular Chaperone-Like Protein of
Rhodobacter sphaeroides
f. sp.
denitrificans
That Is Homologous to the Dipeptide Transport Protein DppA of
Escherichia coli
Authors: Matsuzaki Masahiro , Kiso Yuso , Yamamoto Isamu , Satoh Toshio 
Published: December 2019

ABSTRACT

A periplasmic protein has been found to prevent aggregation of the acid-unfolded dimethyl sulfoxide reductase (DMSOR), the periplasmic terminal reductase of dimethyl sulfoxide respiration in the phototroph
Rhodobacter sphaeroides
f. sp.
denitrificans
, in a manner similar to that of the
Escherichia coli
chaperonin GroEL (Matsuzaki et al., Plant Cell Physiol. 37:333–339, 1996). The protein was isolated from the periplasm of the phototroph. It had a molecular mass of 58 kDa and had no subunits. The sequence of 14 amino-terminal residues of the protein was completely identical to that of the periplasmic dipeptide transport protein (DppA) of
E. coli
. The 58-kDa protein prevented aggregation to a degree comparable to that of GroEL on the basis of monomer protein. The 58-kDa protein also decreased aggregation of guanidine hydrochloride-denatured rhodanese, a mitochondrial matrix protein, during its refolding upon dilution. The 58-kDa protein is a kind of molecular chaperone and could be involved in maintaining unfolded DMSOR, after secretion of the latter into the periplasm, in a competent form for its correct folding.
Function of Protonatable Residues in the Flagellar Motor of
Escherichia coli
: a Critical Role for Asp 32 of MotB
Authors: Zhou Jiadong , Sharp Leslie L. , Tang H. Lucy , Lloyd Scott A. , Billings Stephanie , Braun Timothy F. , Blair David F. 
Published: December 2019

ABSTRACT
Rotation of the bacterial flagellar motor is powered by a transmembrane gradient of protons or, in some species, sodium ions. The molecular mechanism of coupling between ion flow and motor rotation is not understood. The proteins most closely involved in motor rotation are MotA, MotB, and FliG. MotA and MotB are transmembrane proteins that function in transmembrane proton conduction and that are believed to form the stator. FliG is a soluble protein located on the cytoplasmic face of the rotor. Two other proteins, FliM and FliN, are known to bind to FliG and have also been suggested to be involved to some extent in torque generation. Proton (or sodium)-binding sites in the motor are likely to be important to its function and might be formed from the side chains of acidic residues. To investigate the role of acidic residues in the function of the flagellar motor, we mutated each of the conserved acidic residues in the five proteins that have been suggested to be involved in torque generation and measured the effects on motility. None of the conserved acidic residues of MotA, FliG, FliM, or FliN proved essential for torque generation. An acidic residue at position 32 of MotB did prove essential. Of 15 different substitutions studied at this position, only the conservative-replacement D32E mutant retained any function. Previous studies, together with additional data presented here, indicate that the proteins involved in motor rotation do not contain any conserved basic residues that are critical for motor rotation per se. We propose that Asp 32 of MotB functions as a proton-binding site in the bacterial flagellar motor and that no other conserved, protonatable residues function in this capacity.
Role of Alternative ς Factor AlgU in Encystment of
Azotobacter vinelandii
Authors: Moreno Soledad , Nájera Rebeca , Guzmán Josefina , Soberón-Chávez Gloria , Espín Guadalupe 
Published: December 2019

ABSTRACT

Alginate is essential for encystment in
Azotobacter vinelandii
. Transcription of the
algD
gene, which codes for GDP-mannose dehydrogenase, a key enzyme in the alginate biosynthetic pathway, is initiated at two promoters, one of which, p2, has ς
E
consensus sequences. AlgU is the
A. vinelandii
alternative ς
E
factor. In this study, we constructed an
algU
mutant (SMU88) which, as expected, is impaired in alginate production, encystment, and transcription of the
algD
gene from the p2 promoter. Plasmid pJMSAT1, carrying the
A. vinelandii algU
gene, restored alginate production and encystment to SMU88 and to strain UW136, a naturally occurring
algU
mutant. Plasmid pSMU865, carrying the
A. vinelandii mucABCD
genes coding for negative regulators of AlgU activity and previously shown to diminish alginate production in the wild-type strain, ATCC 9046, was shown here to impair encystment and transcription of the
algD
gene from the p2
algU
-dependent promoter. Since nonencysting strain ATCC 9046/pSMU865 produced more alginate than some encysting strains, such as UW136/pJMSAT1, we propose an AlgU role in encystment, independent of the structural role that alginate plays in mature cysts.
Expression of the Transposase Gene
tnpA
of Tn
4652
Is Positively Affected by Integration Host Factor
Authors: Hõrak Rita , Kivisaar Maia 
Published: December 2019

ABSTRACT

Tn
4652
is a derivative of the toluene degradation transposon Tn
4651
that belongs to the Tn
3
family of transposons (M. Tsuda and T. Iino, Mol. Gen. Genet. 210:270–276, 1987). We have sequenced the transposase gene
tnpA
of transposon Tn
4652
and mapped its promoter to the right end of the element. The deduced amino acid sequence of
tnpA
revealed 96.2% identity with the putative transposase of Tn
5041
. Homology with other Tn
3
family transposases was only moderate (about 20 to 24% identity), suggesting that Tn
4652
and Tn
5041
are distantly related members of the Tn
3
family. Functional analysis of the
tnpA
promoter revealed that it is active in
Pseudomonas putida
but silent in
Escherichia coli
, indicating that some
P. putida
-specific factor is required for the transcription from this promoter. Additionally,
tnpA
promoter activity was shown to be modulated by integration host factor (IHF). The presence of an IHF-binding site upstream of the
tnpA
promoter enhanced the promoter activity. The positive role of IHF was also confirmed by the finding that the enhancing effect of IHF was not detected in the
P. putida ihfA
-deficient strain A8759. Moreover, the Tn
4652
terminal sequences had a negative effect on transcription from the
tnpA
promoter in the
ihfA
-defective strain. This finding suggests that IHF not only enhances transcription from the
tnpA
promoter but also alleviates the negative effect of terminal sequences of Tn
4652
on the promoter activity. Also, an in vitro binding assay demonstrated that both ends of Tn
4652
bind IHF from a cell lysate of
E. coli.
Effects of Iron and Temperature on Expression of the
Pseudomonas aeruginosa tolQRA
Genes: Role of the Ferric Uptake Regulator
Authors: Lafontaine Eric R. , Sokol Pamela A. 
Published: December 2019

ABSTRACT

The
tolQRA
genes have been recently identified in
Pseudomonas aeruginosa
PAO. In this study, we examined the effect of iron and temperature on
tolQRA
expression. A promoterless
lacZ
gene was introduced downstream of plasmid-encoded
tolQ
and
tolA
, and expression was monitored by measuring β-galactosidase activity of cultures. Addition of 25 μM FeCl
3
to the culture medium reduced
tolQRA
expression by 50 to 60% in PAO but by only 25% in the
fur
mutant PAO A4. Northern hybridization analysis revealed that iron regulation occurs at the level of transcription and involves the
P. aeruginosa
ferric uptake regulator (Fur). Primer extension analysis was used to identify the proposed transcriptional start site of
tolA
. Although a putative Fur box was identified 20 bp upstream of the proposed start site, purified Fur did not bind to the
tolA
or
tolQR
promoter regions in an in vitro gel retardation assay. Therefore, iron regulation of the
tol
genes appears to involve an intermediate regulatory gene. Expression of
tolQR
and
tolA
was optimal at 37°C and was reduced by 40 to 50% when cultures were grown at either 42 or 25°C. Growth in high-iron medium at 25°C further reduced
tolQR
and
tolA
expression.
Identification and Characterization of
MAE1
, the
Saccharomyces cerevisiae
Structural Gene Encoding Mitochondrial Malic Enzyme
Authors: Boles Eckhard , de Jong-Gubbels Patricia , Pronk Jack T. 
Published: December 2019

ABSTRACT

Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless,
pyk1 pyk2
mutants of
Saccharomyces cerevisiae
devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame
YKL029c
, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression of
YKL029c/MAE1
from the
MET25
promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of
MAE1
alone did not result in a clear phenotype. Regulation of
MAE1
was studied by determining enzyme activities and
MAE1
mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a
MAE1
::
lacZ
fusion. Both in shake flask cultures and in carbon-limited chemostat cultures,
MAE1
was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in
S. cerevisiae
is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.
d-Alanylcardiolipin, a Major Component of the Unique Lipid Pattern ofVagococcus fluvialis
Authors: Fischer Werner , Arneth-Seifert Doris 
Published: December 2019

ABSTRACTMotile group N streptococci, classified asVagococcus fluvialis, have been isolated from cows’ udders, human and animal feces, river water, and seawater. They possess an unusual membrane lipid and fatty acid pattern. We isolated and characterized 13 polar lipids, 8 of them also found in other gram-positive bacteria: mono- and dihexosyldiacylglycerol, an acylated and a glycerophosphate-substituted derivative of the latter, cardiolipin, phosphatidylglycerol,d-alanylphosphatidylglycerol, andl-lysylphosphatidylglycerol. Besides them, we characterized two rare compounds, bis(acylglycero)phosphate and α-d-glucopyranosylcardiolipin, and two compounds so far not detected in nature,d-alanylbis(acylglycero)phosphate andd-alanylcardiolipin. The concomitant occurrence of four aminoacyl phospholipids in one organism is another unique finding. Substituted cardiolipins represent a novel lipid class: in vagococci,d-alanylcardiolipin is a major membrane lipid component, contributing 11 and 26 mol% of total lipids in the exponential and stationary phases of growth, respectively. The vagococcal lipids contain even-numbered straight-chain saturated andcis-monounsaturated fatty acids, but thecis-monoenic acids belong to the ω-9 series and not the ω-7 series, found in enterococci, lactococci, and streptococci.
Characterization of Chemotactic Responses and Flagella of
Hyphomicrobium
Strain W1-1B
Authors: Tuhela Laura , Robinson Jayne B. , Tuovinen Olli H. 
Published: December 2019

ABSTRACT

Motile swarmer cells of
Hyphomicrobium
strain W1-1B displayed positive chemotactic responses toward methylamine, dimethylamine, and trimethylamine but did not display significant chemotactic responses towards methanol and arginine. Electron micrographs of negatively stained intact flagellar filaments indicated a novel striated surface pattern. The flagella were composed of two proteins of 39 and 41 kDa. Neither protein was a glycoprotein as determined by Schiff’s staining and by enzyme immunoassay. Protein fingerprints visualized from silver-stained polyacrylamide gels and Western blots of protease-digested samples indicated that the two proteins were similar but not identical. Monoclonal antibodies prepared to the complex flagella of
Rhizobium meliloti
cross-reacted with the striated flagella of
Hyphomicrobium
strain W1-1B; however, these antibodies did not cross-react with smooth-surface flagella. These results suggest that complex and striated flagella possess homologous epitope regions.
Analysis of
cis
-Acting Elements Required for
bfpA
Expression in Enteropathogenic
Escherichia coli
Authors: Bustamante Victor H. , Calva Edmundo , Puente Jose Luis 
Published: December 2019

ABSTRACT

bfpA
expression in enteropathogenic
Escherichia coli
is regulated by growth medium, temperature, and ammonium concentration and requires the BfpT protein (also called PerA), a member of the AraC family of transcriptional activators. Site-directed and PCR random mutagenesis, as well as deletion analysis of the
bfpA
upstream regulatory region, supported assignment of the promoter elements and demonstrated that the
cis
-acting elements that mediate BfpT-dependent regulation of
bfpA
are located between positions −85 and −46. Interestingly, this region shares 73% identity with a 40-bp-long AT-rich tract located upstream of the
bfpT
gene, which is essential for
bfpT
autoregulation.
Changes in Ribosomal Activity of
Escherichia coli
Cells during Prolonged Culture in Sea Salts Medium
Authors: Kalpaxis Dimitrios L. , Karahalios Panagiotis , Papapetropoulou M. 
Published: December 2019

ABSTRACT

The activity of ribosomes from a clinical isolate of
Escherichia coli
, exposed to starvation for 7 days in sea salts medium, was investigated by measuring the kinetic parameters of ribosomal peptidyltransferase, by using the puromycin reaction as a model reaction. No alterations in the extent of peptide bond formation were observed during starvation. In contrast, a 50% reduction in the
k
max
/

K
s

ratio could be seen after 24 h of starvation; an additional 6 days of starvation resulted in a progressive but less abrupt decline in the
k
max
/

K
s

value. {
k
max
is the apparent catalytic rate constant of peptidyl transferase, and

K
s

is the dissociation constant of the encounter complex between acetyl (Ac)[
3
H]Phe-tRNA-poly(U)-ribosome and puromycin.} Although the distribution of ribosomal particles remained constant, a substantial decrease in the number of ribosomes per starved cell and a clear decline in the ability of ribosomes to bind AcPhe-tRNA were observed, particularly during the first day of starvation. Further analysis indicated that rRNA in general, but especially 23S rRNA, was rapidly degraded during the starvation period. In addition, the L12/L7 molar ratio decreased from 1.5 to 1 during the initial phase of starvation (up to 24 h) but remained constant during the subsequent starvation period. Ribosomes isolated from 24-h-starved cells, when artificially depleted of L7/L12 protein and reconstituted with L7/L12 protein from mid-logarithmic-phase cells, regenerated an L12/L7 molar ratio of 1.5 and restored the peptidyltransferase activity to a substantial level. An analogous effect of reconstitution on the efficiency of ribosomes in binding AcPhe-tRNA was evident not only during the initial phase but throughout the starvation period.
OpaR, a Homolog of
Vibrio harveyi
LuxR, Controls Opacity of
Vibrio parahaemolyticus
Authors: McCarter Linda L. 
Published: December 2019

ABSTRACT

Vibrio parahaemolyticus
is an organism well adapted to communal life on surfaces. When grown on a surface or in a viscous layer, the bacterium induces a large gene system and differentiates to swarmer cells capable of movement over and colonization of surfaces.
V. parahaemolyticus
displays additional phenotypic versatility manifested as variable colony morphology, switching between translucent and opaque colony types. Although not itself luminescent,
V. parahaemolyticus
produces autoinducer molecules capable of inducing luminescence in
Vibrio harveyi
. To examine the role of quorum signaling in the lifestyles of
V. parahaemolyticus
, the functional homolog of the gene encoding the
V. harveyi
autoinducer-controlled transcriptional regulatory protein LuxR was cloned. Sequence analysis of the clone predicted an open reading frame with a deduced product 96% identical to LuxR. Introduction of the clone carrying the
luxR
-like locus into
V. parahaemolyticus
dramatically affected colony morphology, converting a translucent strain to an opaque one. When the coding sequence for the
luxR
homolog was placed under the control of the Ptac promoter, conversion to the opaque phenotype became inducible by isopropyl-β-
d
-thiogalactopyranoside. Allelic disruption of the
luxR
-like gene on the chromosome of an opaque strain produced a translucent strain proficient in swarming ability. Primer extension mapping demonstrated
opaR
transcription in opaque but not translucent cell types. It is postulated that this gene, which has been named
opaR
, encodes a transcription factor controlling cell type. The underlying genetic basis for opaque-translucent variation may be the consequence of a genomic alteration detected in the
opaR
locus of opaque and translucent strains.
Multiple Transcriptional Control of the
Lactococcus lactis trp
Operon
Authors: Raya Raul , Bardowski Jacek , Andersen Paal S. , Ehrlich S. Dusko , Chopin Alain 
Published: December 2019

ABSTRACT

The
Lactococcus lactis trpEGDCFBA
operon is preceded by a noncoding leader region. Transcriptional studies of the
trp
operon revealed three transcripts with respective sizes of 8 kb (encompassing the entire operon), 290 bases, and 160 bases (corresponding to parts of the leader region). These transcripts most likely result from initiation at the unique P

trp

promoter, transcription termination at either T1 (upstream of the
trp
operon) or T2 (downstream of the
trp
operon), and/or processing. Three parameters were shown to differentially affect the amount of these transcripts: (i) following tryptophan depletion, the amount of the 8-kb transcript increases 300- to 500-fold; (ii) depletion in any amino acid increased transcription initiation about fourfold; and (iii) upon entry into stationary phase the amount of the 8-kb transcript decreases abruptly. The tryptophan-dependent transcription control is exerted through transcription antitermination.
Cloning, Sequencing, and Characterization of the
recA
Gene from
Rhodopseudomonas viridis
and Construction of a
recA
Strain
Authors: Chen I-Peng , Michel Hartmut 
Published: December 2019

ABSTRACT

A recombination-deficient strain of the phototrophic bacterium
Rhodopseudomonas viridis
was constructed for the homologous expression of modified photosynthetic reaction center genes. The
R. viridis recA
gene was cloned and subsequently deleted from the
R. viridis
genome. The cloned
R. viridis recA
gene shows high identity to known
recA
genes and was able to complement the Rec
−
phenotype of a
Rhizobium meliloti recA
strain. The constructed
R. viridis recA
strain showed the general Rec
−
phenotype, i.e., increased sensitivity to DNA damage and severely impaired recombination ability. The latter property of this strain will be of advantage in particular for expression of modified, nonfunctional photosynthetic reaction centers which are not as yet available.